Charge-modified designed repeating domains and uses thereof

Abstract

The present invention relates to designed ankyrin repeat domains having a reduced isoelectric point (pI) and / or having a reduced number of basic amino acid residues. The invention also provides such repeat domains linked to a drug moiety, such as a radionuclide or cytotoxic agent. The invention also provides methods for producing such repeat domains, and the use of such repeat domains in therapeutic and / or diagnostic methods. Furthermore, the invention provides recombinant proteins comprising such repeat domains, nucleic acids encoding such repeat domains or recombinant proteins, pharmaceutical compositions comprising such repeat domains, recombinant protein nucleic acids, recombinant expression vectors and host cells, and the use of such proteins, nucleic acids or pharmaceutical compositions in methods for the treatment of diseases such as cancer.

Description

[0001] Cross-references to related patent applications

[0002] This application claims the benefit of priority to EP22188160 filed on August 1, 2022; EP23151023 filed on January 10, 2023; and EP23168056 filed on April 14, 2023. The disclosures of these patent applications are incorporated herein by reference in their entirety for all purposes. Technical Field

[0003] The present invention relates to designed ankyrin repeat domains having a reduced isoelectric point (pI) and / or having a reduced number of basic amino acid residues. The present invention also provides such repeat domains connected to a drug moiety, such as a radionuclide or a cytotoxic agent. The present invention also provides methods for producing such repeat domains, and the use of such repeat domains in treatment and / or diagnosis methods. In addition, the present invention provides recombinant proteins comprising such repeat domains, nucleic acids encoding such repeat domains or recombinant proteins, pharmaceutical compositions comprising such repeat domains, recombinant protein nucleic acids, recombinant expression vectors and host cells, and the use of such proteins, nucleic acids or pharmaceutical compositions in methods for treating diseases such as cancer. Background Art

[0004] Targeted radiopharmaceuticals have become promising tools in the diagnosis and treatment of cancer. Such radiopharmaceuticals are usually composed of radioactive agents (e.g., radionuclides) connected to binding molecules (e.g., antibodies or fragments thereof, engineered protein scaffolds, peptides, or small molecules). By combining the specificity of biological targets with ionizing radiation sources, radiopharmaceuticals can focus radiation emission near the biomarker of interest. High target selectivity and tumor retention, low uptake in non-tumor organs and tissues, and rapid clearance are the features required for accurate cancer diagnosis (e.g., radioactive agents show tumor location) and / or treatment (e.g., radioactive agents damage tumors) with radiopharmaceuticals.

[0005] Early work in radioimmunotherapy emphasized the important disadvantages of using radiolabeled antibodies, such as due to its size (150kDa), the slow extravasation and slow removal of complete antibodies from the blood. Using antibody-based radiopharmaceuticals, the optimal tumor background ratio can usually only be reached after a few days, thus inducing indirect damage to radiosensitive organs and tissues such as bone marrow. Subsequently, alternative conjugates with lower molecular weights were developed to improve pharmacokinetics and increase the dose ratio of tumors to normal tissues. In the case of radiolabeled peptides (including small antibody fragments), drug clearance is mainly driven by renal excretion involving glomerular filtration, active tubular secretion and tubular reabsorption. Glomerular filtration ensures that the valuable macromolecular components of circulating cells and plasma are selectively retained based on molecular size. Molecules with a weight greater than 70kDa or a radius greater than 4.2nm and those combined with plasma proteins (such as albumin) experience negligible glomerular filtration (Parihar, AS et al., Translational Oncology 15.1 (2022): 101295).

[0006] Several radiopharmaceuticals are retained in the kidney due to their inherent properties, thereby contributing to the increase in the radiation absorption dose of the kidney. Although miniaturized binding molecules with lower molecular weight can provide the combined advantages of rapid targeting and rapid clearance and minimal uptake in normal tissues or organs in the early stage after injection, their use also induces undesirable high kidney radioactivity accumulation, thereby hindering their wider clinical application. The selection of radionuclides and the properties of binding molecules may affect the severity of nephrotoxicity caused by such high radioactivity accumulation in the kidney (Chigoho, DM et al., Currentopinion in chemical biology 63 (2021): 219-228).

[0007] The key factor of nephrotoxicity is the process of renal reabsorption. Radioactive labeled molecules of low to medium molecular weight are easily filtered through the glomerulus, then attracted to the negatively charged phospholipid bilayer of proximal tubular cells via charge and reabsorbed, and then decomposed in the cell. After proteolytic degradation in lysosomes, radioactive labeled catabolites are released and are freely washed out of the cell (non-residual radionuclides) or retained in the cell (residual radionuclides) according to their physical properties. From the viewpoint of tumor cell toxicity, residual radionuclides are generally favorable, but due to off-target positioning in normal tissues, toxicity characteristics may be increased.

[0008] To mitigate such renal retention, pharmacological and / or physicochemical approaches have been attempted, such as inhibiting the reabsorption of the radiotracer from the negatively charged proximal tubule membrane by co-injection of positively charged amino acids and / or modulating the net charge of the binding protein used in the conjugate by acylation, respectively.99m Renal clearance and tumor targeting of Tc-labeled humanized anti-Tac monoclonal antibody Fab can be enhanced by acylation to neutralize the positive charge of Fab. In addition, co-injection of lysine further improved the blockade of renal uptake (Kim, MK et al., Nuclear medicine and biology, 29.2 (2002): 139-146). In addition, the effect of single amino acid substitutions on renal radioactivity accumulation has been studied by Akizawa et al. in a very short 8 amino acid peptide ( 111 In-DTPA conjugated octreotide derivatives) were studied in the context of the study, but no larger binding domains or proteins were studied (Akizawa, H. et al., Nuclear medicine and biology 28.7 (2001): 761-768).

[0009] Pharmacological approaches to reduce the uptake of peptide- and protein-based radiopharmaceuticals in the kidney may help circumvent the efforts required to modulate the structural properties of the radiolabeled compounds. However, administration of such mitigating compounds, which act on various parts of the reabsorption system in the kidney, has been shown not to be effective for all radiopharmaceuticals. 99m Studies of renal uptake of Tc-labeled designed ankyrin repeat proteins (DARPins) showed that common clinical strategies were not effective for reducing renal uptake of these radiolabeled DARPins in mice. More specifically, co-injection of lysine or Gelofusine did not reduce renal uptake. Pre-administration of high doses of maleate or fructose (which inhibit ATP-mediated endocytosis) resulted in reduced renal uptake of these protein scaffolds, but the doses required were not suitable for clinical application. None of the other compounds were effective. Thus, according to the authors, the study suggests that 99m Renal uptake of Tc-labeled DARPins occurs via a mechanism that is independent of DARPin structure and binding site composition. (Altai, M. et al., EJNMMI research 10.1 (2020): 1-8). Therefore, despite attempts to reduce renal uptake and mitigate nephrotoxicity using pharmacological and / or physicochemical methods, this cannot be consistently achieved and nephrotoxicity remains an obstacle to the use of radiopharmaceuticals in radiopharmaceutical therapy or diagnosis. Similar considerations apply to drugs in which miniaturized target-specific binders are linked to cytotoxic agents rather than radionuclides, such as in protein conjugates using antibody mimics or small binding moieties (Richards, DADrug Discovery Today: Technologies 30 (2018): 35-46).

[0010] In summary, there remains a need for improved target-specific binders with beneficial properties that, when administered to a mammal, result in reduced renal accumulation, particularly reduced renal accumulation of a linked drug moiety such as a radionuclide or cytotoxic agent. Such improved target-specific binders may be used in therapeutic or diagnostic applications, for example, for radiopharmaceutical or cytotoxic drug conjugates. Summary of the invention

[0011] The present invention provides engineered DARPins with a reduced number of basic amino acids (positive charge) and / or a lower isoelectric point (pi) compared to conventional DARPins. It has been surprisingly found that these properties reduce renal accumulation of attached drug moieties (e.g., radionuclides) following in vivo administration of the drug moiety-attached DARPins of the present invention.

[0012] DARPins are small engineered scaffold proteins (approximately 14 kDa for a single designed repeat domain) that can be selected to bind a given target protein with high affinity and specificity. Their use in the context of radiopharmaceutical therapy or diagnosis has begun to be investigated, for example in studies involving radiopharmaceuticals for breast cancer imaging. 99m In a Phase I clinical trial of Tc-labeled DARPin (Bragina, O. et al., Journal of Nuclear Medicine 63.4 (2022): 528-535). However, as with other radiopharmaceuticals based on low molecular weight conjugates, solutions to nephrotoxicity are needed to fully utilize the potential of DARPin-based radiopharmaceuticals. For example, modification of the net charge of DARPin has been described in WO2016023898 (in this case, in a method for producing a DARPin that can pass through the lipophilic membrane barrier of a cell, the DARPin is modified to be less negative), but modification of the charge properties or pI of DARPin has not been evaluated in the context of renal accumulation.

[0013] The present invention relates to designed ankyrin repeat proteins (DARPins) having structural modifications that improve their pharmacokinetic properties, particularly resulting in reduced renal accumulation. More specifically, the present invention provides designed ankyrin repeat domains having a reduced number of basic amino acid residues and / or a reduced isoelectric point (pI) compared to the commonly described designed ankyrin repeat domains. The present invention also provides such improved repeat domains connected to a drug moiety, such as a radionuclide or a cytotoxic agent. The present invention also provides methods for producing such repeat domains, and the use of such repeat domains in treatment and / or diagnostic methods. In addition, the present invention provides recombinant proteins comprising such repeat domains, nucleic acids encoding such repeat domains or recombinant proteins, pharmaceutical compositions comprising such repeat domains, recombinant protein nucleic acids, recombinant expression vectors and host cells, and the use of such proteins, nucleic acids or pharmaceutical compositions in methods for treating diseases such as cancer in mammals (including humans).

[0014] When linked to a radioactive or cytotoxic agent, the DARPins of the invention exhibit reduced accumulation of the agent in the kidney when administered in vivo. Thus, the improved repeat domains and recombinant proteins provided in the present invention open the door to new therapeutic and diagnostic applications, for example in the field of nuclear medicine, such as in radiopharmaceutical therapy, or in cancer therapy using proteins conjugated to cytotoxic agents.

[0015] In one aspect, the present invention provides a designed ankyrin repeat domain, the designed ankyrin repeat domain comprising an N-terminal capping module, at least one internal repeat module and a C-terminal capping module, wherein the repeat domain has at least one feature selected from the following list of features:

[0016] (a) an isoelectric point (pI) equal to or lower than pH 4.07;

[0017] (b) a percentage of basic amino acid residues of 7.0% or less among all amino acid residues contained in the repeat domain; and

[0018] (c) Among the framework residues contained in the repeat domain, the percentage of basic amino acid residues is equal to or lower than 6.1%.

[0019] In another aspect, the present invention provides a method for producing a modified ankyrin repeat domain, the method comprising steps (a) and (b),

[0020] wherein step (a) is to provide an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.07, and / or a percentage of basic amino acid residues higher than 7.0% among all amino acid residues contained in the repeat domain, and / or a percentage of basic amino acid residues higher than 6.1% among framework residues contained in the repeat domain; and

[0021] wherein step (b) is (1) replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; and / or

[0022] (2) replacing at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid;

[0023] wherein the modified ankyrin repeat domain has at least one feature selected from the following list of features:

[0024] (i) an isoelectric point (pI) equal to or lower than pH 4.07;

[0025] (ii) a percentage of basic amino acid residues of equal to or lower than 7.0% among all amino acid residues contained in the repeat domain; and

[0026] (iii) Among the framework residues contained in the repeat domain, the percentage of basic amino acid residues is equal to or lower than 6.1%.

[0027] In another aspect, the present invention provides a designed ankyrin repeat domain, comprising an N-terminal capping module, at least one internal repeat module and a C-terminal capping module, wherein the repeat domain is linked to a drug moiety, and wherein the repeat domain has at least one feature selected from the following list of features:

[0028] (a) an isoelectric point (pI) equal to or lower than pH 4.6;

[0029] (b) a percentage of basic amino acid residues equal to or lower than 12.0% among all amino acid residues contained in the repeat domain;

[0030] (c) a percentage of Arg and Lys residues of 8.0% or less among all the amino acid residues contained in the repeat domain;

[0031] (d) among the framework residues comprised in the repeat domain, the percentage of basic amino acid residues is equal to or lower than 9.6%; and

[0032] (e) Among the framework residues contained in the repeat domain, the percentage of Arg and Lys residues is equal to or lower than 6.7%.

[0033] In another aspect, the present invention provides a method of producing a modified ankyrin repeat domain linked to a drug moiety, the method comprising steps (a) and (b):

[0034] wherein step (a) is to provide an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.6, and / or a percentage of basic amino acid residues higher than 12.0% among all amino acid residues contained in the repeat domain, and / or a percentage of Arg and Lys residues higher than 8.0% among all amino acid residues contained in the repeat domain, and / or a percentage of basic amino acid residues higher than 9.6% among framework residues contained in the repeat domain, and / or a percentage of Arg and Lys residues higher than 6.7% among the framework residues contained in the repeat domain; and

[0035] wherein step (b) is (1) replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; and / or

[0036] (2) replacing at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid;

[0037] Wherein the modified repeat domain has at least one feature selected from the following list of features:

[0038] (i) an isoelectric point (pI) equal to or lower than pH 4.6;

[0039] (ii) a percentage of basic amino acid residues equal to or lower than 12.0% among all amino acid residues contained in the repeat domain;

[0040] (iii) a percentage of Arg and Lys residues equal to or lower than 8.0% among all the amino acid residues contained in the repeat domain;

[0041] (iv) among the framework residues contained in the repeat domain, the percentage of basic amino acid residues is equal to or lower than 9.6%; and

[0042] (v) Among the framework residues contained in the repeat domain, the percentage of Arg and Lys residues is equal to or lower than 6.7%.

[0043] In another aspect, the present invention provides a designed ankyrin repeat domain obtainable by or by a method of the present invention.

[0044] In another aspect, the present invention provides recombinant proteins comprising such designed repeat domains of the present invention.

[0045] In another aspect, the present invention provides isolated nucleic acids encoding the designed repeat domains of the present invention or encoding the recombinant proteins of the present invention, recombinant expression vectors comprising such nucleic acids, host cells comprising such expression vectors, and pharmaceutical compositions comprising the designed repeat proteins, recombinant proteins, nucleic acids and / or recombinant expression vectors of the present invention and, optionally, at least one pharmaceutically acceptable carrier or diluent.

[0046] In another aspect, the invention provides a method for treating and / or diagnosing a medical condition, the method comprising the step of administering to a patient in need thereof a therapeutically and / or diagnostically effective amount of a designed repeat domain, recombinant protein, nucleic acid or pharmaceutical composition of the invention. In a specific aspect, the medical condition is cancer.

[0047] In another aspect, the invention provides a designed repeat domain, recombinant protein, nucleic acid or pharmaceutical composition of the invention for use in a method of treating a medical condition. In a specific aspect, the medical condition is cancer.

[0048] Based on the disclosure provided herein, those skilled in the art will recognize or be able to determine using only routine experimentation many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be covered by the following embodiment (E):

[0049] E1. A designed ankyrin repeat domain, comprising an N-terminal capping module, at least one internal repeating module and a C-terminal capping module, wherein the repeating domain has at least one feature selected from the following list of features:

[0050] (a) an isoelectric point (pI) equal to or lower than pH 4.07;

[0051] (b) a percentage of basic amino acid residues of 7.0% or less among all amino acid residues contained in the repeat domain; and

[0052] (c) Among the framework residues contained in the repeat domain, the percentage of basic amino acid residues is equal to or lower than 6.1%.

[0053] E2. The repeat domain according to E1, wherein the repeat domain has at least two features selected from features (a), (b) and (c).

[0054] E3. A repeat domain according to any one of E1 to E2, wherein the repeat domain has at least features (a) and (b), (a) and (c), or (b) and (c).

[0055] E4. A repeat domain according to any one of E1 to E3, wherein the repeat domain has features (a), (b) and (c).

[0056] E5. The repeat domain according to any one of E1 to E4, wherein the repeat domain specifically binds to a target.

[0057] E6. The repeat domain according to E5, wherein the repeat domain has a length of less than 10 -7 The dissociation constant (K D ) binds to the target.

[0058] E7. The repeat domain of any one of E1 to E6, wherein the repeat domain comprises one internal repeat module, two internal repeat modules, three internal repeat modules or four internal repeat modules.

[0059] E8. A repeat domain according to any one of E1 to E7, wherein the repeat domain has a KR / DE ratio equal to or lower than 0.44, and / or wherein the repeat domain has a KR / DE ratio equal to or lower than 0.36 in the framework residues, and / or wherein the repeat domain has a KR / DE ratio equal to or lower than 0.66 in all residues of the N-terminal capping module.

[0060] E9. A repeat domain according to any one of E1 to E8, wherein each internal repeat module independently comprises a sequence selected from the group consisting of: (1) SEQ ID NO:

[0061] 131 to 142, and (2) a sequence in which up to 9 framework residues in any one of SEQ ID NOs: 131 to 142 are substituted with another amino acid, and / or the N-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 25 and 115 to 12 ...

[0062] The C-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NO: 123 to 130, and (2) a sequence in which up to 9 framework residues in any one of SEQ ID NO: 123 to 130 are substituted with another amino acid. E10. The repeat domain according to any one of E1 to E9, wherein each internal repeat module independently comprises a sequence selected from the group consisting of: (1) SEQ ID NO:

[0063] 31, and (2) a sequence in which up to 5 framework residues other than positions 1, 10, 13, 17, 19, 21, 22 and 26 in SEQ ID NO:31 are substituted with another amino acid, and / or the N-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NO:19, and (2) a sequence in which up to 5 framework residues other than positions 5, 17, 20 and 23 in SEQ ID NO:19 are substituted with another amino acid, and / or the C-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NO:40, and (2) a sequence in which up to 5 framework residues other than positions 10, 11, 17, 18, 19, 22 and 26 in SEQ ID NO:40 are substituted with another amino acid.

[0064] E11. A repeat domain according to any one of E1 to E10, wherein each internal repeat module independently comprises a member selected from SEQ ID NOs: 31 to 35, 61 to 62, 92 to 94

[0065] and 131 to 142, and / or the N-terminal capping module comprises a sequence selected from SEQ ID NOs: 19 to 25, 57 to 60, 65 to 81 and 115 to 122, and / or the C-terminal capping module comprises a sequence selected from SEQ ID NOs: 40 to 42,

[0066] Sequences 63 to 64, 82 to 91 and 123 to 130.

[0067] E12. The repeat domain according to any one of E1 to E11, wherein each internal repeat module independently comprises a sequence selected from the group consisting of: (1) SEQ ID NO:

[0068] 26 to 30 and 47 to 48, and (2) a sequence in which up to 9 amino acids in any one of SEQ ID NOs: 26 to 30 and 47 to 48 are substituted by another amino acid, and / or the N-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 15 to 18, 43 and 99 to 101, and (2) a sequence in which up to 9 amino acids in any one of SEQ ID NOs: 15 to 18, 43 and 99 to 101 are substituted by another amino acid, and / or the C-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 36 to 39, 54 and 102 to 103, and (2) a sequence in which up to 9 amino acids in any one of SEQ ID NOs: 36 to 39, 54 and 102 to 103

[0069] In some embodiments, the substitution is according to Table 3. In another embodiment, the substitution is according to Table 3.

[0070] E12a. A repeat domain according to any one of E1 to E12, wherein each internal repeat module independently comprises a sequence selected from the group consisting of: (1) SEQ ID NO:

[0071] 26 to 30 and 47 to 48, and (2) a sequence in which at most 9 amino acids, at most 8 amino acids, at most 7 amino acids, at most 6 amino acids, at most 5 amino acids, at most 4 amino acids, at most 3 amino acids, at most 2 amino acids or at most 1 amino acid in any one of SEQ ID NOs: 26 to 30 and 47 to 48 is substituted with another amino acid, and / or the N-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 15 to 18, 43 and 99 to 101, and (2) a sequence in which at most 9 amino acids, at most 8 amino acids, at most 7 amino acids, at most 6 amino acids, at most 5 amino acids, at most 4 amino acids, at most 3 amino acids, at most 2 amino acids or at most 1 amino acid in any one of SEQ ID NOs: 26 to 30 and 47 to 48 is substituted with another amino acid. NO: 15 to 18, 43 and 99 to 101, up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids or up to 1 amino acid sequence is substituted with another amino acid, and / or the C-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NO: 36 to 39, 54 and 102 to 103, and (2) wherein any of SEQ ID NO: 36 to 39, 54 and 102 to 103 has a maximum of 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids or up to 1 amino acid sequence substituted with another amino acid. In certain embodiments, the substitution is a substitution according to Table 3. In another embodiment, the substitution is a conservative substitution according to Table 3.

[0072] E12b. A repeat domain according to any one of E1 to E12a, wherein each internal repeat module independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 26 to 30 and 47 to 48, and (2) a sequence in which at most 9 amino acids, at most 8 amino acids, at most 7 amino acids, at most 6 amino acids, at most 5 amino acids, at most 4 amino acids, at most 3 amino acids, at most 2 amino acids or at most 1 amino acid in any one of SEQ ID NOs: 26 to 30 and 47 to 48 is substituted by another amino acid.

[0073] E12c. The repeat domain according to any one of E1 to E12b, wherein the N-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NO:

[0074] 15 to 18, 43 and 99 to 101, and (2) sequences in which up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids or up to 1 amino acid in any one of SEQ ID NOs: 15 to 18, 43 and 99 to 101 are substituted by another amino acid.

[0075] E12d. The repeat domain according to any one of E1 to E12c, wherein the C-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NO:

[0076] 36 to 39, 54 and 102 to 103, and (2) sequences in which up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids, or up to 1 amino acid in any one of SEQ ID NOs: 36 to 39, 54 and 102 to 103 is substituted by another amino acid.

[0077] E13. The repeat domain according to any one of E1 to E12, wherein the repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NO:

[0078] 104, 108, 109 and 112 to 114, and (2) sequences having at least 80% amino acid sequence identity in the framework residues of any one of SEQ ID NOs: 104, 108, 109 and 112 to 114.

[0079] E14. The repeat domain according to any one of E1 to E13, wherein the repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NO: 8

[0080] to 9 and 95 to 98, and (2) a sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 8 to 9 and 95 to 98.

[0081] E15. A method for producing a modified ankyrin repeat domain, the method comprising steps (a) and (b),

[0082] wherein step (a) is to provide an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.07, and / or a percentage of basic amino acid residues higher than 7.0% among all amino acid residues contained in the repeat domain, and / or a percentage of basic amino acid residues higher than 6.1% among framework residues contained in the repeat domain; and

[0083] wherein step (b) is (1) replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; and / or

[0084] (2) replacing at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid;

[0085] wherein the modified ankyrin repeat domain has at least one feature selected from the following list of features:

[0086] (i) an isoelectric point (pI) equal to or lower than pH 4.07;

[0087] (ii) a percentage of basic amino acid residues of equal to or lower than 7.0% among all amino acid residues contained in the repeat domain; and

[0088] (iii) Among the framework residues contained in the repeat domain, the percentage of basic amino acid residues is equal to or lower than 6.1%.

[0089] E16. The method according to E15, wherein the method comprises steps (a) and (b),

[0090] wherein step (a) is to provide an ankyrin repeat domain, wherein the ankyrin repeat domain has an isoelectric point (pI) higher than pH 4.07; and

[0091] wherein step (b) is (1) replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; and / or

[0092] (2) replacing at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid;

[0093] wherein the modified ankyrin repeat domain has an isoelectric point (pi) equal to or lower than pH 4.07.

[0094] E17. The method according to E15, wherein the method comprises steps (a) and (b),

[0095] wherein step (a) is to provide an ankyrin repeat domain, said ankyrin repeat domain having a percentage of basic amino acid residues higher than 7.0% among all amino acid residues contained in said repeat domain; and

[0096] wherein step (b) comprises replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue;

[0097] wherein the modified ankyrin repeat domain has a percentage of basic amino acid residues equal to or lower than 7.0% among all amino acid residues comprised in the repeat domain.

[0098] E18. The method according to E15, wherein the method comprises steps (a) and (b),

[0099] wherein step (a) is to provide an ankyrin repeat domain having a percentage of basic amino acid residues higher than 6.1% among the framework residues contained in the repeat domain; and

[0100] wherein step (b) comprises replacing at least one basic amino acid residue in the framework residues of the repeat domain of step (a) with a neutral or acidic amino acid residue;

[0101] wherein the modified ankyrin repeat domain has a percentage of basic amino acid residues among the framework residues comprised in the repeat domain that is equal to or lower than 6.1%.

[0102] E19. The method according to any one of E15 to E18, wherein the repeat domain in step (a) has a KR / DE ratio higher than 0.44 and the modified repeat domain has a KR / DE ratio equal to or lower than 0.44, and / or wherein the repeat domain in step (a) has a KR / DE ratio higher than 0.36 in the framework residues and the modified repeat domain has a KR / DE ratio equal to or lower than 0.36, and / or wherein the repeat domain in step (a) has a KR / DE ratio higher than 0.66 in all residues of the N-terminal capping module and the modified repeat domain has a KR / DE ratio equal to or lower than 0.66 in all residues of the N-terminal capping module.

[0103] E20. The method according to any one of E15 to E19, wherein the repeating domain of step (a) is optionally present in an amount of less than 10 -7 The dissociation constant (K D ) specifically binds to the target.

[0104] E21. The method according to E20, wherein the modified repeat domain has a length of less than 10 -7 M of K D Specifically binds to the target, preferably wherein the modified repeat domain is expressed as follows: D Binds to the target: (1) approximately equal to the K of the repeat domain binding to the target in step (a) D , or (2) the K of the repeat domain of step (a) binding to the targetD less than 100 times, less than 10 times, less than 5 times or less than 2 times.

[0105] E22. The method according to any one of E15 to E21, wherein the modified repeat domain comprises one internal repeat module, two internal repeat modules, three internal repeat modules or four internal repeat modules.

[0106] E23. A designed ankyrin repeat domain, comprising an N-terminal capping module, at least one internal repeat module and a C-terminal capping module, wherein the repeat domain is linked to a drug moiety, and wherein the repeat domain has at least one feature selected from the following list of features:

[0107] (a) an isoelectric point (pI) equal to or lower than pH 4.6;

[0108] (b) a percentage of basic amino acid residues of 12.0% or less among all amino acid residues contained in the repeat domain;

[0109] (c) a percentage of Arg and Lys residues of 8.0% or less among all the amino acid residues contained in the repeat domain;

[0110] (d) among the framework residues comprised in the repeat domain, the percentage of basic amino acid residues is equal to or lower than 9.6%; and

[0111] (e) Among the framework residues contained in the repeat domain, the percentage of Arg and Lys residues is equal to or lower than 6.7%.

[0112] E24. The repeat domain according to E23, wherein the isoelectric point (pi) is in the range between pH 3.0 and pH 5.0, preferably between pH 3.0 and pH 4.6, and more preferably between pH 3.50 and pH 4.53.

[0113] E25. The repeat domain according to any one of E23 to E24, wherein the repeat domain has at least feature (b), and wherein the percentage in (b) is optionally equal to or lower than 8.7%.

[0114] E26. The repeat domain of any one of E23 to E25, wherein the repeat domain has at least feature (c), and wherein the percentage in (c) is optionally equal to or lower than 4.7%.

[0115] E27. The repeat domain of any one of E23 to E26, wherein the repeat domain has at least feature (d), and wherein the percentage in (d) is optionally equal to or lower than 6.7%.

[0116] E28. The repeat domain of any one of E23 to E27, wherein the repeat domain has at least feature (e), and wherein the percentage in (e) is optionally equal to or lower than 5.7%.

[0117] E29. A repeat domain according to any one of E23 to E28, wherein the repeat domain has a KR / DE ratio equal to or lower than 0.44, and / or wherein the repeat domain has a KR / DE ratio equal to or lower than 0.36 in the framework residues, and / or wherein the repeat domain has a KR / DE ratio equal to or lower than 0.66 in all residues of the N-terminal capping module.

[0118] E30. A repeat domain according to any one of E23 to E29, wherein the repeat domain has a total number of basic amino acid residues equal to or lower than n, wherein n=4+5R, wherein R is the number of internal repeat modules contained in the repeat domain.

[0119] E31. A repeat domain according to any one of E23 to E30, wherein the repeat domain has a total number of Arg and Lys residues equal to or lower than m, where m=5+2R, where R is the number of internal repeat modules contained in the repeat domain.

[0120] E32. A repeat domain according to any one of E23 to E31, wherein the repeat domain does not have Arg residues and Lys residues within the framework residue positions.

[0121] E33. The repeat domain of any one of E23 to E32, wherein the repeat domain comprises one internal repeat module, two internal repeat modules, three internal repeat modules or four internal repeat modules.

[0122] E34. A repeat domain according to any one of E23 to E33, wherein the repeat domain specifically binds to a target.

[0123] E35. The repeat domain according to E34, wherein the repeat domain has a length of less than 10 -7 The dissociation constant (K D ) binds to the target.

[0124] E36. A repeat domain according to any one of E23 to E35, wherein the drug moiety is a therapeutic moiety or a diagnostic moiety.

[0125] E37. A repeat domain according to any one of E23 to E36, wherein the drug moiety is a toxin.

[0126] E38. A repeat domain according to E37, wherein the toxin is a radionuclide.

[0127] E39. The repeat domain of E37, wherein the toxin is a cytotoxin.

[0128] E40. The repeat domain according to any one of E23 to E39, wherein the repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NO:

[0129] 104 to 114, and (2) a sequence having at least 80% amino acid sequence identity in the framework residues of any one of SEQ ID NOs: 104 to 114.

[0130] E41. The repeat domain according to any one of E23 to E40, wherein the repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NO:

[0131] 104 and 112, and (2) a sequence having at least 80% amino acid sequence identity in the framework residues of either of SEQ ID NOs: 104 and 112.

[0132] E42. The repeat domain according to any one of E23 to E41, wherein the repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NO:

[0133] 8 to 10, 12 and 95 to 98, and (2) a sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 8 to 10, 12 and 95 to 98.

[0134] E43. A designed ankyrin repeat domain linked to a drug moiety according to any one of E23 to E42 for use in a method for treating and / or diagnosing a medical condition, the method comprising the step of administering a therapeutically and / or diagnostically effective amount of the repeat domain to a patient in need thereof, and wherein optionally the administration is not oral administration.

[0135] E44. A designed ankyrin repeat domain linked to a drug moiety for use in a method of treating and / or diagnosing a medical condition according to E43, wherein the medical condition is cancer.

[0136] E45. A method for producing a modified ankyrin repeat domain linked to a drug moiety, the method comprising steps (a) and (b),

[0137] wherein step (a) is to provide an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.6, and / or a percentage of basic amino acid residues higher than 12.0% among all amino acid residues contained in the repeat domain, and / or a percentage of Arg and Lys residues higher than 8.0% among all amino acid residues contained in the repeat domain, and / or a percentage of basic amino acid residues higher than 9.6% among framework residues contained in the repeat domain, and / or a percentage of Arg and Lys residues higher than 6.7% among the framework residues contained in the repeat domain; and

[0138] wherein step (b) is (1) replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; and / or

[0139] (2) replacing at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid;

[0140] Wherein the modified repeat domain has at least one feature selected from the following list of features:

[0141] (i) an isoelectric point (pI) equal to or lower than pH 4.6;

[0142] (ii) a percentage of basic amino acid residues equal to or lower than 12.0% among all amino acid residues contained in the repeat domain;

[0143] (iii) a percentage of Arg and Lys residues equal to or lower than 8.0% among all the amino acid residues contained in the repeat domain;

[0144] (iv) among the framework residues contained in the repeat domain, the percentage of basic amino acid residues is equal to or lower than 9.6%; and

[0145] (v) Among the framework residues contained in the repeat domain, the percentage of Arg and Lys residues is equal to or lower than 6.7%.

[0146] E46. The method according to E45, wherein the method comprises steps (a) and (b),

[0147] wherein step (a) is to provide an ankyrin repeat domain, wherein the ankyrin repeat domain has an isoelectric point (pI) higher than pH 4.6; and

[0148] wherein step (b) is (1) replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; and / or

[0149] (2) replacing at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid;

[0150] wherein the modified repeat domain has an isoelectric point (pi) equal to or lower than pH 4.6.

[0151] E47. The method according to E45, wherein the method comprises steps (a) and (b),

[0152] wherein step (a) is to provide an ankyrin repeat domain having a percentage of basic amino acid residues higher than 12.0% among all amino acid residues contained in the repeat domain; and

[0153] wherein step (b) comprises replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue;

[0154] wherein the modified repeat domain has a percentage of basic amino acid residues equal to or lower than 12.0% among all amino acid residues contained in the repeat domain.

[0155] E48. The method according to E45, wherein the method comprises steps (a) and (b),

[0156] wherein step (a) is to provide an ankyrin repeat domain having a percentage of Arg and Lys residues higher than 8.0% among all amino acid residues contained in the repeat domain; and

[0157] wherein step (b) comprises replacing at least one Arg or Lys residue of the repeat domain of step (a) with a neutral or acidic amino acid residue;

[0158] wherein the modified repeat domain has a percentage of Arg and Lys residues equal to or lower than 8.0% of all amino acid residues contained in the repeat domain. E49. The method according to E45, wherein the method comprises steps (a) and (b),

[0159] wherein step (a) is to provide an ankyrin repeat domain having a percentage of basic amino acid residues higher than 9.6% among the framework residues contained in the repeat domain; and

[0160] wherein step (b) comprises replacing at least one basic amino acid residue in the framework residues of the repeat domain of step (a) with a neutral or acidic amino acid residue;

[0161] wherein the modified repeat domain has a percentage of basic amino acid residues in the framework residues contained in the repeat domain that is equal to or lower than 9.6%. E50. The method according to E45, wherein the method comprises steps (a) and (b),

[0162] wherein step (a) is to provide an ankyrin repeat domain having a percentage of Arg and Lys residues higher than 6.7% among the framework residues comprised in the repeat domain; and

[0163] wherein step (b) comprises replacing at least one Arg or Lys residue in the framework residues of the repeat domain of step (a) with a neutral or acidic amino acid residue;

[0164] wherein the modified repeat domain has a percentage of Arg and Lys residues equal to or lower than 6.7% in the framework residues contained in the repeat domain. E51. The method according to any one of E45 to E50, wherein the repeat domain in step (a) has a KR / DE ratio higher than 0.44, and the modified repeat domain has a KR / DE ratio equal to or lower than 0.44, and / or wherein the repeat domain in step (a) has a KR / DE ratio higher than 0.36 in the framework residues, and the modified repeat domain has a KR / DE ratio equal to or lower than 0.36, and / or wherein the repeat domain in step (a) has a KR / DE ratio higher than 0.66 in all residues of the N-terminal capping module, and the modified repeat domain has a KR / DE ratio equal to or lower than 0.66 in all residues of the N-terminal capping module.

[0165] E52. The method according to any one of E45 to E51, wherein the method further comprises the step of linking a drug moiety to the modified repeat domain.

[0166] E53. The method according to any one of E45 to E52, wherein the repeating domain of step (a) is optionally present in an amount of less than 10 -7 The dissociation constant (K D ) specifically binds to the target.

[0167] E54. A method according to E53, wherein the modified repeat domain has a length of less than 10 - 7 M of K DSpecifically binds to the target, preferably wherein the modified repeat domain is expressed as follows: D Binds to the target: (1) approximately equal to the K of the repeat domain binding to the target in step (a) D , or (2) the K of the repeat domain of step (a) binding to the target D less than 100 times, less than 10 times, less than 5 times or less than 2 times.

[0168] E55. The method according to any one of E45 to E54, wherein the modified repeat domain comprises one internal repeat module, two internal repeat modules, three internal repeat modules or four internal repeat modules.

[0169] E56. A designed ankyrin repeat domain, said designed ankyrin repeat domain being obtainable by or by the method according to any one of E15 to E22 or E45 to E55.

[0170] E57. A recombinant protein comprising the proteins according to E1 to E14, E23 to

[0171] The designed repeat domain of any one of E42 and E56.

[0172] E58. An isolated nucleic acid encoding a designed repeat domain according to any one of E1 to E14, E23 to E42 and E56 or a recombinant protein according to E57.

[0173] E59. A recombinant expression vector comprising the nucleic acid according to E58.

[0174] E60. A host cell, comprising the recombinant expression vector according to E59.

[0175] E61. A pharmaceutical composition comprising one or more of the following: (i) a repeat domain according to any one of E1 to E140, E23 to E42 and E56, (ii) a recombinant protein according to E57, (iii) a nucleic acid according to E58, and / or (iv) a recombinant expression vector according to E59, and optionally at least one pharmaceutically acceptable carrier or diluent.

[0176] E62. A method for treating and / or diagnosing a medical condition, the method comprising the step of administering to a patient in need thereof a therapeutically and / or diagnostically effective amount of a repeat domain according to any one of E1 to E14, E23 to E42 and E56, a recombinant protein according to E57, a nucleic acid according to E58 or a pharmaceutical composition according to E61.

[0177] E63. The method according to E62, wherein the medical condition is cancer.

[0178] E64. A method according to E1 to E14, for use in a method of treating and / or diagnosing a medical condition.

[0179] The repeat domain of any one of E23 to E42 and E56, the recombinant protein according to E57, the nucleic acid according to E58, or the pharmaceutical composition according to E61.

[0180] E65. The repeat domain, the recombinant protein, the nucleic acid or the pharmaceutical composition for use according to E64, wherein the medical condition is cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0181] Figure 1: shows the sequences and properties of representative DARPin variants, parental DARPins, negative control DARPin variants (DARPin07) and consensus DARPins according to the present invention. Bold and underlined sequence names indicate the parental DARPins on which the engineered variants were based. Figure 1A Some features of DARPins calculated over the entire repeat domain (i.e. by considering all residues contained in the repeat domain) or only on the framework positions (i.e. by considering all residues contained in the repeat domain except for potential target-interacting residues) are shown. The symbol "#" used, for example, in #(Arg+Lys) means the total number of Arg and Lys residues. The symbol "%" used, for example, in %(Arg+Lys) means the percentage of Arg and Lys residues contained in the repeat domain or in the indicated framework position. Figure 1B As shown, an exemplary DARPin variant comprises an N-terminal capping module, a C-terminal capping module, and two internal repeat modules. Figure 1B is an alignment of all DARPin variant sequences, where the randomized positions are indicated in the consensus line by an underlined “ X " marks (the full sequence is divided into different boxes only for practical reasons). Some positions have a strong preference for a certain acidic / basic residue based on the consensus sequence (such as position 10 in internal repeat module 1), but have been found to be different for the indicated variants (e.g. due to mutations that increase affinity).

[0182] Figure 2: Size Exclusion Chromatography (SEC) profiles of the parent DARPin and DARPin variants tested. Graphs 1 to 12 show the SEC profiles of DARPin01 to DARPin12 (SEQ ID NOs: 1 to 12, respectively), each of which further comprises a C-terminal GSGSC tag (SEQ ID NO: 14). Due to the partial formation of disulfide-linked dimers (C-terminal Cys), all SEC profiles exhibit dimer peaks before the main monomer peak.

[0183] Figure 3 : A graphical overview of the method for preparing radiolabeled DARPins according to the present invention. DARPin variants are expressed in E. coli and purified by IMAC (immobilized metal affinity chromatography) and GF (gel filtration). The construct is cleaved by recombinant TEV protease to cut off the His tag. Subsequently, the uncleaved DARPin variant and the His-tagged TEV protease are removed by reverse IMAC, and the flow-through is collected and loaded onto a SEC column. The purified DARPin is reduced and coupled with the chelator DTPA. The chelated DARPin is then loaded with the radionuclide Indium-111 (also known as 111 In).

[0184] Figure 4 : Single trace SPR profile of DTPA-coupled DARPin (also referred to as construct, see Table 7) against biotinylated full-length HER2. For reference, curves 1 and 2 are shown in Table 7. Figure 2 The HER2 binding profiles of the parent DARPin06 (SEQ ID NO: 6) and DARPin11 (SEQ ID NO: 11) are shown respectively. Figure 3 ,curve Figure 4 ,curve Figure 5 , graph 6 and graph Figure 7 The spectra of construct 6 (DARPin06-GSGSC-DTPA), construct 7 (DARPin07-GSGSC-DTPA), construct 8 (DARPin08-GSGSC-DTPA), construct 9 (DARPin09-GSGSC-DTPA) and construct 10 (DARPin10-GSGSC-DTPA) are shown respectively. Fig. 9Construct 11 (DARPin11-GSGSC-DTPA) and construct 12 (DARPin12-GSGSC-DTPA) are shown, respectively. The GSGSC tag (SEQ ID NO: 14) was fused to the C-terminus of the DARPin. All analytes (500 nM) were injected continuously for 120 s and dissociation was recorded for 180 s (25 μl / min of PBS-Tween20 (0.005%)). Each injection was followed by a regeneration step of 60 s with glycine pH 2.0. The data were double referenced (control point and buffer injection) and fitted to a 1:1 Langmuir model.

[0185] Figure 5 : Renal uptake of indium-labeled DARPins. Radiolabeled DARPin01 to DARPin12 were injected in wild-type mice. Data are shown as average injected activity % / tissue mass grams (%IA / g). Measurements were taken 4 hours after injection. Error bars show standard deviation. Bars represented in black correspond to the parent DARPin. DARPin07 is a negative control with a higher pI and a higher percentage of basic amino acids in framework residues compared to its parent DARPin06.

[0186] Figure 6: 111 Biodistribution study of In-labeled DARPins in mice bearing HER2-expressing SKOV3ip tumors. Two DARPins with binding specificity for HER2 (i.e., DARPin06 and DARPin08) and two DARPins that do not bind to HER2 (i.e., DARPin01 and DARPin02) were tested in this study. Mice were injected with radiolabeled DARPins (1 mg / kg, approximately 150 KBq) two weeks (n=6 per group) or three weeks (n=6 per group) after SKOV3ip tumor cell implantation. Measurements were taken 4 hours after injection. Data are presented as mean % injected activity per gram of tissue mass (%IA / g, n=12 per group combined). 6A: In-labeled DARPins with 1 mg / kg, approximately 150 KBq, were injected with radiolabeled DARPins (1 mg / kg, approximately 150 KBq, respectively) two weeks (n=6 per group) or three weeks (n=6 per group) after SKOV3ip tumor cell implantation. Measurements were taken 4 hours after injection. Data are presented as mean % injected activity per gram of tissue mass (%IA / g, n=12 per group combined). 111 Compared with the parental DARPin labeled with In, 111In radiolabeled pI engineered DARPins renal accumulation. Significant reductions in renal accumulation of the engineered variants (DARPin02 and DARPin08) were observed compared to the corresponding parental DARPins (DARPin01 and DARPin06, respectively). More specifically, 90% (DARPin02 vs. DARPin01) and 78% (DARPin08 vs. DARPin06) of the reductions were caused by pI engineering of the DARPin variants. 6B: DARPin uptake in tumors. Significant accumulation of HER2-specific DARPins in tumors was observed, and accumulation of DARPin08 in tumors was comparable to that of its parental DARPin06. Only negligible nonspecific accumulation of non-binding DARPin01 and DARPin02 was observed in tumors. In 6A and 6B, results corresponding to engineered variants are represented by white bars and results corresponding to parental DARPins are represented by black bars. 6C: Accumulation of DARPins in other tissues or organs (blood, heart, lung, spleen, liver, small intestine, large intestine, muscle, bone, and tail) was measured. Overall, the accumulation of the engineered variants (DARPin02 and DARPin08) was similar to the corresponding parental DARPins (DARPin01 and DARPin06, respectively). Error bars show SD.

[0187] Figure 7 : In mice bearing HER2-expressing SKOV3ip tumors 111 Time course biodistribution study of In-labeled DARPins (DARPin06 and DARPin08). Mice were injected with radiolabeled DARPin (1 mg / kg, approximately 150 KBq) and the tumor volume was approximately 350 mm 3 . The evolution of radioactivity in isolated kidney, tumor and blood samples was measured at 1h, 4h, 24h, 48h and 96h after injection. Data are shown as mean injected activity % / tissue mass grams (%IA / g, n=4 per group). The area under the curve (AUC) of charge-engineered DARPin08 in kidney was reduced by 76% compared to parental DARPin06. The AUCs of the two DARPins in tumor and blood remained comparable.

[0188] FIG. 8 : Sequences and properties of other exemplary DARPin variants according to the invention derived from the parent DARPin06. Fig. 8A Features of DARPins calculated over the entire repeat domain (i.e. by considering all residues contained in the repeat domain) or only on framework positions (i.e. by considering all residues contained in the repeat domain except potential target-interacting residues) are shown. Figure 8BAs shown, these other exemplary DARPin variants comprise an N-terminal capping module, a C-terminal capping module, and two internal repeat modules. The randomized positions are indicated in the consensus row by an underlined “ X ” markings (the full sequence is divided into different boxes only for practical reasons). The definition and legend of Fig. 8 correspond to those of Fig. 1.

[0189] Fig. 9 : Multi-trace SPR profiles of DTPA-conjugated DARPins (also referred to as constructs, see Tables 7 and 16) against biotinylated full-length HER2. Fig. 9 A. Fig. 9 B. Fig. 9 C. Fig. 9 D. Fig. 9 E and Fig. 9 F shows the profiles of construct 6 (DARPin06-GSGSC-DTPA), construct 8 (DARPin08-GSGSC-DTPA), construct 13 (DARPin13-GSGSC-DTPA), construct 14 (DARPin14-GSGSC-DTPA), construct 15 (DARPin15-GSGSC-DTPA) and construct 16 (DARPin16-GSGSC-DTPA), respectively. The GSGSC tag (SEQ ID NO: 14) was fused to the C-terminus of the DARPin. Three-fold dilutions of the analyte (50 nM, 16.667 nM, 5.556 nM, 1.852 nM and 0.617 nM) were injected for 300 s and the dissociation was recorded for 1500 s (25 μl / min). Each injection was followed by a regeneration step of 60 s with glycine pH 2.0. The dissociation constant (K) was calculated from the fully fitted association and dissociation rates using a standard 1:1-Langemuir model. D )(See Table 17).

[0190] Figure 10: 111 Biodistribution study of In-labeled DARPins in mice bearing HER2-expressing SKOV3ip tumors. All DARPins tested had binding specificity for HER2. DARPin06 is the parental DARPin, and DARPin08, DARPin13, DARPin14, DARPin15, and DARPin16 are charge engineered variants according to the present invention. Mice were injected with radiolabeled DARPins (1 mg / kg, approximately 150 KBq) with tumor volumes of approximately 350 mm 3 The measurements were performed 4 hours after injection. Data are presented as the average injected activity % / tissue mass gram (%IA / g, n=4 per group). 111 Compared with the parental DARPin06 labeled with In,111 In radiolabeled charge engineered DARPin08, DARPin13, DARPin14, DARPin15 and DARPin16 renal accumulation. A significant reduction in renal accumulation of the engineered variants was observed compared to the parental DARPin. More specifically, 82% (DARPin08 vs. DARPin06), 90% (DARPin13 vs. DARPin06), 85% (DARPin14 vs. DARPin06), 93% (DARPin15 vs. DARPin06) and 87% (DARPin16 vs. DARPin06) of the reductions were caused by charge engineering of the DARPin variants. 10B: DARPin uptake in tumors. Accumulation of HER2-specific DARPins in tumors was observed, and the accumulation of charge engineered DARPin variants in tumors was comparable to that of parental DARPin06. In 10A and 10B, the results corresponding to the engineered variants are represented by white bars and the results corresponding to the parental DARPin are represented by black bars. Error bars show SD. DETAILED DESCRIPTION

[0191] The designed ankyrin repeat domain is the structural unit of the designed ankyrin repeat protein. Designed repeat protein library (including designed ankyrin repeat protein library) (WO2002020565; Binz et al., Nat.Biotechnol.22,575-582,2004; Stumpp et al., Drug Discov.Today 13,695-701,2008) can be used to select the designed repeat domain of target specificity that is bound to its target with high affinity. Such target-specific designed repeat domains can then be used as valuable components of recombinant binding proteins for treating and / or diagnosing diseases.

[0192] Designed ankyrin repeat protein is a class of binding molecules, which has the potential to overcome the limitations of monoclonal antibodies, thereby allowing novel treatments and / or diagnostic methods. Such ankyrin repeat proteins can include a single designed ankyrin repeat domain, or can include two, three, four, five or more combinations of designed ankyrin repeat domains with the same or different target specificities (Stumpp et al., Drug Discov. Today 13,695-701, 2008; U.S. Patent No. 9,458,211). Ankyrin repeat proteins comprising only a single designed ankyrin repeat domain are small proteins (14 kDa), which can be selected to bind to a given target protein with high affinity and specificity. These features and the possibility of combining two, three, four, five or more designed ankyrin repeat domains in a protein make designed ankyrin repeat proteins ideal agonism, antagonism and / or inhibitory drug candidates. In addition, such ankyrin repeat proteins can be engineered to carry various effector functions, such as cytotoxic agents or half-life extenders, thereby realizing brand-new drug forms. In summary, designed ankyrin repeat proteins are examples of next-generation protein therapeutics with the potential to surpass existing antibody drugs.

[0193] The inventors of the present invention have surprisingly found that the pharmacokinetic properties of designed ankyrin repeat domains can be improved by reducing the number of positive charges contained in such repeat domains and / or by lowering the isoelectric point (pI) of such repeat domains. More specifically, it was found that such designed repeat domains of the present invention exhibit reduced accumulation in the kidney when administered in vivo. In addition, when the designed repeat domains of the present invention are linked to a drug moiety, the drug moiety exhibits reduced accumulation in the kidney when administered in vivo. For example, when the designed repeat domains of the present invention are linked to a radioactive agent, the accumulation of radioactivity in the kidney is reduced when administered in vivo. This property is very desirable because renal accumulation of toxic drug moieties in the kidney leads to nephrotoxicity. For this reason, renal radioactivity accumulation limits the use of radiolabeled DARPins in treatment or diagnosis, and similarly, renal accumulation of cytotoxic agents limits the use of cytotoxic agent-conjugated DARPins. The biophysical properties and target-specific accumulation of the designed ankyrin repeat proteins of the present invention in tumors expressing targets are not significantly affected by charge engineering. Therefore, the designed ankyrin repeat domains of the present invention provide an improved tumor-to-kidney ratio. Therefore, the improved repeat domains and recombinant proteins provided in the present invention open the door to new therapeutic and / or diagnostic applications, for example in the field of nuclear medicine, such as in radiopharmaceutical therapy or in the field of cancer treatment using proteins conjugated to cytotoxic agents.

[0194] definition

[0195] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. In addition, unless the context otherwise requires, singular terms shall include the plural, and plural terms shall include the singular. Generally speaking, the nomenclature used in conjunction with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry described herein, and the techniques thereof are those well known and commonly used in the art.

[0196] Unless otherwise stated, the terms "comprising," "having," "including," and "containing" are to be understood as open-ended terms. If aspects of the invention are described as "comprising" features, embodiments "consisting of" or "consisting essentially of" the features are also contemplated. Unless otherwise stated, any and all examples or exemplary language (e.g., "such as") provided herein are intended merely to better illustrate the disclosure and are not intended to limit the scope of the disclosure. No language in the specification should be construed to indicate that any unclaimed element is necessary to implement the disclosure. Unless otherwise stated, the term "about" as used herein is equivalent to ±10% of a given value.

[0197] Recitation of ranges of values ​​herein are merely intended to serve as a shorthand method of referring individually to each separate value and endpoint falling within the range, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.

[0198] The term "nucleic acid" refers to a polynucleotide molecule, which can be a single-stranded or double-stranded ribonucleic acid (RNA) molecule or a deoxyribonucleic acid (DNA) molecule, including modified and artificial forms of DNA or RNA. The nucleic acid can exist in an isolated form or be contained in a recombinant nucleic acid molecule or a vector.

[0199] In the context of the present invention, the term "protein" refers to a molecule comprising polypeptides, wherein at least a portion of the polypeptides has or is capable of acquiring a defined three-dimensional arrangement by forming secondary, tertiary and / or quaternary structures within a single polypeptide chain and / or between multiple polypeptide chains. If the protein comprises two or more polypeptide chains, the individual polypeptide chains may be non-covalently linked or covalently linked, for example, by a disulfide bond between two polypeptides. The portion of a protein that alone has or is capable of acquiring a defined three-dimensional arrangement by forming secondary and / or tertiary structures is referred to as a "protein domain". Such protein domains are well known to those skilled in the art.

[0200] The term "recombinant" used in recombinant proteins, recombinant polypeptides, etc. means that the protein or polypeptide is produced by using recombinant DNA technology well known to those skilled in the art. For example, a recombinant DNA molecule encoding a polypeptide (e.g., produced by gene synthesis) can be cloned into a bacterial expression plasmid (e.g., pQE30, QIAgen), a yeast expression plasmid, a mammalian expression plasmid, or a plant expression plasmid, or a DNA capable of in vitro expression. If, for example, such a recombinant bacterial expression plasmid is inserted into appropriate bacteria (e.g., Escherichia coli), these bacteria can produce polypeptides encoded by the recombinant DNA. The correspondingly produced polypeptide or protein is referred to as a recombinant polypeptide or recombinant protein.

[0201] 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 via peptide bonds. Preferably, a polypeptide consists of eight or more amino acids linked via peptide bonds. The term "polypeptide" also includes multiple chains of amino acids linked together by SS bridges of cysteine. Polypeptides are well known to those skilled in the art.

[0202] The term "target" refers to a single molecule, such as a nucleic acid, a polypeptide or a protein, a carbohydrate or any other naturally or non-naturally occurring molecule or part, including any part of such a single molecule, or a complex of two or more such molecules. The target can be a whole cell or a tissue sample. Preferably, the target is a naturally occurring or non-natural polypeptide or a polypeptide containing a chemical modification (e.g., modified by natural or non-natural phosphorylation, acetylation or methylation).

[0203] Patent application WO2002020565 and Forrer et al., 2003 (Forrer, P., Stumpp, MT, Binz, HK, Plückthun, A., 2003. FEBS Letters 539, 2-6) contain a general description of repeat protein characteristics and repeat domain characteristics, technology and applications. The term "repeat protein" refers to a protein comprising one or more repeat domains. Preferably, the repeat protein comprises one, two, three, four, five or six repeat domains. In addition, the repeat protein may comprise additional non-repeat protein domains, polypeptide tags and / or peptide linkers.

[0204] The term "repeat domain" refers to a protein domain comprising two or more consecutive repeat modules as structural units, wherein the repeat modules have structural and sequence homology. Preferably, the repeat domain also comprises an N-terminal and / or C-terminal capping module. For clarity, the capping module can be a repeat module. Such repeat domains, repeat modules and capping modules, sequence motifs and structural and sequence homologies are familiar to practitioners in the art based on the following examples: ankyrin repeat domains (Binz et al., J. Mol. Biol. 332, 489-503, 2003; Binz et al., 2004, loc. Cit.; WO2002020565; WO2012069655), leucine-rich repeat domains (WO2002020565), triangular tetratricopeptide repeat domains (Main, ER, Xiong, Y., Cocco, MJ, D'Andrea, L., Regan, L., Structure 11(5), 497-508, 2003) and armadillo repeat domains (WO2009040338). It is also well known to those skilled in the art that such repeat domains are different from proteins comprising repeat amino acid sequences, wherein each repeat amino acid sequence is capable of forming a single domain (eg, the FN3 domain of fibronectin).The repeat domain may be a binding domain.

[0205] The term "ankyrin repeat domain" refers to a repeat domain comprising two or more consecutive ankyrin repeat modules as structural units, wherein the ankyrin repeat modules have structural homology and sequence homology.

[0206] As used in designed repeat proteins, designed repeat domains, etc., the term "designed" refers to the property that such repeat proteins and repeat domains, respectively, are artificial and do not exist in nature. The binding domain of the present invention is a designed repeat domain. Preferably, the designed repeat domain of the present invention is a designed ankyrin repeat domain.

[0207] The term "repeat module" refers to the repeated amino acid sequence and structural unit of the designed repeat domain, which is originally derived from the repeat unit of naturally occurring repeat protein.Each repeat module contained in the repeat domain is derived from one or more repeat units of naturally occurring repeat protein family or subfamily (preferably ankyrin repeat protein family).In addition, each repeat module contained in the repeat domain can include "repetitive sequence motif" derived from homologous repeat modules, and the homologous repeat modules are obtained from the repeat domain selected on the target and have the same target specificity.Repeat modules as used in the present invention cover internal repeat modules and end-capping modules, such as N-terminal and C-terminal end-capping modules." internal repeat module " refers to the repeat module of two repeat modules flanked.In other words, the internal repeat module is flanked by one repeat module at the N-terminal and another repeat module at the C-terminal.

[0208] Therefore, the term "ankyrin repeat module" refers to a repeating module of a repeating unit originally derived from a naturally occurring ankyrin repeat protein. Ankyrin repeat proteins are well known to those skilled in the art. Designed ankyrin repeat proteins have been previously described; see, for example, International Patent Publications WO2002020565, WO2010060748, WO2011135067, WO2012069654, WO2012069655, WO2014001442, WO2014191574, WO2014083208, WO2016156596 and WO2018054971, all of which are incorporated by reference in their entirety. Typically, ankyrin repeat modules comprise about 31 to 33 amino acid residues that form two alpha helices separated by a loop.

[0209] Repeat modules can include positions with amino acid residues that have not been randomized in the library for the purpose of selecting target-specific repeat domains ("non-randomized positions" or "fixed positions" are used interchangeably herein) and positions with amino acid residues that have been randomized in the library for the purpose of selecting target-specific repeat domains ("randomized positions"). Non-randomized positions include framework residues and may also include target interaction residues. Randomized positions include target interaction residues. "Randomized" means that two or more amino acids are allowed at the amino acid positions of the repeat module, for example, wherein any one of the twenty naturally occurring amino acids is allowed, or wherein most of the twenty naturally occurring amino acids are allowed, such as amino acids other than cysteine, or amino acids other than glycine, cysteine ​​and proline. For the purposes of this patent application, amino acid residues 3, 4, 6, 11, 14 and 15 of SEQ ID NOs: 26 to 35, 46 to 52, 61 to 62, 92 to 94 and 131 to 142, amino acid residues 4, 8, 11 and 12 of SEQ ID NOs: 15 to 25, 43 to 45, 57 to 60, 65 to 81, 99 to 101 and 115 to 122 and amino acid residues 3, 4, 6, 14 and 15 of SEQ ID NOs: 36 to 42, 53 to 56, 63 to 64, 82 to 91, 102 to 103 and 123 to 130 are randomized positions of the ankyrin repeat module of the present invention.

[0210] The term "repetitive sequence motif" refers to an amino acid sequence derived from one or more repeat modules. Preferably, the repeat modules are from repeat domains with binding specificity to the same target. Such repeat motifs comprise framework residue positions and target interaction residue positions. The framework residue positions correspond to the framework residue positions of the repeat modules. Likewise, the target interaction residue positions correspond to the positions of the target interaction residues of the repeat modules. The repeat motif comprises non-randomized positions and randomized positions.

[0211] The term "repeat unit" refers to an amino acid sequence comprising one or more sequence motifs of naturally occurring proteins, wherein the "repeat unit" is present in multiple copies and exhibits a defined folding topology common to all of the motifs that determine 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.

[0212] Residue or amino acid residue refers to the amino acid contained in the peptide chain.Term " target interaction residue " refers to the amino acid residue of the repeat module that contributes to direct interaction with target.The contribution of such residues can be tested, for example, in binding assay (for example, in mutagenesis study), to identify the required, sufficient and / or necessary residues of the repeat domain with its original binding affinity or amount (i.e. its binding affinity or amount in the absence of any mutation) binding target.Target interaction residue can also be determined by structural analysis of the repeat domain combined with target.

[0213] 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 the interaction with adjacent modules. Such contributions may be interactions with other residues in the repeat module, or an influence on the conformation of the polypeptide backbone as present in α-helices or β-sheets, or amino acid stretches that participate in the formation of linear polypeptides or loops. Specifically, for the purpose of calculating (1) the percentage of basic amino acid residues among the framework residues contained in the designed ankyrin repeat domain of the present invention, or (2) the percentage of Arg and Lys residues together among the framework residues contained in the designed ankyrin repeat domain of the present invention, the term "framework residues" includes amino acid residues located at positions within the designed ankyrin repeat domain corresponding to the positions of the representative N-terminal capping module (i.e., positions 1 to 3, 5 to 7, 9, 10, and 13 to 30 of SEQ ID NO: 43), the representative internal repeat module (i.e., positions 1, 2, 5, 7 to 10, 12, 13, and 16 to 33 of SEQ ID NO: 48), and the representative C-terminal capping module (i.e., positions 1, 2, 5, 7 to 13, and 16 to 28 of SEQ ID NO: 54) listed in Table 4. The term "framework residues" does not include amino acid residues located at positions within the designed ankyrin repeat domain, which positions correspond to the positions of the representative N-terminal capping module (i.e., positions 4, 8, 11 and 12 of SEQ ID NO: 43), the representative internal repeat module (i.e., positions 3, 4, 6, 11, 14 and 15 of SEQ ID NO: 48), and the representative C-terminal capping module (i.e., positions 3, 4, 6, 14 and 15 of SEQ ID NO: 54) listed in Table 5.

[0214] Such framework and target interacting 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 and relevant structural information well known to practitioners in the fields of structural biology and / or bioinformatics.

[0215] The terms "binding specificity", "having binding specificity for a target", "specifically binds to a target", "binds to a target with high specificity", "specific for a target" or "target specificity" and the like mean that a binding protein or binding domain binds to a target with a lower dissociation constant (i.e., it binds with a higher affinity) than it binds to an unrelated protein such as E. coli maltose binding protein (MBP). Preferably, the dissociation constant ("K D ”) is at least 10 times greater than the corresponding dissociation constant of MBP 2 times, more preferably at least 10 3 times, more preferably at least 10 4 times or more preferably at least 10 5 Methods for determining the dissociation constant of a protein-protein interaction, such as techniques based on surface plasmon resonance (SPR) (e.g., SPR equilibrium analysis) or isothermal titration calorimetry (ITC) are well known to those skilled in the art. If the measurement is performed under different conditions (e.g., salt concentration, pH), the measured K for a specific protein-protein interaction may be different. D The K values ​​may vary. Therefore, it is preferred to use a standardized protein solution and a standardized buffer (such as PBS) for K determination. D The measurement of the value.

[0216] The binding of any molecule to another molecule is governed by two forces, the binding rate (k on ) and dissociation rate (k off The affinity of any binder [B] for the target [T] can be expressed by the equilibrium dissociation constant K D It means that k off / k on Business.

[0217]

[0218] k on is the second-order rate constant of the binding reaction, in M -1 s -1 , and the dissociation reaction k off is the first-order rate constant, in s -1 It is clear from this that the association reaction depends on the concentration of the reactants, whereas the dissociation is independent of concentration, following a simple exponential decay function.

[0219] A variety of methods for measuring binding affinity are known in the art, any of which may be used for the purposes of the present invention. For example, as exemplified herein, the binding affinity of a particular binding moiety to a drug molecule target may be expressed as K D The value K is the dissociation constant between the binding moiety and the drug molecule target. D is the dissociation rate (also called the “dissociation rate (koff )”) and the association rate or “binding rate (k on )”. Therefore, K D Equal to k off / k on and expressed as molar concentration (M), and K D The smaller it is, the stronger the binding affinity.

[0220] K D The value can be determined using any suitable method. D An exemplary method is surface plasmon resonance (SPR) (see, e.g., Nguyen et al., Sensors (Basel). 2015 May 5; 15(5): 10481-510). D The value can be obtained by using SPR biosensor systems such as BIAcore kinetic analysis includes, for example, analysis of the binding and dissociation of an antigen to a chip having molecules (e.g., molecules comprising an epitope binding domain) immobilized on its surface. D Another method is by using biolayer interferometry (see, e.g., Shah et al., J Vis Exp. 2014; (84): 51383). D Values ​​can be used Technology (Octet Qke system, ForteBio). Alternatively or additionally, it is also possible to use (kinetic exclusion assay) assay, available from Sapidyne Instruments (Boise, supra). Any method suitable for assessing the binding affinity between two binding partners is contemplated herein. Surface plasmon resonance (SPR) is particularly preferred. Most preferably, K is determined in PBS and by SPR D value.

[0221] The term "isoelectric point" or "pi" refers to the pH value at which a macromolecule, such as a protein, carries no net charge. There may be many charged groups in a protein, and at the isoelectric point the sum of all these charges is zero. At a pH above the isoelectric point, the total net charge of the polypeptide will be negative, while at a pH below the isoelectric point, the total net charge of the polypeptide will be positive. The isoelectric point can be determined experimentally or the isoelectric point of a polypeptide can be calculated based on the primary sequence. Technicians know methods for determining the isoelectric point of a protein. Most commonly, the isoelectric point of a protein is calculated based on the amino acid sequence of the protein. Many tools that allow the calculation of the isoelectric point of a protein are available online. One such preferred tool is "ExPASy Compute pI / Mw" (https: / / web.expasy.org / compute_pi / ); see Protein Identification and Analysis Tools on the ExPASy server; Gasteiger E., Hoogland C., Gattiker A., ​​Duvaud S., Wilkins MR, Appel RD, Bairoch A.; (In) John M. Walker (ed.): The Proteomics Protocols Handbook, Humana Press (2005), pp. 571-607. This "ExPASy Compute pI / Mw" tool is preferably used to determine the pI of the ankyrin repeat domain of the present invention. Any N-terminal or C-terminal tag comprising one or more amino acids (which may be fused to the repeat domain for production or other purposes) and any N-terminal or C-terminal peptide linker are not considered for calculating the pI of the repeat domain of the present invention. Such tags or linkers are well known in the art and include, for example, a His6-TEV tag (N-terminus), a GS residue (N-terminus) of SEQ ID NO: 13, and a GSGSC tag (C-terminus) of SEQ ID NO: 14, such as Figure 3 shown.

[0222] The term "basic amino acid" refers to a hydrophilic amino acid with a positively charged side chain at physiological pH. Of the 20 common amino acids, His (H), Arg (R), and Lys (K) are basic amino acids (see Table 1). The term "acidic amino acid" refers to a hydrophilic amino acid with a negatively charged side chain at physiological pH. Of the 20 common amino acids, Asp (D) and Glu (E) are acidic amino acids (see Table 1). Basic amino acids and acidic amino acids may also be collectively referred to as charged amino acids because their side chains are ionized at physiological pH. "Neutral amino acids" refer to amino acids that are neither basic nor acidic and are therefore effectively non-ionized under physiological conditions. 15 of the 20 common amino acids are neutral amino acids (see Table 1). Such considerations are well known to those skilled in the art.

[0223] The term "drug moiety" refers to a chemical moiety that is linked to or suitable for linking to a protein, particularly a designed repeat domain or a designed repeat protein, and includes any therapeutic or diagnostic agent having a desired therapeutic or diagnostic property, respectively. Drug moieties as used herein encompass the terms "therapeutic moiety" and "diagnostic moiety". Such moieties can be linked to the repeat domain or repeat protein of the invention using methods available in the art or, for example, as described in Example 3.

[0224] The term "therapeutic moiety" refers to a chemical moiety that, when administered or otherwise provided to a patient or subject, can act as a therapeutic agent (or perform a therapeutic function), such as for treating a disease or disorder.

[0225] The term "diagnostic moiety" refers to a chemical moiety that, when administered or otherwise provided to a patient or subject, can be used as a diagnostic agent (or perform a diagnostic function), such as for diagnosing a disease or disorder.

[0226] The term "linked" or "linkage" refers to any covalent or non-covalent linkage between a chemical moiety and a protein, such as a designed repeat domain or a designed repeat protein.

[0227] The term "toxin" refers to any agent that is detrimental to the growth, proliferation and / or survival of cells and can act to reduce, inhibit, kill and / or destroy cells or malignancies. The term encompasses radionuclides and cytotoxic agents that may be toxic, for example, due to their radioactivity.

[0228] The term "cytotoxic agent" or "cytotoxin" refers to a substance that causes cell death or toxicity mainly by interfering with the life processes of cells (such as gene expression activity, DNA replication, cell division and / or cell survival). Non-limiting examples of cytotoxins include chemotherapeutic agents, mitotic inhibitors, growth inhibitors, enzymes and fragments thereof (such as nucleolytic enzymes), antibiotics, toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin, auristatin, calicheamicin, maytansinoid and camptothecin analogs. Other non-limiting examples of cytotoxins are cytotoxins that can be used in antibody-drug conjugates as described in, for example, Drago, Joshua Z., Shanu Modi and Sarat Chandarlapaty. Nature Reviews Clinical Oncology 18.6 (2021).

[0229] The term "radionuclide" or "radioisotope" refers to an isotope of natural or artificial origin having an unstable neutron to proton ratio that decomposes with the emission of particulate matter, i.e., protons (α-radiation) or electrons (β-radiation) or electromagnetic radiation (γ-radiation). In other words, the radionuclide undergoes radioactive decay. Such radionuclides include, but are not limited to 94 Tc, 99m Tc, 90 ln, 111 ln, 67 Ga, 68 Ga, 86 Y. 90 Y. 177 Lu, 151 Tb, 223 Ra, 186 Re, 188 Re, 64 Cu, 67 Cu, 55 Co. 57 Co. 43 Sc, 44 Sc, 47 Sc, 235 Ac, 213 Bi, 212 Bi, 212 Pb, 227 Th, 153 Sm, 166 Ho, 152 Gd, 153 Gd, 157 Gd, 225 Ac or 166 The selection of an appropriate radionuclide may depend on the chemical structure and chelating capacity of the chelating agent, as well as the intended application (eg, diagnostics versus therapy) of the resulting drug.

[0230] The term "chelator" refers to a multidentate (multiple bonded) ligand capable of forming two or more separate coordination bonds with ("coordinate") a central (metal) ion. Specifically, such molecules or molecules sharing one electron pair may also be referred to as "Lewis bases". The central (metal) ion is typically coordinated with the chelator by two or more electron pairs. Typically, the electron pairs of the chelator form coordination bonds with a single central (metal) ion; however, in some examples, the chelator may form coordination bonds with more than one metal ion, wherein a variety of binding modes are possible. The term "coordination" refers to a coordination bond ("coordination") in which one of the multi-electron pair donors is coordinated with a central (metal) ion, i.e., an interaction in which two or more unshared electron pairs are shared with a central (metal) ion. Chelators are preferably selected based on their ability to coordinate the desired central (metal) ion (typically a radionuclide as specified herein).

[0231] For example, the term "percentage" used in the percentage of basic amino acid residues means the value obtained by dividing the number of basic residues contained in the sequence under consideration (e.g., (1) the entire sequence of the repeat domain or (2) all framework residues contained in the repeat domain) by the total number of amino acid residues contained in the sequence under consideration, and multiplying the result by 100 to obtain the percentage. Any N-terminal or C-terminal tag comprising one or more amino acids (which may be fused to the repeat domain for production or other purposes) and any N-terminal or C-terminal peptide linker are not considered for calculating such percentages. Such tags or linkers are well known in the art and include, for example, the His6-TEV tag (N-terminus) of SEQ ID NO: 13, the GS residue (N-terminus) and the GSGSC tag (C-terminus) of SEQ ID NO: 14, such as Figure 3 shown.

[0232] The term "physiological conditions" refers to conditions that normally exist in mammals. Thus, for example, for humans, physiological conditions mean a pH between 7.35 and 7.45, with an average of 7.40, and a temperature between 36.1°C and 37.2°C, with an average of 37°C.

[0233] Repeat domains of the present invention

[0234] DARPin (designed ankyrin repeat protein) is a repeat protein comprising a designed ankyrin repeat domain, which generally comprises one or more internal repeat modules that are identical or similar except for their randomized positions and flanked by N-terminal and C-terminal capping repeat modules (see, e.g., Binz et al. 2003, supra). As used herein, the term "repeat module" encompasses internal repeat modules and terminal repeat modules (C-capping modules and N-capping modules). Of the 33 amino acid positions of a typical internal repeat module, 27 amino acid positions are highly conserved, while the other 6 amino acid positions are less conserved and are largely responsible for the specific interaction of the ankyrin repeat domain with its target (Binz et al. 2003, supra). The paratope of the ankyrin repeat domain is formed by a continuous surface formed primarily by these variable positions of the internal repeat modules and sometimes the capping repeat modules. DARPin also encompasses proteins comprising multiple designed ankyrin repeat domains connected together by appropriate linkers. Such linkers are known to those skilled in the art.

[0235] Representative designed ankyrin repeat domains according to the present invention have been generated and characterized as described in Examples 1 to 8 and are shown in FIGS. 1 and 8 .

[0236] Thus, in one aspect, the present invention provides a designed ankyrin repeat domain having a reduced number of basic amino acid residues and / or a reduced pI compared to a conventional designed ankyrin repeat domain. As will be described in more detail below, the designed ankyrin repeat domain of the present invention can be obtained by substituting basic amino acids, and methods for performing such substitutions are well known in the art and include mutagenesis of a cDNA encoding the repeat domain.

[0237] The ankyrin repeat domains described herein generally comprise a core scaffold that provides structure, and target interaction residues that bind to a target. The structural core comprises conserved amino acid residues (also referred to as framework residues), and the target binding surface comprises target interaction residues that vary depending on the target.

[0238] Table 1 shows typical properties of common amino acids considered in this specification.

[0239] Table 1

[0240] Amino Acids category Charge (under physiological conditions) Arginine, Arg, R Alkaline just Lysine, Lys, K Alkaline just Histidine, His, H Alkaline just Glutamate, Glu, E Acidic burden Aspartic acid, Asp, D Acidic burden Glycine, Gly, G neutral none Alanine, Ala, A neutral none Proline, Pro, P neutral none Valine, V neutral none Leucine, Leu, L neutral none Isoleucine, Ile, I neutral none Methionine, Met, M neutral none Phenylalanine, Phe, F neutral none Tyrosine, Tyr, Y neutral none Tryptophan, Trp, W neutral none Serine, Ser, S neutral none Threonine, Thr, T neutral none Cysteine, Cys, C neutral none Asparagine, Asn, N neutral none Glutamine, Gln, Q neutral none

[0241] The categories of charge-modifying amino acid residue substitutions considered in this specification and their effects on charge are shown in Table 2.

[0242] Table 2

[0243] initial replace Effect on charge Alkaline neutral reduce neutral Acidic reduce Alkaline Acidic Maximum reduction

[0244] Examples of conservative and other exemplary amino acid residue substitutions that may occur in the repeat domains and proteins of the invention are shown in Table 3.

[0245] Table 3

[0246] Initial residue Conservative substitution Exemplary substitutions Ala(A) Val Val; Leu; Ile Arg(R) Lys Lys; Gln; Asn Asn(N) Gln Gln; His; Asp, Lys; Arg Asp(D) Glu Glu; Asn Cys(C) Ser Ser; Ala Gln(Q) Asn Asn;Glu Glu(E) Asp Asp; Gln Gly(G) Ala Ala His(H) Arg Asn; Gln; Lys; Arg Ile(I) Leu Leu; Val; Met; Ala; Phe; Norleucine Leu(L) Ile Norleucine; Ile; Val; Met; Ala; Phe Lys(K) Arg Arg; Gln; Asn Met(M) Leu Leu; Phe; Ile Phe(F) Tyr Leu; Val; Ile; Ala; Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr Tyr; Phe Tyr(Y) Phe Trp; Phe; Thr; Ser Val(V) Leu Ile; Leu; Met; Phe; Ala; norleucine

[0247] In some embodiments, the designed ankyrin repeat domain of the invention has a pi of equal to or lower than pH 4.9, equal to or lower than pH 4.8, equal to or lower than pH 4.7, equal to or lower than pH 4.6, equal to or lower than pH 4.5, equal to or lower than pH 4.4, equal to or lower than pH 4.3, equal to or lower than pH 4.2, equal to or lower than pH 4.1, equal to or lower than pH 4.0, equal to or lower than pH 3.9, equal to or lower than pH 3.8, equal to or lower than pH 3.7, equal to or lower than pH 3.6, or equal to or lower than pH 3.5.

[0248] In some embodiments, the designed ankyrin repeat domains of the invention have a pH of at or below pH 4.53, at or below pH 4.52, at or below pH 4.50, at or below pH 4.48, at or below pH 4.46, at or below pH 4.44, at or below pH 4.42, at or below pH 4.40, at or below pH 4.38, at or below pH 4.36, at or below pH 4.34, at or below pH 4.32, at or below pH 4.30, at or below pH 4.28, at or below pH 4.26, at or below pH 4.24, at or below pH 4.22, at or below pH 4.20, at or below pH 4.18, at or below pH 4.16, at or below pH 4.14, at or below pH 4.12, at or below pH 4.10, at or below pH 4.14, at or below pH 4.15, at or below pH 4.16 4.08, equal to or lower than pH 4.06, equal to or lower than pH 4.04, equal to or lower than pH 4.02, equal to or lower than pH 4.00, equal to or lower than pH 3.98, equal to or lower than pH 3.96, equal to or lower than pH 3.94, equal to or lower than pH 3.92, equal to or lower than pH 3.90, equal to or lower than pH 3.88, equal to or lower than pH 3.86, equal to or lower than pH 3.84, equal to or lower than pH 3.82, equal to or lower than pH 3.80, equal to or lower than pH 3.78, equal to or lower than pH 3.76, equal to or lower than pH 3.74, equal to or lower than pH 3.72, equal to or lower than pH 3.70, equal to or lower than pH 3.68, equal to or lower than pH 3.66, equal to or lower than pH 3.64, equal to or lower than pH 3.62, equal to or lower than pH 3.60, equal to or lower than pH 3.58, at or below pH 3.56, at or below pH 3.54, at or below pH 3.52, or at or below pH 3.50.

[0249] In some embodiments, the designed ankyrin repeat domains of the invention have a pH between pH 3.00 and pH 4.60, between pH 3.10 and pH 4.60, between pH 3.20 and pH 4.60, between pH 3.30 and pH 4.60, between pH 3.40 and pH 4.60, between pH 3.50 and pH 4.60, between pH 3.50 and pH 4.58, between pH 3.50 and pH 4.56, between pH 3.50 and pH 4.54, between pH 3.50 and pH 4.53, between pH 3.50 and pH 4.52, between pH 3.50 and pH 4.50, between pH 3.50 and pH 4.48, between pH 3.50 and pH 4.46, between pH 3.50 and pH 4.44, between pH 3.50 and pH 4.42, between pH 3.50 and pH 4.40, between pH 3.50 and pH 4.38, between pH 3.50 and pH 4.36, between pH 3.50 and pH 4.34, between pH 3.50 and pH 4.32, between pH 3.50 and pH 4.30, between pH 3.60 and pH 4.60, between pH 3.60 and pH 4.58, between pH 3.60 and pH 4.56, between pH 3.60 and pH 4.54, between pH 3.60 and pH 4.53, between pH 3.60 and pH 4.52, between pH 3.60 and pH 4.50, between pH 3.60 and pH 4.48, between pH 4.42, between pH 3.60 and pH 4.40, between pH 3.60 and pH 4.38, between pH 3.60 and pH 4.36, between pH 3.60 and pH 4.34, between pH 3.60 and pH 4.32, between pH 3.60 and pH 4.30, between pH 3.70 and pH 4.60, between pH 3.70 and pH 4.58, between pH 3.70 and pH 4.56, between pH 3.70 and pH 4.54, between pH 3.70 and pH 4.53, between pH 3.70 and pH 4.52, between pH 3.70 and pH 4.64, between pH 3.70 and pH 4.70, between pH 3.70 and pH 4.80, between pH 3.70 and pH 4.90, between pH 3.81 and pH 4.91, between pH 3.82 and pH 4.92, between pH 3.83 and pH 4.93 between pH 3.70 and pH 4.48, between pH 3.70 and pH 4.46, between pH 3.70 and pH 4.44, between pH 3.70 and pH 4.42, between pH 3.70 and pH 4.40, between pH 3.70 and pH 4.38, between pH 3.70 and pH 4.36, between pH 3.70 and pH 4.34, between pH 3.70 and pH 4.32, or in a range between pH 3.70 and pH 4.30. .

[0250] In some embodiments, the designed ankyrin repeat domain of the invention has a percentage of basic amino acid residues of equal to or less than 12.0%, equal to or less than 11.5%, equal to or less than 11.0%, equal to or less than 10.5%, equal to or less than 10.0%, equal to or less than 9.5%, or equal to or less than 9.0%.

[0251] In some embodiments, the designed ankyrin repeat domain of the invention has an affinity for 5-10% tantalum products, an affinity for 5-10% tantalum products, an affinity for 5-10% tantalum products, an affinity for 5-10% tantalum products, an affinity for 5-10% tantalum products, an affinity for 5-10% tantalum products, an affinity for 5-10% tantalum products, an affinity for 5-10% tantalum products, an affinity for 5-10% tantalum products, an affinity for 5-10% tantalum products, an affinity for 5-10% tantalum products, an affinity for 5-10% tantalum products, an affinity for 5-10% tantalum products, an affinity for 5-10% tantalum products, an affinity for 5-10% tantalum products, an affinity for 5-10% tantalum products, an affinity for 5-10% tantalum products, an affinity for 5-10% tantalum products, an affinity for 5-10% tantalum products, an affinity for 5-10% tantalum products, The percentage of basic amino acid residues is less than 6.90%, equal to or less than 6.80%, equal to or less than 6.70%, equal to or less than 6.60%, equal to or less than 6.50%, equal to or less than 6.40%, equal to or less than 6.30%, equal to or less than 6.20%, equal to or less than 6.10%, equal to or less than 6.00%, equal to or less than 5.90%, equal to or less than 5.80%, equal to or less than 5.70%, equal to or less than 5.60%, equal to or less than 5.50%, equal to or less than 5.40%, equal to or less than 5.30%, equal to or less than 5.20%, equal to or less than 5.10%, equal to or less than 5.00%, equal to or less than 4.90% or equal to or less than 4.80%.

[0252] Histidine residues are usually partially positively charged under physiological conditions. The effect of charge reduction caused by mutation of His to neutral or acidic residues is not as obvious as mutation of Lys or Arg residues to neutral or acidic residues. Unless otherwise specifically stated, basic amino acid residues include Arg, Lys and His.

[0253] In some embodiments, the designed ankyrin repeat domains of the invention have a percentage of Arg and Lys residues equal to or less than 8.0%, equal to or less than 7.5%, equal to or less than 7.0%, equal to or less than 6.5%, or equal to or less than 6.0%.

[0254] For the sake of clarity, the term "percentage of Arg and Lys residues" used in the present disclosure shall refer to the percentage of Arg residues and Lys residues together.

[0255] In some embodiments, the designed ankyrin repeat domain of the invention has a molecular weight of 5.65% or less, 5.60% or less, 5.50% or less, 5.40% or less, 5.30% or less, 5.20% or less, 5.10% or less, 5.00% or less, 4.90% or less, 4.80% or less, 4.70% or less, 4.60% or less, 4.5 0%, 4.40% or less, 4.30% or less, 4.20% or less, 4.10% or less, 4.00% or less, 3.90% or less, 3.80% or less, 3.70% or less, 3.60% or less, 3.50% or less, 3.40% or less, 3.30% or less, 3.20% or less, 3.10% or less, 3.00% or less , 2.90% or less, 2.80% or less, 2.70% or less, 2.60% or less, 2.50% or less, 2.40% or less, 2.30% or less, 2.20% or less, 2.10% or less, 2.00% or less, 1.90% or less, 1.80% or less, 1.70% or less, 1.60% or less, 1.50% or less, etc. The percentage of Arg and Lys residues of 1.40% or less, 1.30% or less, 1.20% or less, 1.10% or less, 1.00% or less, 0.90% or less, 0.80% or less, 0.70% or less, 0.60% or less, 0.50% or less, 0.40% or less, 0.30% or less, 0.20% or less, or 0.10% or less.

[0256] The number of basic amino acid residues in the designed ankyrin repeat domain of the present invention can be selectively limited according to the position of the basic amino acid residues in the repeat domain. In some embodiments, the number of basic amino acids is considered only in the framework residues of the repeat domain. The position of the framework residues can be determined by those skilled in the art. Preferably, the framework residues should correspond to residues occupying specific positions in the repeat module as described in Table 4:

[0257] Table 4

[0258]

[0259]

[0260] In some embodiments, the designed ankyrin repeat domain of the invention has a percentage of basic amino acid residues among the framework residues of 9.6% or less, 9.4% or less, 9.2% or less, 9.0% or less, 8.8% or less, 8.6% or less, 8.4% or less, 8.2% or less, 8.0% or less, 7.8% or less.

[0261] In some embodiments, the designed ankyrin repeat domain of the invention has equal to or less than 7.77%, equal to or less than 7.70%, equal to or less than 7.60%, equal to or less than 7.50%, equal to or less than 7.40%, equal to or less than 7.30%, equal to or less than 7.20%, equal to or less than 7.10%, equal to or less than 7.00%, equal to or less than 6.90%, equal to or less than 6.80%, equal to or less than 6.70%, equal to or less than 6.60%, equal to or less than 6.50%, equal to or less than 6.40%, equal to or less than 6.30%, equal to or less than 6. The percentage of basic amino acid residues of 6.20% or less, 6.10% or less, 6.00% or less, 5.90% or less, 5.80% or less, 5.70% or less, 5.60% or less, 5.50% or less, 5.40% or less, 5.30% or less, 5.20% or less, 5.10% or less, 5.00% or less, 4.90% or less, 4.80% or less, 4.70% or less, 4.60% or less, or 4.50% or less.

[0262] In some embodiments, the designed ankyrin repeat domain of the invention has a percentage of Arg and Lys residues among framework residues that is equal to or lower than 6.7%, equal to or lower than 6.6%, equal to or lower than 6.5%, equal to or lower than 6.4%, equal to or lower than 6.3%, equal to or lower than 6.2%, equal to or lower than 6.1%, equal to or lower than 6.0%, or equal to or lower than 5.9%.

[0263] In some embodiments, the designed ankyrin repeat domains of the invention have 5.83% or less, 5.80% or less, 5.70% or less, 5.60% or less, 5.50% or less, 5.40% or less, 5.30% or less, 5.20% or less, 5.10% or less, 5.00% or less, 4.90% or less, 4.80% or less, 4.70% or less in framework residues. %, 4.60% or less, 4.50% or less, 4.40% or less, 4.30% or less, 4.20% or less, 4.10% or less, 4.00% or less, 3.90% or less, 3.80% or less, 3.70% or less, 3.60% or less, 3.50% or less, 3.40% or less, 3.30% or less, 3.20% or less, 3. 10%, equal to or less than 3.00%, equal to or less than 2.90%, equal to or less than 2.80%, equal to or less than 2.70%, equal to or less than 2.60%, equal to or less than 2.50%, equal to or less than 2.40%, equal to or less than 2.30%, equal to or less than 2.20%, equal to or less than 2.10%, equal to or less than 2.00%, equal to or less than 1.90%, equal to or less than 1.80%, equal to or less than 1.70%, equal to or less than 1.60%, equal to or less than 1.50%, 1.40%, 1.30%, 1.20%, 1.10%, 1.00%, 0.90%, 0.80%, 0.70%, 0.60%, 0.50%, 0.40%, 0.30%, 0.20%, or 0.10% Arg and Lys residues.

[0264] The designed ankyrin repeat domain of the present invention may comprise an N-terminal capping module, a C-terminal capping module and at least one internal repeating module. Preferably, the repeating domain of the present invention comprises 1, 2, 3, 4, 5, 6, 7, 8 or 9 internal repeating modules. More preferably, the repeating domain of the present invention comprises 1, 2, 3 or 4 internal repeating modules.

[0265] In some embodiments, the total number of basic amino acid residues contained in the designed ankyrin repeat domain of the present invention can be expressed as a function based on the number of repeat modules contained in the repeat domain. In one embodiment, the ankyrin repeat domain of the invention has a total number of basic amino acid residues equal to or lower than n, wherein n=R, n=1+R, n=2+R, n=3+R, n=4+R, n=5+R, n=6+R, n=7+R, n=8+R, n=9+R, n=2R, n=1+2R, n=2+2R, n=3+2R, n=4+2R, n=5+2R, n=6+2R, n=7+2R, n=8+2R, n=9+2R, n=3R, n=1+3R, n=2+3R, n=3+3R, n=4+3R, n=5+3R, n=6+3R, n=7+3R, n=8+3R, n=9 In some embodiments, the invention relates to a repeat domain in which the plurality of repeat modules are present in an inner region of the repetitive structure. In some embodiments, the plurality of repeat domains are present in an inner region of the repetitive structure. In some embodiments, the plurality of repeat domains are present in an inner region of the repetitive structure. In some embodiments, the plurality of repeat domains are present in an inner region of the repetitive structure. In some embodiments, the plurality of repeat domains are present in an inner region of the repetitive structure.

[0266] In another embodiment, the total number of Arg and Lys residues contained in the designed ankyrin repeat domain of the present invention is equal to or lower than m, wherein m=R, m=1+R, m=2+R, m=3+R, m=4+R, m=5+R, m=6+R, m=7+R, m=8+R, m=9+R, m=2R, m=1+2R, m=2+2R, m=3+2R, m=4+2R, m=5+2R, m=6+2R, m=7+2R, m=8+2R , m=9+2R, m=3R, m=1+3R, m=2+3R, m=3+3R, m=4+3R, m=5+3R, m=6+3R, m=7+3R, m=8+3R, m=9+3R, m=4R, m=1+4R, m=2+4R, m=3+4R, m=4+4R, m=5+4R, m=6+4R, m=7+4R, m=8+4R or m=9+4R, wherein R is the number of internal repeat modules contained in the repeat domain.

[0267] In some embodiments, the designed ankyrin repeat domain of the present invention has a total number of basic amino acid residues in the framework residues that is equal to or lower than x, wherein x=R, x=1+R, x=2+R, x=3+R, x=4+R, x=5+R, x=6+R, x=7+R, x=2R, x=1+2R, x=2+2R, x=3+2R, x=4+2R, x=5+2R, x=6+2R, x=7+2R, x=3R, x=1+3R, x=2+3R, x=3+3R, x=4+3R, x=5+3R, x=6+3R or x=7+3R, wherein R is the number of internal repeat modules contained in the repeat domain.

[0268] In other embodiments, the designed ankyrin repeat domain of the present invention has a total number of Arg and Lys residues in the framework residues that is equal to or less than y, wherein y=1+R, y=2+R, y=3+R, y=4+R, y=5+R, y=6+R, y=2R, y=1+2R, y=2+2R, y=3+2R, y=4+2R, y=5+2R or y=6+2R, wherein R is the number of internal repeat modules contained in the repeat domain.

[0269] Preferably, the designed ankyrin repeat domain of the present invention specifically binds to the target. In a preferred embodiment, the repeat domain of the present invention is about 10 -5 M or smaller, about 10 -6 M or smaller, about 10 -7 M or smaller, about 10 -8 M or smaller, about 10 -9 M or smaller, about 10 -10 M or smaller, about 10 -11 M or smaller, about 10 -12 M or smaller, about 10 -13 M or smaller, about 10 -14 M or less dissociation constant (K D ) binds to the target.

[0270] In some embodiments, the target is HER2. Therefore, in specific embodiments, the designed ankyrin repeat domain of the present invention specifically binds to HER2. HER2 as used herein relates to human epidermal growth factor receptor 2, also known as Neu, ErbB-2, CD340 (differentiation cluster 340) or p185. HER2 is a member of the epidermal growth factor receptor (EGFR / ErbB) family. In humans, HER2 is encoded by ERBB2, a known proto-oncogene located on the long arm of human chromosome 17 (17q12). HER2 has UniProtKB / Swiss-Prot number P04626.

[0271] In one embodiment, the present invention provides a designed ankyrin repeat domain, the designed ankyrin repeat domain comprising an N-terminal capping module, at least one internal repeat module and a C-terminal capping module, and wherein the repeat domain has at least one feature selected from the following list of features:

[0272] (a) an isoelectric point (pI) equal to or lower than pH 4.6;

[0273] (b) a percentage of basic amino acid residues of 12.0% or less among all amino acid residues contained in the repeat domain;

[0274] (c) a percentage of Arg and Lys residues of 8.0% or less among all the amino acid residues contained in the repeat domain;

[0275] (d) among the framework residues comprised in the repeat domain, the percentage of basic amino acid residues is equal to or lower than 9.6%; and

[0276] (e) Among the framework residues contained in the repeat domain, the percentage of Arg and Lys residues is equal to or lower than 6.7%.

[0277] In preferred embodiments, the repeat domain is linked to a drug moiety.

[0278] The present invention encompasses embodiments in which the repeat domain has any combination of the above features (a) to (e). Thus, in some embodiments, the repeat domain has at least features (a), (b), (c), (d) or (e). In other embodiments, the repeat domain has at least features (a) and (b), (a) and (c), (a) and (d), (a) and (e), (b) and (c), (b) and (d), (b) and (e), (c) and (d), (c) and (e) or (d) and (e). In other embodiments, the repeat domain has at least features (a), (b), and (c); (a), (b), and (d); (a), (b), and (e); (a), (c), and (d); (a), (c), and (e); (a), (d), and (e); (b), (c), and (d); (b), (c), and (e); (b), (d), and (e) or (c), (d), and (e). In other embodiments, the repeat domain has at least features (a), (b), (c), and (d); (a), (b), (c), and (e); (a), (b), (d), and (e); (a), (c), (d), and (e) or (b), (c), (d), and (e). In other embodiments, the repeat domain has features (a), (b), (c), (d), and (e). In some preferred embodiments, the repeat domain has an isoelectric point (pI) in the range of between pH 3.0 and pH 4.6, preferably between pH 3.3 and pH 4.6, and more preferably between pH 3.50 and pH 4.53. In some preferred embodiments, the percentage in (b) is equal to or lower than 8.7%. In some most preferred embodiments, the percentage in (b) is equal to or lower than 8.06%. In some preferred embodiments, the percentage in (c) is equal to or lower than 4.7%. In some most preferred embodiments, the percentage in (c) is equal to or lower than 4.03%. In some preferred embodiments, the percentage in (d) is equal to or lower than 6.7%. In some most preferred embodiments, the percentage in (d) is equal to or lower than 6.1%. In some preferred embodiments, the percentage in (e) is equal to or lower than 5.7%. In some most preferred embodiments, the percentage in (e) is equal to or lower than 4.85%.

[0279] In some embodiments, the repeat domain has a total number of basic amino acid residues equal to or less than n, wherein n=R, n=1+R, n=2+R, n=3+R, n=4+R, n=5+R, n=6+R, n=7+R, n=8+R, n=9+R, n=2R, n=1+2R, n=2+2R, n=3+2R, n=4+2R, n=5+2R, n=6+2R, n=7+2R, n=8+2R, n=9+2R, n=3R, n=1+3R, n=2+3R, n=3+3R, n=4+3R, n=5+3R, n=6+3R, n=7+3R, n=8+3R, n=9+3 R, n=4R, n=1+4R, n=2+4R, n=3+4R, n=4+4R, n=5+4R, n=6+4R, n=7+4R, n=8+4R, n=9+4R, n=5R, n=1+5R, n=2+5R, n=3+5R, n=4+5R, n=5+5R, n=6+5R, n=7+5R, n=8+5R, n=9+5R, n=6R, n=1+6R, n=2+6R, n=3+6R, n=4+6R, n=5+6R, n=6+6R, n=7+6R, n=8+6R or n=9+6R, wherein R is the number of internal repeat modules contained in the repeat domain. In some more specific embodiments, n=4+5R. In such embodiments, "xR" means x times R.

[0280] In some embodiments, the repeat domain has a total number of Arg and Lys residues equal to or less than m, wherein m=R, m=1+R, m=2+R, m=3+R, m=4+R, m=5+R, m=6+R, m=7+R, m=8+R, m=9+R, m=2R, m=1+2R, m=2+2R, m=3+2R, m=4+2R, m=5+2R, m=6+2R, m=7+2R, m=8+2R, m=9+ In some specific embodiments, m=5+2R, m=3R, m=1+3R, m=2+3R, m=3+3R, m=4+3R, m=5+3R, m=6+3R, m=7+3R, m=8+3R, m=9+3R, m=4R, m=1+4R, m=2+4R, m=3+4R, m=4+4R, m=5+4R, m=6+4R, m=7+4R, m=8+4R or m=9+4R, wherein R is the number of internal repeat modules contained in the repeat domain. In some more specific embodiments, m=5+2R.

[0281] In some embodiments, the repeat domain has a total number of basic amino acid residues in the framework residues that is equal to or lower than x, wherein x=R, x=1+R, x=2+R, x=3+R, x=4+R, x=5+R, x=6+R, x=7+R, x=2R, x=1+2R, x=2+2R, x=3+2R, x=4+2R, x=5+2R, x=6+2R, x=7+2R, x=3R, x=1+3R, x=2+3R, x=3+3R, x=4+3R, x=5+3R, x=6+3R or x=7+3R, wherein R is the number of internal repeat modules contained in the repeat domain.

[0282] In some embodiments, the repeat domain has a total number of Arg and Lys residues in the framework residues that is equal to or less than y, where y=1+R, y=2+R, y=3+R, y=4+R, y=5+R, y=6+R, y=2R, y=1+2R, y=2+2R, y=3+2R, y=4+2R, y=5+2R or y=6+2R, where R is the number of internal repeat modules contained in the repeat domain.

[0283] In some embodiments, the repeat domains do not have Arg residues and Lys residues within the framework residue positions.

[0284] In some embodiments, the repeat domain comprises two internal repeat modules. In some embodiments, the repeat domain comprising two internal repeat modules may have a total number of basic amino acid residues equal to or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. Preferably, the total number of basic residues is equal to or less than 14. More preferably, the total number of basic residues is equal to or less than 10. In another specific embodiment, the repeat domain comprising two internal repeat modules may have a total number of Arg and Lys residues equal to or less than 1, 2, 3, 4, 5, 6, 7, 8 or 9. Preferably, the total number of Arg and Lys residues is equal to or less than 9. More preferably, the total number of Arg and Lys residues is equal to or less than 5. In another specific embodiment, the repeat domain comprising two internal repeat modules may have a total number of Arg and Lys residues equal to or less than 1, 2, 3, 4, 5 or 6 in framework residues. Preferably, the total number of said Arg and Lys residues in the framework residues is equal to or lower than 6. More preferably, the total number of said Arg and Lys residues in the framework residues is equal to or lower than 5.

[0285] In another embodiment, the present invention provides a designed ankyrin repeat domain, the designed ankyrin repeat domain comprising an N-terminal capping module, at least one internal repeat module and a C-terminal capping module, wherein the repeat domain has at least one feature selected from the following list of features:

[0286] (a) an isoelectric point (pI) equal to or lower than pH 4.07;

[0287] (b) a percentage of basic amino acid residues of 7.0% or less among all amino acid residues contained in the repeat domain; and

[0288] (c) Among the framework residues contained in the repeat domain, the percentage of basic amino acid residues is equal to or lower than 6.1%.

[0289] The present invention encompasses embodiments in which the repeat domain has any combination of the above features (a) to (c). Thus, in some embodiments, the repeat domain has at least feature (a), (b) or (c). In other embodiments, the repeat domain has at least features (a) and (b), (a) and (c) or (b) and (c). In other embodiments, the repeat domain has features (a), (b) and (c).

[0290] In some embodiments, the repeat domain is approximately 10 -5 M or smaller, about 10 -6 M or smaller, about 10 -7 M or smaller, about 10 -8 M or smaller, about 10 -9 M or smaller, about 10 -10 M or smaller, about 10 -11 M or smaller, about 10 -12 M or smaller, about 10 -13 M or less or about 10 -14 M or less dissociation constant (K D ) binds to the target. In a preferred embodiment, the repeat domain is less than 10 -7 The dissociation constant (K D ) binds to the target.

[0291] The charge characteristics of the designed ankyrin repeat domain according to the present invention may alternatively or additionally be defined by the ratio between the sum of Arg (R) and Lys (K) residues and the sum of Asp (D) and Glu (E) residues contained in the designed ankyrin repeat domain (also referred to as the KR / DE ratio). The KR / DE ratio may be calculated based on the amino acid sequence of the designed ankyrin repeat domain of the present invention.

[0292] Thus, in some embodiments, the designed ankyrin repeat domains of the invention have a KR / DE ratio of equal to or lower than 0.44, equal to or lower than 0.42, equal to or lower than 0.40, equal to or lower than 0.38, equal to or lower than 0.36, equal to or lower than 0.34, equal to or lower than 0.32, equal to or lower than 0.30, equal to or lower than 0.28, equal to or lower than 0.26, equal to or lower than 0.24, equal to or lower than 0.22, equal to or lower than 0.20, equal to or lower than 0.18, equal to or lower than 0.16, equal to or lower than 0.14, equal to or lower than 0.12, equal to or lower than 0.10, or equal to or lower than 0.08. Preferably, the KR / DE ratio is equal to or lower than 0.3.

[0293] Unless otherwise stated, the KR / DE ratio is determined by taking into account all residues contained in the repeat domain. In other embodiments, the KR / DE ratio is calculated only in the framework residues of the repeat domain. The framework residues are defined in Table 4 as residues in the repeat module corresponding to specific positions within the repeat module of the reference sequence. Therefore, in some embodiments, the designed ankyrin repeat domain of the present invention has a KR / DE ratio of equal to or lower than 0.36, equal to or lower than 0.34, equal to or lower than 0.32, equal to or lower than 0.30, equal to or lower than 0.28, equal to or lower than 0.26, equal to or lower than 0.24, equal to or lower than 0.22, equal to or lower than 0.20, equal to or lower than 0.18, equal to or lower than 0.16, equal to or lower than 0.14, equal to or lower than 0.12, equal to or lower than 0.10 or equal to or lower than 0.08 in the framework residues. Preferably, the KR / DE ratio in the framework residues is equal to or lower than 0.25.

[0294] In other embodiments, the KR / DE ratio is calculated only among all residues included in the N-terminal capping module. Thus, in some embodiments, the designed ankyrin repeat domain of the invention has a KR / DE ratio of 0.66, 0.64, 0.62, 0.60, 0.58, 0.56, 0.54, 0.52, 0.50, 0.48, 0.46, 0.44, 0.42, 0.4 ...50 Preferably, the KR / DE ratio of all residues in the N-terminal capping module is equal to or lower than 0.5.

[0295] In some embodiments, the designed ankyrin repeat domain of the present invention comprises one or more internal repeat modules, wherein each internal repeat module independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 131 to 142, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in any one of SEQ ID NOs: 131 to 142 is substituted by another amino acid.

[0296] In one embodiment, the designed ankyrin repeat domain of the present invention comprises one or more internal repeat modules, wherein each internal repeat module independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 131, 135 and 139, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in any one of SEQ ID NOs: 131, 135 and 139 is substituted by another amino acid.

[0297] In another embodiment, the designed ankyrin repeat domain of the present invention comprises one or more internal repeat modules, wherein each internal repeat module independently comprises a sequence selected from the group consisting of: (1) SEQ ID NO: 131, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in SEQ ID NO: 131 are substituted by another amino acid.

[0298] As an alternative or in addition to the internal repeat module defined above, in some embodiments, the designed ankyrin repeat domain of the present invention comprises an N-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID NOs: 25 and 115 to 122, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in any one of SEQ ID NOs: 25 and 115 to 122 are substituted by another amino acid.

[0299] In one embodiment, the N-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 115 and 121, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in any of SEQ ID NOs: 115 and 121 are substituted with another amino acid.

[0300] In another embodiment, the N-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NO: 115, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in SEQ ID NO: 115 is substituted with another amino acid.

[0301] As an alternative or in addition to the internal repeat module and / or N-terminal capping module defined above, in some embodiments, the designed ankyrin repeat domain of the present invention comprises a C-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID NOs: 123 to 130, and (2) a sequence in which at most 9 framework residues, at most 8 framework residues, at most 7 framework residues, at most 6 framework residues, at most 5 framework residues, at most 4 framework residues, at most 3 framework residues, at most 2 framework residues, or at most 1 framework residue in any one of SEQ ID NOs: 123 to 130 is substituted by another amino acid.

[0302] In one embodiment, the C-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 123 and 126, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in any of SEQ ID NOs: 123 and 126 is substituted with another amino acid.

[0303] In another embodiment, the C-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NO: 123, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in SEQ ID NO: 123 is substituted with another amino acid.

[0304] In a specific embodiment, the designed ankyrin repeat domain of the present invention comprises:

[0305] (a) an N-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0306] NO: 115 and 121, and (2) wherein at most 9 framework residues, at most 8 framework residues, at most 7 framework residues, at most 6 framework residues, at most 5 framework residues, at most 4 framework residues, at most 3 framework residues, at most 2 framework residues, or at most 1 framework residue in any of SEQ ID NO: 115 and 121 is replaced by another

[0307] The sequence of amino acid substitutions, and

[0308] (b) a C-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0309] NO:123 and 126, and (2) sequences in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in any of SEQ ID NO:123 and 126 is substituted by another amino acid.

[0310] In another embodiment, the designed ankyrin repeat domain of the present invention comprises:

[0311] (a) an N-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0312] NO:115, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in SEQ ID NO:115 is substituted with another amino acid, and (b) a C-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID NO:123, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in SEQ ID NO:123 is substituted with another amino acid.

[0313] In one embodiment, the designed ankyrin repeat domain of the present invention comprises:

[0314] (a) an N-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0315] NO: 115 and 121, and (2) wherein at most 9 framework residues, at most 8 framework residues, at most 7 framework residues, at most 6 framework residues, at most 5 framework residues, at most 4 framework residues, at most 3 framework residues, at most 2 framework residues, or at most 1 framework residue in any of SEQ ID NO: 115 and 121 is replaced by another

[0316] The sequence of amino acid substitutions, and

[0317] (b) one or more internal repeating modules, wherein each internal repeating module independently comprises a selected

[0318] A sequence selected from the group consisting of: (1) SEQ ID NOs: 131, 135 and 139, and (2) sequences in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in any of SEQ ID NOs: 131, 135 and 139 is substituted by another amino acid.

[0319] In another embodiment, the designed ankyrin repeat domain of the present invention comprises:

[0320] (a) an N-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0321] NO:115, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues or up to 1 framework residue in SEQ ID NO:115 is substituted with another amino acid, and (b) one or more internal repeat modules, wherein each internal repeat module independently comprises selected

[0322] A sequence selected from the group consisting of: (1) SEQ ID NO: 131, and (2) sequences in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in SEQ ID NO: 131 are substituted by another amino acid.

[0323] In a specific embodiment, the designed ankyrin repeat domain of the present invention comprises:

[0324] (a) a C-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0325] NO: 123 and 126, and (2) wherein at most 9 framework residues, at most 8 framework residues, at most 7 framework residues, at most 6 framework residues, at most 5 framework residues, at most 4 framework residues, at most 3 framework residues, at most 2 framework residues, or at most 1 framework residue in any of SEQ ID NO: 123 and 126 is replaced by another

[0326] The sequence of amino acid substitutions, and

[0327] (b) one or more internal repeating modules, wherein each internal repeating module independently comprises a selected

[0328] A sequence selected from the group consisting of: (1) SEQ ID NOs: 131, 135 and 139, and (2) sequences in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in any of SEQ ID NOs: 131, 135 and 139 is substituted by another amino acid.

[0329] In another embodiment, the designed ankyrin repeat domain of the present invention comprises:

[0330] (a) a C-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0331] NO:123, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues or up to 1 framework residue in SEQ ID NO:123 is substituted with another amino acid, and (b) one or more internal repeat modules, wherein each internal repeat module independently comprises selected

[0332] A sequence selected from the group consisting of: (1) SEQ ID NO: 131, and (2) sequences in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in SEQ ID NO: 131 are substituted by another amino acid.

[0333] In a specific embodiment, the designed ankyrin repeat domain of the present invention comprises:

[0334] (a) an N-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0335] NO: 115 and 121, and (2) wherein at most 9 framework residues, at most 8 framework residues, at most 7 framework residues, at most 6 framework residues, at most 5 framework residues, at most 4 framework residues, at most 3 framework residues, at most 2 framework residues, or at most 1 framework residue in any of SEQ ID NO: 115 and 121 is replaced by another

[0336] the sequence of amino acid substitutions; and

[0337] (b) one or more internal repeating modules, wherein each internal repeating module independently comprises a selected

[0338] A sequence of the group consisting of: (1) SEQ ID NOs: 131, 135 and 139, and (2) wherein up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in any of SEQ ID NOs: 131, 135 and 139 is substituted with another amino acid.

[0339] sequence; and

[0340] (c) a C-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0341] NO:123 and 126, and (2) sequences in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in any of SEQ ID NO:123 and 126 is substituted by another amino acid.

[0342] In one embodiment, the designed ankyrin repeat domain of the invention comprises:

[0343] (a) an N-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0344] NO:115, and (2) sequences in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in SEQ ID NO:115 are substituted with another amino acid; and

[0345] (b) one or more internal repeating modules, wherein each internal repeating module independently comprises a selected

[0346] A sequence of the group consisting of: (1) SEQ ID NO: 131, and (2) wherein at most 9 framework residues, at most 8 framework residues, at most 7 framework residues, at most 6 framework residues, at most 5 framework residues, at most 4 framework residues, at most 3 framework residues, at most 2 framework residues, or at most 1 of SEQ ID NO: 131

[0347] A sequence in which a framework residue is substituted with another amino acid; and

[0348] (c) a C-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0349] NO:123, and (2) sequences in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues or up to 1 framework residue in SEQ ID NO:123 are substituted by another amino acid.

[0350] In one embodiment, the designed ankyrin repeat domain of the invention comprises:

[0351] (a) an N-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0352] NO:115, and (2) wherein up to 9 framework residues in SEQ ID NO:115 are

[0353] another amino acid substituted sequence; and

[0354] (b) one or more internal repeating modules, wherein each internal repeating module independently comprises a selected

[0355] A sequence consisting of the group consisting of: (1) SEQ ID NO: 131, and (2) a sequence in which up to 9 framework residues in SEQ ID NO: 131 are substituted by another amino acid.

[0356] Columns; and

[0357] (c) a C-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0358] NO:123, and (2) sequences in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues or up to 1 framework residue in SEQ ID NO:123 are substituted by another amino acid.

[0359] In one embodiment, the designed ankyrin repeat domain of the invention comprises:

[0360] (a) an N-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0361] NO:115, and (2) wherein up to 5 framework residues in SEQ ID NO:115 are

[0362] another amino acid substituted sequence; and

[0363] (b) one or more internal repeating modules, wherein each internal repeating module independently comprises a selected

[0364] A sequence consisting of the group consisting of: (1) SEQ ID NO: 131, and (2) a sequence in which up to 5 framework residues in SEQ ID NO: 131 are substituted by another amino acid.

[0365] Columns; and

[0366] (c) a C-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0367] NO:123, and (2) sequences in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues or up to 1 framework residue in SEQ ID NO:123 are substituted by another amino acid.

[0368] In one embodiment, the designed ankyrin repeat domain of the invention comprises:

[0369] (a) an N-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0370] NO:115, and (2) wherein up to 3 framework residues in SEQ ID NO:115 are

[0371] another amino acid substituted sequence; and

[0372] (b) one or more internal repeating modules, wherein each internal repeating module independently comprises a selected

[0373] A sequence consisting of the group consisting of: (1) SEQ ID NO: 131, and (2) a sequence in which up to 3 framework residues in SEQ ID NO: 131 are substituted by another amino acid

[0374] Columns; and

[0375] (c) a C-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0376] NO:123, and (2) sequences in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues or up to 1 framework residue in SEQ ID NO:123 are substituted by another amino acid.

[0377] In one embodiment, the designed ankyrin repeat domain of the invention comprises:

[0378] (a) an N-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0379] NO:115, and (2) wherein up to 9 framework residues in SEQ ID NO:115 are

[0380] another amino acid substituted sequence; and

[0381] (b) one or more internal repeating modules, wherein each internal repeating module independently comprises a selected

[0382] A sequence of the group consisting of: (1) SEQ ID NO: 131, and (2) wherein at most 9 framework residues, at most 8 framework residues, at most 7 framework residues, at most 6 framework residues, at most 5 framework residues, at most 4 framework residues, at most 3 framework residues, at most 2 framework residues, or at most 1 of SEQ ID NO: 131

[0383] A sequence in which a framework residue is substituted with another amino acid; and

[0384] (c) a C-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0385] NO: 123, and (2) a sequence in which up to 9 framework residues in SEQ ID NO: 123 are substituted with another amino acid.

[0386] In one embodiment, the designed ankyrin repeat domain of the invention comprises:

[0387] (a) an N-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0388] NO:115, and (2) wherein up to 5 framework residues in SEQ ID NO:115 are

[0389] another amino acid substituted sequence; and

[0390] (b) one or more internal repeating modules, wherein each internal repeating module independently comprises a selected

[0391] A sequence of the group consisting of: (1) SEQ ID NO: 131, and (2) wherein at most 9 framework residues, at most 8 framework residues, at most 7 framework residues, at most 6 framework residues, at most 5 framework residues, at most 4 framework residues, at most 3 framework residues, at most 2 framework residues, or at most 1 of SEQ ID NO: 131

[0392] A sequence in which a framework residue is substituted with another amino acid; and

[0393] (c) a C-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0394] NO: 123, and (2) a sequence in which up to 5 framework residues in SEQ ID NO: 123 are substituted with another amino acid.

[0395] In one embodiment, the designed ankyrin repeat domain of the invention comprises:

[0396] (a) an N-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0397] NO:115, and (2) wherein up to 3 framework residues in SEQ ID NO:115 are

[0398] another amino acid substituted sequence; and

[0399] (b) one or more internal repeating modules, wherein each internal repeating module independently comprises a selected

[0400] A sequence of the group consisting of: (1) SEQ ID NO: 131, and (2) wherein at most 9 framework residues, at most 8 framework residues, at most 7 framework residues, at most 6 framework residues, at most 5 framework residues, at most 4 framework residues, at most 3 framework residues, at most 2 framework residues, or at most 1 of SEQ ID NO: 131

[0401] A sequence in which a framework residue is substituted with another amino acid; and

[0402] (c) a C-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0403] NO: 123, and (2) a sequence in which up to 3 framework residues in SEQ ID NO: 123 are substituted by another amino acid.

[0404] In one embodiment, the designed ankyrin repeat domain of the invention comprises:

[0405] (a) an N-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0406] NO:115, and (2) wherein at most 2 framework residues in SEQ ID NO:115 are

[0407] another amino acid substituted sequence; and

[0408] (b) one or more internal repeating modules, wherein each internal repeating module independently comprises a selected

[0409] A sequence of the group consisting of: (1) SEQ ID NO: 131, and (2) wherein at most 9 framework residues, at most 8 framework residues, at most 7 framework residues, at most 6 framework residues, at most 5 framework residues, at most 4 framework residues, at most 3 framework residues, at most 2 framework residues, or at most 1 of SEQ ID NO: 131

[0410] A sequence in which a framework residue is substituted with another amino acid; and

[0411] (c) a C-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0412] NO: 123, and (2) a sequence in which up to 2 framework residues in SEQ ID NO: 123 are substituted by another amino acid.

[0413] In one embodiment, the designed ankyrin repeat domain of the invention comprises:

[0414] (a) an N-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0415] NO:115, and (2) wherein at most one framework residue in SEQ ID NO:115 is replaced

[0416] another amino acid substituted sequence; and

[0417] (b) one or more internal repeating modules, wherein each internal repeating module independently comprises a selected

[0418] A sequence of the group consisting of: (1) SEQ ID NO: 131, and (2) wherein at most 9 framework residues, at most 8 framework residues, at most 7 framework residues, at most 6 framework residues, at most 5 framework residues, at most 4 framework residues, at most 3 framework residues, at most 2 framework residues, or at most 1 of SEQ ID NO: 131

[0419] A sequence in which a framework residue is substituted with another amino acid; and

[0420] (c) a C-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0421] NO: 123, and (2) sequences in which at most one framework residue in SEQ ID NO: 123 is substituted by another amino acid.

[0422] In one embodiment, the designed ankyrin repeat domain of the invention comprises:

[0423] (a) an N-terminal capping module having a sequence of SEQ ID NO: 115; and

[0424] (b) one or more internal repeating modules, wherein each internal repeating module independently comprises a selected

[0425] A sequence of the group consisting of: (1) SEQ ID NO: 131, and (2) wherein at most 9 framework residues, at most 8 framework residues, at most 7 framework residues, at most 6 framework residues, at most 5 framework residues, at most 4 framework residues, at most 3 framework residues, at most 2 framework residues, or at most 1 of SEQ ID NO: 131

[0426] A sequence in which a framework residue is substituted with another amino acid; and

[0427] (c) a C-terminal capping module having the sequence of SEQ ID NO: 123.

[0428] In one embodiment, the designed ankyrin repeat domain of the invention comprises:

[0429] (a) an N-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0430] NO:115, and (2) sequences in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in SEQ ID NO:115 are substituted with another amino acid; and

[0431] (b) one or more internal repeating modules, wherein each internal repeating module independently comprises a selected

[0432] A sequence consisting of the group consisting of: (1) SEQ ID NO: 131, and (2) a sequence in which up to 9 framework residues in SEQ ID NO: 131 are substituted by another amino acid.

[0433] Columns; and

[0434] (c) a C-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0435] NO: 123, and (2) a sequence in which up to 9 framework residues in SEQ ID NO: 123 are substituted with another amino acid.

[0436] In one embodiment, the designed ankyrin repeat domain of the invention comprises:

[0437] (a) an N-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0438] NO:115, and (2) sequences in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in SEQ ID NO:115 are substituted with another amino acid; and

[0439] (b) one or more internal repeating modules, wherein each internal repeating module independently comprises a selected

[0440] A sequence consisting of the group consisting of: (1) SEQ ID NO: 131, and (2) a sequence in which up to 5 framework residues in SEQ ID NO: 131 are substituted by another amino acid.

[0441] Columns; and

[0442] (c) a C-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0443] NO: 123, and (2) a sequence in which up to 5 framework residues in SEQ ID NO: 123 are substituted by another amino acid.

[0444] In one embodiment, the designed ankyrin repeat domain of the invention comprises:

[0445] (a) an N-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0446] NO:115, and (2) sequences in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in SEQ ID NO:115 are substituted with another amino acid; and

[0447] (b) one or more internal repeat modules, wherein each internal repeat module independently comprises a sequence selected from the group consisting of: (1) SEQ ID NO: 131, and (2) a sequence in which up to 3 framework residues in SEQ ID NO: 131 are substituted with another amino acid; and

[0448] (c) a C-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID

[0449] NO: 123, and (2) a sequence in which up to 3 framework residues in SEQ ID NO: 123 are substituted by another amino acid.

[0450] In other embodiments, the designed ankyrin repeat domain of the present invention comprises one or more internal repeat modules independently comprising a sequence selected from the group consisting of: (1) SEQ ID NO: 31, and (2) a sequence wherein at most 5, 4, 3, 2 or 1 framework residues other than positions 1, 10, 13, 17, 19, 21, 22 and 26 in SEQ ID NO: 31 are substituted with another amino acid, and / or the N-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NO: 19, and (2) a sequence wherein at most 5, 4, 3, 2 or 1 framework residues other than positions 5, 17, 20 and 23 in SEQ ID NO: 19 are substituted with another amino acid, and / or the C-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NO: 40, and (2) a sequence wherein at most 5, 4, 3, 2 or 1 framework residues other than positions 5, 17, 20 and 23 in SEQ ID NO: 41 are substituted with another amino acid. A sequence in which at most 5, 4, 3, 2 or 1 framework residues other than positions 10, 11, 17, 18, 19, 22 and 26 in NO:40 are substituted with another amino acid.

[0451] In a specific embodiment, the designed ankyrin repeat domain of the invention comprises a sequence selected from SEQ ID NOs: 19 to 25, 57 to 60, 65 to 81, 31 to 35, 61 to 62, 92 to 94, 40 to 42, 63 to 64, and 82 to 91.

[0452] In a specific embodiment, each internal repeat module comprised in the designed ankyrin repeat domain of the present invention may independently comprise a sequence selected from the group consisting of SEQ ID NOs: 31 to 35, 61 to 62, and 92 to 94. Alternatively or additionally, the N-terminal capping module comprised in the designed ankyrin repeat domain of the present invention may comprise a sequence selected from the group consisting of SEQ ID NOs: 19 to 25, 57 to 60, and 65 to 81. Alternatively or additionally, the C-terminal capping module comprised in the ankyrin repeat domain of the present invention may comprise a sequence selected from the group consisting of SEQ ID NOs: 40 to 42, 63 to 64, and 82 to 91.

[0453] In a specific embodiment, each internal repeat module comprised in the designed ankyrin repeat domain of the present invention independently comprises a sequence selected from the group consisting of SEQ ID NOs: 31 to 35, 61 to 62, 92 to 94, and 131 to 142. Alternatively or additionally, the N-terminal capping module comprised in the designed ankyrin repeat domain of the present invention comprises a sequence selected from the group consisting of SEQ ID NOs: 19 to 25, 57 to 60, 65 to 81, and 115 to 122. Alternatively or additionally, the C-terminal capping module comprised in the ankyrin repeat domain of the present invention comprises a sequence selected from the group consisting of SEQ ID NOs: 40 to 42, 63 to 64, 82 to 91, and 123 to 130.

[0454] In other embodiments, each internal repeat module contained in the designed ankyrin repeat domain of the present invention independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 31 to 35, 61 to 62, and 92 to 94, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in any one of SEQ ID NOs: 31 to 35, 61 to 62, and 92 to 94 are substituted by another amino acid. Alternatively or additionally, the N-terminal capping module comprised in the designed ankyrin repeat domain of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 19 to 25, 57 to 60 and 65 to 81, and (2) a sequence in which at most 9 framework residues, at most 8 framework residues, at most 7 framework residues, at most 6 framework residues, at most 5 framework residues, at most 4 framework residues, at most 3 framework residues, at most 2 framework residues or at most 1 framework residue in any one of SEQ ID NOs: 19 to 25, 57 to 60 and 65 to 81 are substituted by another amino acid. Alternatively or additionally, the C-terminal capping module comprised in the designed ankyrin repeat domain of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 40 to 42, 63 to 64 and 82 to 91, and (2) a sequence in which at most 9 framework residues, at most 8 framework residues, at most 7 framework residues, at most 6 framework residues, at most 5 framework residues, at most 4 framework residues, at most 3 framework residues, at most 2 framework residues or at most 1 framework residue in any one of SEQ ID NOs: 40 to 42, 63 to 64 and 82 to 91 are substituted by another amino acid.

[0455] In a specific embodiment, each internal repeat module contained in the designed ankyrin repeat domain of the present invention independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 31 to 32, and (2) a sequence in which at most 9 framework residues, at most 8 framework residues, at most 7 framework residues, at most 6 framework residues, at most 5 framework residues, at most 4 framework residues, at most 3 framework residues, at most 2 framework residues, or at most 1 framework residue in any one of SEQ ID NOs: 31 to 32 is substituted by another amino acid. Alternatively or additionally, the N-terminal capping module comprised in the designed ankyrin repeat domain of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 19 to 20, and (2) a sequence in which at most 9 framework residues, at most 8 framework residues, at most 7 framework residues, at most 6 framework residues, at most 5 framework residues, at most 4 framework residues, at most 3 framework residues, at most 2 framework residues or at most 1 framework residue in any one of SEQ ID NOs: 19 to 20 is substituted by another amino acid. Alternatively or additionally, the C-terminal capping module comprised in the designed ankyrin repeat domain of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 40 to 41, and (2) a sequence in which at most 9 framework residues, at most 8 framework residues, at most 7 framework residues, at most 6 framework residues, at most 5 framework residues, at most 4 framework residues, at most 3 framework residues, at most 2 framework residues or at most 1 framework residue in any one of SEQ ID NOs: 40 to 41 are substituted by another amino acid.

[0456] In other embodiments, each internal repeat module included in the designed ankyrin repeat domain of the present invention may independently comprise a sequence selected from the group consisting of: (1) SEQ ID NOs: 26 to 30, and (2) a sequence in which 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids in any one of SEQ ID NOs: 26 to 30 are substituted by another amino acid. Alternatively or additionally, the N-terminal capping module included in the designed ankyrin repeat domain of the present invention may comprise a sequence selected from the group consisting of: (1) SEQ ID NOs: 15 to 18, and (2) a sequence in which 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids in any one of SEQ ID NOs: 15 to 18 are substituted by another amino acid. Alternatively or additionally, the C-terminal capping module comprised in the designed ankyrin repeat domain of the present invention may comprise a sequence selected from the group consisting of: (1) SEQ ID NOs: 36 to 39, and (2) a sequence in which 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids in any one of SEQ ID NOs: 36 to 39 are substituted by another amino acid.

[0457] In other embodiments, each internal repeat module included in the designed ankyrin repeat domain of the present invention may also independently include a sequence selected from the group consisting of: (1) SEQ ID NOs: 46 to 52, and (2) a sequence in which 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids in any one of SEQ ID NOs: 46 to 52 are substituted by another amino acid. Alternatively or additionally, the N-terminal capping module included in the designed ankyrin repeat domain of the present invention may also include a sequence selected from the group consisting of: (1) SEQ ID NOs: 43 to 45, and (2) a sequence in which 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids in any one of SEQ ID NOs: 43 to 45 are substituted by another amino acid. Alternatively or additionally, the C-terminal capping module comprised in the ankyrin repeat domain of the present invention may also comprise a sequence selected from the group consisting of: (1) SEQ ID NOs: 53 to 56, and (2) a sequence in which 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids in any one of SEQ ID NOs: 53 to 56 are substituted by another amino acid.

[0458] In other embodiments, each internal repeat module contained in the designed ankyrin repeat domain of the present invention independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 26 to 30 and 46 to 52, and (2) a sequence in which up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids, or up to 1 amino acid in any one of SEQ ID NOs: 26 to 30 and 46 to 52 is substituted by another amino acid. Alternatively or additionally, the N-terminal capping module comprised in the designed ankyrin repeat domain of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 15 to 18, 43 to 45 and 99 to 101, and (2) a sequence in which at most 9 amino acids, at most 8 amino acids, at most 7 amino acids, at most 6 amino acids, at most 5 amino acids, at most 4 amino acids, at most 3 amino acids, at most 2 amino acids or at most 1 amino acid in any one of SEQ ID NOs: 15 to 18, 43 to 45 and 99 to 101 is substituted by another amino acid. Alternatively or additionally, the C-terminal capping module comprised in the designed ankyrin repeat domain of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 36 to 39, 53 to 56 and 102 to 103, and (2) a sequence in which at most 9 amino acids, at most 8 amino acids, at most 7 amino acids, at most 6 amino acids, at most 5 amino acids, at most 4 amino acids, at most 3 amino acids, at most 2 amino acids or at most 1 amino acid in any one of SEQ ID NOs: 36 to 39, 53 to 56 and 102 to 103 is substituted by another amino acid.

[0459] In other embodiments, each internal repeat module contained in the designed ankyrin repeat domain of the present invention independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 26 to 30 and 47 to 48, and (2) a sequence in which up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids, or up to 1 amino acid in any one of SEQ ID NOs: 26 to 30 and 47 to 48 is substituted by another amino acid. Alternatively or additionally, the N-terminal capping module comprised in the designed ankyrin repeat domain of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 15 to 18, 43 and 99 to 101, and (2) a sequence in which at most 9 amino acids, at most 8 amino acids, at most 7 amino acids, at most 6 amino acids, at most 5 amino acids, at most 4 amino acids, at most 3 amino acids, at most 2 amino acids or at most 1 amino acid in any one of SEQ ID NOs: 15 to 18, 43 and 99 to 101 is substituted by another amino acid. Alternatively or additionally, the C-terminal capping module comprised in the designed ankyrin repeat domain of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 36 to 39, 54 and 102 to 103, and (2) a sequence in which at most 9 amino acids, at most 8 amino acids, at most 7 amino acids, at most 6 amino acids, at most 5 amino acids, at most 4 amino acids, at most 3 amino acids, at most 2 amino acids or at most 1 amino acid in any one of SEQ ID NOs: 36 to 39, 54 and 102 to 103 is substituted by another amino acid.

[0460] In other embodiments, each internal repeat module contained in the designed ankyrin repeat domain of the present invention independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 47 to 48, and (2) a sequence in which up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids, or up to 1 amino acid in any one of SEQ ID NOs: 47 to 48 is substituted by another amino acid. Alternatively or additionally, the N-terminal capping module comprised in the designed ankyrin repeat domain of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NO: 43, 100, 99 and 101, and (2) a sequence in which at most 9 amino acids, at most 8 amino acids, at most 7 amino acids, at most 6 amino acids, at most 5 amino acids, at most 4 amino acids, at most 3 amino acids, at most 2 amino acids or at most 1 amino acid in any one of SEQ ID NO: 43, 100, 99 and 101 is substituted by another amino acid. Alternatively or additionally, the C-terminal capping module comprised in the designed ankyrin repeat domain of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 54 and 102 to 103, and (2) a sequence in which at most 9 amino acids, at most 8 amino acids, at most 7 amino acids, at most 6 amino acids, at most 5 amino acids, at most 4 amino acids, at most 3 amino acids, at most 2 amino acids or at most 1 amino acid in any one of SEQ ID NOs: 54 and 102 to 103 is substituted by another amino acid.

[0461] In some embodiments, each internal repeat module contained in the designed ankyrin repeat domain of the present invention comprises one or more residues numbered relative to SEQ ID NO:48 selected from the group consisting of: (i) Gln at position 1, and (ii) Glu at position 26.

[0462] In some embodiments, the N-terminal capping module included in the designed ankyrin repeat domain of the present invention comprises one or more residues numbered relative to SEQ ID NO:43 selected from the group consisting of: (i) Thr at position 4, (ii) Gln at position 5, (iii) Leu, Val, Ile, Ala or Met at position 15, and (iv) Gln or Ala at position 23.

[0463] In some embodiments, the C-terminal capping module included in the designed ankyrin repeat domain of the present invention comprises one or more residues numbered relative to SEQ ID NO:54 selected from the group consisting of: (i) Thr or Asn at position 3, (ii) Ser at position 4, (iii) Thr or Ser at position 6, (iv) Asn at position 17, (v) Glu at position 18, and (vi) Asp at position 19.

[0464] In some embodiments, the designed ankyrin repeat domain of the present invention comprises (1) an N-terminal capping module comprising one or more residues numbered relative to SEQ ID NO:43 selected from the group consisting of: (i) Thr at position 4, (ii) Gln at position 5, (iii) Leu, Val, Ile, Ala or Met at position 15, and (iv) Gln or Ala at position 23, and / or (2) one or more internal repeat modules, wherein each internal repeat module independently comprises one or more residues numbered relative to SEQ ID NO:48 selected from the group consisting of: (i) Gln at position 1, and (ii) Glu at position 26, and / or (3) a C-terminal capping module comprising one or more residues numbered relative to SEQ ID NO:48 selected from the group consisting of: (i) Gln at position 1, and (ii) Glu at position 26. Residues numbered in NO:54: (i) Thr or Asn at position 3, (ii) Ser at position 4, (iii) Thr or Ser at position 6, (iv) Asn at position 17, (v) Glu at position 18, and (vi) Asp at position 19.

[0465] Thus, in an exemplary embodiment, a designed ankyrin repeat domain of the invention comprises (1) an N-terminal capping module comprising Thr at position 4, Gln at position 5, Leu at position 15, and Ala at position 23, numbered relative to SEQ ID NO:43, (2) two internal repeat modules, each of which comprises Glu at position 26, numbered relative to SEQ ID NO:48, and (3) a C-terminal capping module comprising Thr at position 3, Ser at position 4, Ser at position 6, Asn at position 17, Glu at position 18, and Asp at position 19, numbered relative to SEQ ID NO:54.

[0466] In another exemplary embodiment, the designed ankyrin repeat domain of the present invention comprises (1) an N-terminal capping module comprising Thr at position 4, Gln at position 5, Leu at position 15, and Gln at position 23, numbered relative to SEQ ID NO:43, (2) two internal repeat modules, each of which comprises Gln at position 1 and Glu at position 26, numbered relative to SEQ ID NO:48, and (3) a C-terminal capping module comprising Asn at position 17, Glu at position 18, and Asp at position 19, numbered relative to SEQ ID NO:54.

[0467] In other embodiments, the designed ankyrin repeat domain of the invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 104 to 114, and (2) a sequence having 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%, or at least 99% amino acid sequence identity in the framework residues of any one of SEQ ID NOs: 104 to 114. Preferably, the designed ankyrin repeat domain of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 104 and 112, and (2) a sequence having 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% or at least 99% amino acid sequence identity in the framework residues of any one of SEQ ID NOs: 104 and 112. More preferably, the designed ankyrin repeat domain of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NO: 104, and (2) a sequence having 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% or at least 99% amino acid sequence identity in the framework residues of SEQ ID NO: 104 substituted by another amino acid.

[0468] In a specific embodiment, the designed ankyrin repeat domain of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 2 to 5, 8 to 10, 12, and 95 to 98, and (2) a sequence having 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%, or at least 99% amino acid sequence identity to any one of SEQ ID NOs: 2 to 5, 8 to 10, 12, and 95 to 98. Therefore, in a more specific embodiment, the designed repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 2 to 5, 8 to 10, 12 and 95 to 98, and (2) a sequence having at least 80% amino acid sequence identity with any one of SEQ ID NOs: 2 to 5, 8 to 10, 12 and 95 to 98.

[0469] In a specific embodiment, the designed ankyrin repeat domain of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 8 to 10, 12, and 95 to 98, and (2) a sequence having 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%, or at least 99% amino acid sequence identity to any one of SEQ ID NOs: 8 to 10, 12, and 95 to 98. Therefore, in a more specific embodiment, the designed repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 8 to 10, 12 and 95 to 98, and (2) a sequence having at least 80% amino acid sequence identity with any one of SEQ ID NOs: 8 to 10, 12 and 95 to 98.

[0470] In a specific embodiment, the designed ankyrin repeat domain of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 8 to 10 and 95 to 98, and (2) a sequence having 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% or at least 99% amino acid sequence identity to any one of SEQ ID NOs: 8 to 10 and 95 to 98. Thus, in a more specific embodiment, the designed repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 8 to 10 and 95 to 98, and (2) a sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 8 to 10 and 95 to 98.

[0471] In a specific embodiment, the designed ankyrin repeat domain of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 8 to 9 and 95 to 98, and (2) a sequence having 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% or at least 99% amino acid sequence identity to any one of SEQ ID NOs: 8 to 9 and 95 to 98. Thus, in a more specific embodiment, the designed repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 8 to 9 and 95 to 98, and (2) a sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 8 to 9 and 95 to 98.

[0472] In a specific embodiment, the designed ankyrin repeat domain of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 95 to 98, and (2) a sequence having 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%, or at least 99% amino acid sequence identity to any one of SEQ ID NOs: 95 to 98. Thus, in a more specific embodiment, the designed repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 95 to 98, and (2) a sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 95 to 98.

[0473] In a specific embodiment, the designed ankyrin repeat domain of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NO: 12, and (2) a sequence having 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%, or at least 99% amino acid sequence identity to SEQ ID NO: 12. Thus, in a more specific embodiment, the designed repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NO: 12, and (2) a sequence having at least 80% amino acid sequence identity to SEQ ID NO: 12.

[0474] In a specific embodiment, the designed ankyrin repeat domain of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 2 to 5, and (2) a sequence having 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%, or at least 99% amino acid sequence identity to any one of SEQ ID NOs: 2 to 5. Thus, in a more specific embodiment, the designed repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 2 to 5, and (2) a sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 2 to 5.

[0475] In a specific embodiment, the designed ankyrin repeat domain of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 2, 3 and 5, and (2) a sequence having 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%, or at least 99% amino acid sequence identity to any one of SEQ ID NOs: 2, 3 and 5. Thus, in a more specific embodiment, the designed repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 2, 3 and 5, and (2) a sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 2, 3 and 5.

[0476] In other embodiments, the designed ankyrin repeat domain of the invention comprises one or more residues numbered relative to SEQ ID NO:6 selected from the group consisting of: (i) Thr at position 4, (ii) Gln at position 5, (iii) Leu, Val, Ile, Ala or Met at position 15, (iv) Gln or Ala at position 23, (v) Gln at position 31, (vi) Glu at position 56, (vii) Gln at position 64, (viii) Glu at position 89, (ix) Thr or Asn at position 99, (x) Ser at position 100, (xi) Thr or Ser at position 102, (xii) Asn at position 113, (xiii) Glu at position 114, and (xiv) Asp at position 115.

[0477] Thus, in an exemplary embodiment, the designed ankyrin repeat domain of the invention comprises Thr at position 4, Gln at position 5, Leu at position 15, Ala at position 23, Glu at position 56, Glu at position 89, Thr at position 99, Ser at position 100, Ser at position 102, Asn at position 113, Glu at position 114, and Asp at position 115, numbered relative to SEQ ID NO:6.

[0478] In another exemplary embodiment, the designed ankyrin repeat domain of the present invention comprises Thr at position 4, Gln at position 5, Leu at position 15, Gln at position 23, Gln at position 31, Glu at position 56, Gln at position 64, Glu at position 89, Asn at position 113, Glu at position 114, and Asp at position 115, numbered relative to SEQ ID NO:6.

[0479] In addition, the sequence of any repeat domain of the present invention may optionally contain G, S or GS at its N-terminus. In addition, the sequence of any repeat domain of the present invention may optionally have A at the penultimate position replaced by L and / or A at the last position replaced by N.

[0480] In some embodiments, the designed ankyrin repeat domain of the present invention is connected to a drug moiety. In some embodiments, the drug moiety is covalently or non-covalently connected to the designed ankyrin repeat domain of the present invention. In a more specific embodiment, the drug moiety is connected to the designed ankyrin repeat domain of the present invention through a chelator. Suitable chelators can be selected according to the application of the repeat domain of the present invention. In a specific embodiment, the chelator is DTPA. In a more specific embodiment, the drug moiety is a radionuclide. The selection of the radionuclide may depend on the intended application (e.g., diagnosis and treatment) of the designed ankyrin repeat domain of the present invention.

[0481] In a specific embodiment, the drug moiety is a therapeutic moiety. In one embodiment, the therapeutic moiety is a toxin. In one embodiment, the therapeutic moiety is a radionuclide. In a more specific embodiment, the radionuclide is indium-111.

[0482] In another specific embodiment, the drug moiety is a diagnostic moiety. In a more specific embodiment, the diagnostic moiety is a fluorophore, a chromophore, an imaging agent, or a radionuclide.

[0483] In another aspect, the present invention provides a designed ankyrin repeat domain obtainable or obtainable by a method according to the present invention as described hereinafter.

[0484] The production method according to the present invention

[0485] A designed ankyrin repeat domain with a reduced pi and / or a decreased number of basic amino acids according to the present invention can be generated by starting from the sequence of a conventional designed ankyrin repeat domain that does not have a pi and / or a number of basic amino acids as low as or below the threshold values ​​described herein.

[0486] Substitution of basic amino acid residues with neutral or acidic amino acid residues is most important for generating designed ankyrin repeat domains of the present invention with improved pharmacokinetic properties. However, considering that substitution of neutral amino acid residues with acidic amino acid residues in proteins generally also induces a decrease in isoelectric point (pI), such substitutions can be carried out in a complementary manner to substitution of basic amino acid residues with neutral or acidic amino acid residues, where appropriate.

[0487] Thus, in one aspect, the present invention provides a method for producing a modified ankyrin repeat domain, the method comprising steps (a) and (b),

[0488] wherein step (a) is to provide an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.07, and / or a percentage of basic amino acid residues higher than 7.0% among all amino acid residues contained in the repeat domain, and / or a percentage of basic amino acid residues higher than 6.1% among framework residues contained in the repeat domain; and

[0489] wherein step (b) is (1) replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; and / or

[0490] (2) replacing at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid;

[0491] wherein the modified ankyrin repeat domain has at least one feature selected from the following list of features:

[0492] (i) an isoelectric point (pI) equal to or lower than pH 4.07;

[0493] (ii) a percentage of basic amino acid residues of equal to or lower than 7.0% among all amino acid residues contained in the repeat domain; and

[0494] (iii) Among the framework residues contained in the repeat domain, the percentage of basic amino acid residues is equal to or lower than 6.1%.

[0495] For the sake of clarity, the pI and percentage values ​​in step (a) of the method are preferably related to the pI and percentage values ​​in features (i) to (iii), so that if in some embodiments another value is selected in one of features (i) to (iii), the value can be applied to the corresponding feature of the repeat domain of step (a). Thus, if one of features (i) to (iii) refers to a feature that is "equal to or lower than" X, the corresponding feature of step (a) should be "higher than" X.

[0496] For further clarity, at least one of the properties listed for the ankyrin repeat domain provided in step (a) is modified by step (b) in a way that the corresponding features of (i) to (iii) are achieved.

[0497] In a specific embodiment, the method according to the present invention comprises steps (a) and (b),

[0498] wherein step (a) is to provide an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.07, and / or a percentage of basic amino acid residues higher than 7.0% among all amino acid residues contained in the repeat domain, and / or a percentage of basic amino acid residues higher than 6.1% among framework residues contained in the repeat domain; and

[0499] wherein step (b) is (1) replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; and

[0500] (2) replacing at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid;

[0501] wherein the modified ankyrin repeat domain has at least one feature selected from the following list of features:

[0502] (i) an isoelectric point (pI) equal to or lower than pH 4.07;

[0503] (ii) a percentage of basic amino acid residues of equal to or lower than 7.0% among all amino acid residues contained in the repeat domain; and

[0504] (iii) Among the framework residues contained in the repeat domain, the percentage of basic amino acid residues is equal to or lower than 6.1%.

[0505] In another embodiment, the method according to the present invention comprises steps (a) and (b),

[0506] wherein step (a) is to provide an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.07, and / or a percentage of basic amino acid residues higher than 7.0% among all amino acid residues contained in the repeat domain, and / or a percentage of basic amino acid residues higher than 6.1% among framework residues contained in the repeat domain; and

[0507] wherein step (b) is to replace at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue;

[0508] wherein the modified ankyrin repeat domain has at least one feature selected from the following list of features:

[0509] (i) an isoelectric point (pI) equal to or lower than pH 4.07;

[0510] (ii) a percentage of basic amino acid residues of equal to or lower than 7.0% among all amino acid residues contained in the repeat domain; and

[0511] (iii) Among the framework residues contained in the repeat domain, the percentage of basic amino acid residues is equal to or lower than 6.1%.

[0512] In another embodiment, the method according to the present invention comprises steps (a) and (b),

[0513] wherein step (a) is to provide an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.07, and / or a percentage of basic amino acid residues higher than 7.0% among all amino acid residues contained in the repeat domain, and / or a percentage of basic amino acid residues higher than 6.1% among framework residues contained in the repeat domain; and

[0514] wherein step (b) is to replace at least one neutral amino acid residue of the repeat domain of step (a) with an acidic amino acid;

[0515] wherein the modified ankyrin repeat domain has at least one feature selected from the following list of features:

[0516] (i) an isoelectric point (pI) equal to or lower than pH 4.07;

[0517] (ii) a percentage of basic amino acid residues of equal to or lower than 7.0% among all amino acid residues contained in the repeat domain; and

[0518] (iii) Among the framework residues contained in the repeat domain, the percentage of basic amino acid residues is equal to or lower than 6.1%.

[0519] The present invention encompasses embodiments in which the modified repeat domains in the methods of the present invention have any combination of the above features (i) to (iii). Thus, in some embodiments, the modified repeat domains have at least features (i), (ii) or (iii). In other embodiments, the modified repeat domains have at least features (i) and (ii), (i) and (iii) or (ii) and (iii). In other embodiments, the modified repeat domains have features (i), (ii) and (iii).

[0520] In some preferred embodiments, the modified repeat domain has an isoelectric point (pi) in the range between pH 3.0 and pH 4.07, preferably between pH 3.3 and pH 4.07, and more preferably between pH 3.50 and pH 4.07.

[0521] In one embodiment, the method according to the present invention comprises steps (a) and (b),

[0522] wherein step (a) is to provide an ankyrin repeat domain, wherein the ankyrin repeat domain has an isoelectric point (pI) higher than pH 4.07; and

[0523] wherein step (b) is (1) replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; and / or

[0524] (2) replacing at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid;

[0525] wherein the modified ankyrin repeat domain has an isoelectric point (pi) equal to or lower than pH 4.07.

[0526] In a more specific embodiment, the method according to the present invention comprises steps (a) and (b),

[0527] wherein step (a) is to provide an ankyrin repeat domain, wherein the ankyrin repeat domain has an isoelectric point (pI) higher than pH 4.07; and

[0528] wherein step (b) is (1) replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; and

[0529] (2) replacing at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid;

[0530] wherein the modified ankyrin repeat domain has an isoelectric point (pi) equal to or lower than pH 4.07.

[0531] In another specific embodiment, the method according to the present invention comprises steps (a) and (b),

[0532] wherein step (a) is to provide an ankyrin repeat domain, wherein the ankyrin repeat domain has an isoelectric point (pI) higher than pH 4.07; and

[0533] wherein step (b) is to replace at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue;

[0534] wherein the modified ankyrin repeat domain has an isoelectric point (pi) equal to or lower than pH 4.07.

[0535] In another specific embodiment, the method according to the present invention comprises steps (a) and (b),

[0536] wherein step (a) is to provide an ankyrin repeat domain, wherein the ankyrin repeat domain has an isoelectric point (pI) higher than pH 4.07; and

[0537] wherein step (b) is to replace at least one neutral amino acid residue of the repeat domain of step (a) with an acidic amino acid;

[0538] wherein the modified ankyrin repeat domain has an isoelectric point (pi) equal to or lower than pH 4.07.

[0539] In one embodiment, the method according to the present invention comprises steps (a) and (b),

[0540] wherein step (a) is to provide an ankyrin repeat domain having a percentage of basic amino acid residues higher than 7.0% among all amino acid residues contained in the repeat domain; and

[0541] wherein step (b) comprises replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue;

[0542] wherein the modified ankyrin repeat domain has a percentage of basic amino acid residues equal to or lower than 7.0% among all amino acid residues comprised in the repeat domain.

[0543] In a more specific embodiment, the method according to the present invention comprises steps (a) and (b),

[0544] wherein step (a) is to provide an ankyrin repeat domain having a percentage of basic amino acid residues higher than 7.0% among all amino acid residues contained in the repeat domain; and

[0545] wherein step (b) is (1) replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; and

[0546] (2) replacing at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid;

[0547] wherein the modified ankyrin repeat domain has a percentage of basic amino acid residues equal to or lower than 7.0% among all amino acid residues comprised in the repeat domain.

[0548] In another more specific embodiment, the method according to the present invention comprises steps (a) and (b),

[0549] wherein step (a) is to provide an ankyrin repeat domain having a percentage of basic amino acid residues higher than 7.0% among all amino acid residues contained in the repeat domain; and

[0550] wherein step (b) is to replace at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue;

[0551] wherein the modified ankyrin repeat domain has a percentage of basic amino acid residues equal to or lower than 7.0% among all amino acid residues comprised in the repeat domain.

[0552] In one embodiment, the method according to the present invention comprises steps (a) and (b),

[0553] wherein step (a) is to provide an ankyrin repeat domain having a percentage of basic amino acid residues higher than 6.1% among the framework residues contained in the repeat domain; and

[0554] wherein step (b) comprises replacing at least one basic amino acid residue in the framework residues of the repeat domain of step (a) with a neutral or acidic amino acid residue;

[0555] wherein the modified ankyrin repeat domain has a percentage of basic amino acid residues among the framework residues comprised in the repeat domain that is equal to or lower than 6.1%.

[0556] In a more specific embodiment, the method according to the invention comprises steps (a) and (b),

[0557] wherein step (a) is to provide an ankyrin repeat domain having a percentage of basic amino acid residues higher than 6.1% among the framework residues contained in the repeat domain; and

[0558] wherein step (b) is (1) replacing at least one basic amino acid residue among the framework residues of the repeat domain of step (a) with a neutral or acidic amino acid residue; and

[0559] (2) replacing at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid;

[0560] wherein the modified ankyrin repeat domain has a percentage of basic amino acid residues among the framework residues comprised in the repeat domain that is equal to or lower than 6.1%.

[0561] In another more specific embodiment, the method according to the present invention comprises steps (a) and (b),

[0562] wherein step (a) is to provide an ankyrin repeat domain having a percentage of basic amino acid residues higher than 6.1% among the framework residues contained in the repeat domain; and

[0563] wherein step (b) is to replace at least one basic amino acid residue in the framework residues of the repeat domain of step (a) with a neutral or acidic amino acid residue;

[0564] wherein the modified ankyrin repeat domain has a percentage of basic amino acid residues among the framework residues comprised in the repeat domain that is equal to or lower than 6.1%.

[0565] In other embodiments of the method according to the invention, the repeat domain in step (a) has a KR / DE ratio higher than 0.44, and the modified repeat domain has a KR / DE ratio equal to or lower than 0.44.

[0566] In other embodiments of the method according to the invention, the repeat domain in step (a) has a KR / DE ratio in framework residues higher than 0.36, and the modified repeat domain has a KR / DE ratio equal to or lower than 0.36.

[0567] In other embodiments of the method according to the present invention, the repeat domain in step (a) has a KR / DE ratio higher than 0.66 among all residues of the N-terminal capping module, and the modified repeat domain has a KR / DE ratio equal to or lower than 0.66 among all residues of the N-terminal capping module.

[0568] In other embodiments of the method according to the present invention, the repeat domain in step (a) has a KR / DE ratio higher than 0.44, and the modified repeat domain has a KR / DE ratio equal to or lower than 0.44, and / or the repeat domain in step (a) has a KR / DE ratio higher than 0.36 in framework residues, and the modified repeat domain has a KR / DE ratio equal to or lower than 0.36, and / or the repeat domain in step (a) has a KR / DE ratio higher than 0.66 in all residues of the N-terminal capping module, and the modified repeat domain has a KR / DE ratio equal to or lower than 0.66 in all residues of the N-terminal capping module.

[0569] In another aspect, the present invention provides a method for producing a modified ankyrin repeat domain linked to a drug moiety, the method comprising steps (a) and (b),

[0570] wherein step (a) is to provide an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.6, and / or a percentage of basic amino acid residues higher than 12.0% among all amino acid residues contained in the repeat domain, and / or a percentage of Arg and Lys residues higher than 8.0% among all amino acid residues contained in the repeat domain, and / or a percentage of basic amino acid residues higher than 9.6% among framework residues contained in the repeat domain, and / or a percentage of Arg and Lys residues higher than 6.7% among the framework residues contained in the repeat domain; and

[0571] wherein step (b) is (1) replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; and / or

[0572] (2) replacing at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid;

[0573] Wherein the modified repeat domain has at least one feature selected from the following list of features:

[0574] (i) an isoelectric point (pI) equal to or lower than pH 4.6;

[0575] (ii) a percentage of basic amino acid residues equal to or lower than 12.0% among all amino acid residues contained in the repeat domain;

[0576] (iii) a percentage of Arg and Lys residues equal to or lower than 8.0% among all the amino acid residues contained in the repeat domain;

[0577] (iv) among the framework residues contained in the repeat domain, the percentage of basic amino acid residues is equal to or lower than 9.6%; and

[0578] (v) Among the framework residues contained in the repeat domain, the percentage of Arg and Lys residues is equal to or lower than 6.7%.

[0579] For the sake of clarity, the pI and percentage values ​​in step (a) of the method are preferably related to the pI and percentage values ​​in features (i) to (v), so that if in some embodiments another value is selected in one of features (i) to (v), the value can be applied to the corresponding feature of the repeat domain of step (a). Thus, if one of features (i) to (v) refers to a feature that is "equal to or lower than" X, the corresponding feature of step (a) should be "higher than" X.

[0580] For further clarity, at least one of the properties listed for the ankyrin repeat domain provided in step (a) is modified by step (b) in a way that the corresponding features of (i) to (v) are achieved.

[0581] In one embodiment, the method according to the present invention comprises steps (a) and (b),

[0582] wherein step (a) is to provide an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.6, and / or a percentage of basic amino acid residues higher than 12.0% among all amino acid residues contained in the repeat domain, and / or a percentage of Arg and Lys residues higher than 8.0% among all amino acid residues contained in the repeat domain, and / or a percentage of basic amino acid residues higher than 9.6% among framework residues contained in the repeat domain, and / or a percentage of Arg and Lys residues higher than 6.7% among the framework residues contained in the repeat domain; and

[0583] wherein step (b) is (1) replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; and

[0584] (2) replacing at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid;

[0585] Wherein the modified repeat domain has at least one feature selected from the following list of features:

[0586] (i) an isoelectric point (pI) equal to or lower than pH 4.6;

[0587] (ii) a percentage of basic amino acid residues equal to or lower than 12.0% among all amino acid residues contained in the repeat domain;

[0588] (iii) a percentage of Arg and Lys residues equal to or lower than 8.0% among all the amino acid residues contained in the repeat domain;

[0589] (iv) among the framework residues contained in the repeat domain, the percentage of basic amino acid residues is equal to or lower than 9.6%; and

[0590] (v) Among the framework residues contained in the repeat domain, the percentage of Arg and Lys residues is equal to or lower than 6.7%.

[0591] In another embodiment, the method according to the present invention comprises steps (a) and (b),

[0592] wherein step (a) is to provide an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.6, and / or a percentage of basic amino acid residues higher than 12.0% among all amino acid residues contained in the repeat domain, and / or a percentage of Arg and Lys residues higher than 8.0% among all amino acid residues contained in the repeat domain, and / or a percentage of basic amino acid residues higher than 9.6% among framework residues contained in the repeat domain, and / or a percentage of Arg and Lys residues higher than 6.7% among the framework residues contained in the repeat domain; and

[0593] wherein step (b) is to replace at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue;

[0594] Wherein the modified repeat domain has at least one feature selected from the following list of features:

[0595] (i) an isoelectric point (pI) equal to or lower than pH 4.6;

[0596] (ii) a percentage of basic amino acid residues equal to or lower than 12.0% among all amino acid residues contained in the repeat domain;

[0597] (iii) a percentage of Arg and Lys residues equal to or lower than 8.0% among all the amino acid residues contained in the repeat domain;

[0598] (iv) among the framework residues contained in the repeat domain, the percentage of basic amino acid residues is equal to or lower than 9.6%; and

[0599] (v) Among the framework residues contained in the repeat domain, the percentage of Arg and Lys residues is equal to or lower than 6.7%.

[0600] In another embodiment, the method according to the present invention comprises steps (a) and (b),

[0601] wherein step (a) is to provide an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.6, and / or a percentage of basic amino acid residues higher than 12.0% among all amino acid residues contained in the repeat domain, and / or a percentage of Arg and Lys residues higher than 8.0% among all amino acid residues contained in the repeat domain, and / or a percentage of basic amino acid residues higher than 9.6% among framework residues contained in the repeat domain, and / or a percentage of Arg and Lys residues higher than 6.7% among the framework residues contained in the repeat domain; and

[0602] wherein step (b) is to replace at least one neutral amino acid residue of the repeat domain of step (a) with an acidic amino acid;

[0603] Wherein the modified repeat domain has at least one feature selected from the following list of features:

[0604] (i) an isoelectric point (pI) equal to or lower than pH 4.6;

[0605] (ii) a percentage of basic amino acid residues equal to or lower than 12.0% among all amino acid residues contained in the repeat domain;

[0606] (iii) a percentage of Arg and Lys residues equal to or lower than 8.0% among all the amino acid residues contained in the repeat domain;

[0607] (iv) among the framework residues contained in the repeat domain, the percentage of basic amino acid residues is equal to or lower than 9.6%; and

[0608] (v) Among the framework residues contained in the repeat domain, the percentage of Arg and Lys residues is equal to or lower than 6.7%.

[0609] The present invention encompasses embodiments in which the modified repeat domains in the methods of the present invention have any combination of the above features (i) to (v). Thus, in some embodiments, the repeat domains have at least features (i), (ii), (iii), (iv) or (v). In other embodiments, the repeat domains have at least features (i) and (ii), (i) and (iii), (i) and (iv), (i) and (v), (ii) and (iii), (ii) and (iv), (ii) and (v), (iii) and (iv), (iii) and (v) or (iv) and (v). In other embodiments, the repeat domain has at least features (i), (ii) and (iii); (i), (ii) and (iv); (i), (ii) and (v); (i), (iii) and (iv); (i), (iii) and (v); (i), (iv) and (v); (ii), (iii) and (iv); (ii), (iii) and (v); (ii), (iv) and (v) or (iii), (iv) and (v). In other embodiments, the repeat domain has at least features (i), (ii), (iii) and (iv); (i), (ii), (iii) and (v); (i), (ii), (iv) and (v); (i), (iii), (iv) and (v) or (ii), (iii), (iv) and (v). In other embodiments, the modified repeat domain has features (i), (ii), (iii), (iv) and (v).

[0610] In some preferred embodiments, the modified repeat domain has an isoelectric point (pI) in the range of between pH 3.0 and pH 4.6, preferably between pH 3.3 and pH 4.6, and more preferably between pH 3.50 and pH 4.53. In some preferred embodiments, the percentage in (ii) is equal to or lower than 8.7%. In some most preferred embodiments, the percentage in (ii) is equal to or lower than 8.06%. In some preferred embodiments, the percentage in (iii) is equal to or lower than 4.7%. In some most preferred embodiments, the percentage in (iii) is equal to or lower than 4.03%. In some preferred embodiments, the percentage in (iv) is equal to or lower than 6.7%. In some most preferred embodiments, the percentage in (iv) is equal to or lower than 6.1%. In some preferred embodiments, the percentage in (v) is equal to or lower than 5.7%. In some most preferred embodiments, the percentage in (v) is equal to or lower than 4.85%.

[0611] In one embodiment, the method according to the present invention comprises steps (a) and (b),

[0612] wherein step (a) is to provide an ankyrin repeat domain, wherein the ankyrin repeat domain has an isoelectric point (pI) higher than pH 4.6; and

[0613] wherein step (b) is (1) replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; and / or

[0614] (2) replacing at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid;

[0615] wherein the modified repeat domain has an isoelectric point (pi) equal to or lower than pH 4.6.

[0616] In a more specific embodiment, the method according to the present invention comprises steps (a) and (b),

[0617] wherein step (a) is to provide an ankyrin repeat domain, wherein the ankyrin repeat domain has an isoelectric point (pI) higher than pH 4.6; and

[0618] wherein step (b) is (1) replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; and

[0619] (2) replacing at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid;

[0620] wherein the modified repeat domain has an isoelectric point (pi) equal to or lower than pH 4.6.

[0621] In another more specific embodiment, the method according to the present invention comprises steps (a) and (b),

[0622] wherein step (a) is to provide an ankyrin repeat domain, wherein the ankyrin repeat domain has an isoelectric point (pI) higher than pH 4.6; and

[0623] wherein step (b) is to replace at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue;

[0624] wherein the modified repeat domain has an isoelectric point (pi) equal to or lower than pH 4.6.

[0625] In another more specific embodiment, the method according to the present invention comprises steps (a) and (b),

[0626] wherein step (a) is to provide an ankyrin repeat domain, wherein the ankyrin repeat domain has an isoelectric point (pI) higher than pH 4.6; and

[0627] wherein step (b) is to replace at least one neutral amino acid residue of the repeat domain of step (a) with an acidic amino acid;

[0628] wherein the modified repeat domain has an isoelectric point (pi) equal to or lower than pH 4.6.

[0629] In one embodiment, the method according to the present invention comprises steps (a) and (b),

[0630] wherein step (a) is to provide an ankyrin repeat domain having a percentage of basic amino acid residues higher than 12.0% among all amino acid residues contained in the repeat domain; and

[0631] wherein step (b) comprises replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue;

[0632] wherein the modified repeat domain has a percentage of basic amino acid residues equal to or lower than 12.0% among all amino acid residues contained in the repeat domain.

[0633] In a more specific embodiment, the method according to the present invention comprises steps (a) and (b),

[0634] wherein step (a) is to provide an ankyrin repeat domain having a percentage of basic amino acid residues higher than 12.0% among all amino acid residues contained in the repeat domain; and

[0635] wherein step (b) is (1) replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; and

[0636] (2) replacing at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid;

[0637] wherein the modified repeat domain has a percentage of basic amino acid residues equal to or lower than 12.0% among all amino acid residues contained in the repeat domain.

[0638] In another more specific embodiment, the method according to the present invention comprises steps (a) and (b),

[0639] wherein step (a) is to provide an ankyrin repeat domain having a percentage of basic amino acid residues higher than 12.0% among all amino acid residues contained in the repeat domain; and

[0640] wherein step (b) is to replace at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue;

[0641] wherein the modified repeat domain has a percentage of basic amino acid residues equal to or lower than 12.0% among all amino acid residues contained in the repeat domain.

[0642] In one embodiment, the method according to the present invention comprises steps (a) and (b),

[0643] wherein step (a) is to provide an ankyrin repeat domain having a percentage of Arg and Lys residues higher than 8.0% among all amino acid residues contained in the repeat domain; and

[0644] wherein step (b) comprises replacing at least one Arg or Lys residue of the repeat domain of step (a) with a neutral or acidic amino acid residue;

[0645] wherein the modified repeat domain has a percentage of Arg and Lys residues equal to or lower than 8.0% among all amino acid residues comprised in the repeat domain.

[0646] In a more specific embodiment, the method according to the invention comprises steps (a) and (b),

[0647] wherein step (a) is to provide an ankyrin repeat domain having a percentage of Arg and Lys residues higher than 8.0% among all amino acid residues contained in the repeat domain; and

[0648] wherein step (b) is (1) replacing at least one Arg or Lys residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; and

[0649] (2) replacing at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid;

[0650] wherein the modified repeat domain has a percentage of Arg and Lys residues equal to or lower than 8.0% among all amino acid residues comprised in the repeat domain.

[0651] In another more specific embodiment, the method according to the present invention comprises steps (a) and (b),

[0652] wherein step (a) is to provide an ankyrin repeat domain having a percentage of Arg and Lys residues higher than 8.0% among all amino acid residues contained in the repeat domain; and

[0653] wherein step (b) is to replace at least one Arg or Lys residue of the repeat domain of step (a) with a neutral or acidic amino acid residue;

[0654] wherein the modified repeat domain has a percentage of Arg and Lys residues equal to or lower than 8.0% among all amino acid residues comprised in the repeat domain.

[0655] In one embodiment, the method according to the present invention comprises steps (a) and (b),

[0656] wherein step (a) is to provide an ankyrin repeat domain having a percentage of basic amino acid residues higher than 9.6% among the framework residues contained in the repeat domain; and

[0657] wherein step (b) comprises replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue;

[0658] wherein the modified repeat domain has a percentage of basic amino acid residues equal to or lower than 9.6% among the framework residues comprised in the repeat domain.

[0659] In a more specific embodiment, the method according to the present invention comprises steps (a) and (b),

[0660] wherein step (a) is to provide an ankyrin repeat domain having a percentage of basic amino acid residues higher than 9.6% among the framework residues contained in the repeat domain; and

[0661] wherein step (b) is (1) replacing at least one basic amino acid residue among the framework residues of the repeat domain of step (a) with a neutral or acidic amino acid residue; and

[0662] (2) replacing at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid;

[0663] wherein the modified repeat domain has a percentage of basic amino acid residues equal to or lower than 9.6% among the framework residues comprised in the repeat domain.

[0664] In another more specific embodiment, the method according to the present invention comprises steps (a) and (b),

[0665] wherein step (a) is to provide an ankyrin repeat domain having a percentage of basic amino acid residues higher than 9.6% among the framework residues contained in the repeat domain; and

[0666] wherein step (b) is to replace at least one basic amino acid residue in the framework residues of the repeat domain of step (a) with a neutral or acidic amino acid residue;

[0667] wherein the modified repeat domain has a percentage of basic amino acid residues equal to or lower than 9.6% among the framework residues comprised in the repeat domain.

[0668] In one embodiment, the method according to the present invention comprises steps (a) and (b),

[0669] wherein step (a) is to provide an ankyrin repeat domain having a percentage of Arg and Lys residues higher than 6.7% among the framework residues comprised in the repeat domain; and

[0670] wherein step (b) comprises replacing at least one Arg or Lys residue in the framework residues of the repeat domain of step (a) with a neutral or acidic amino acid residue;

[0671] wherein the modified repeat domain has a percentage of Arg and Lys residues equal to or lower than 6.7% among the framework residues comprised in the repeat domain.

[0672] In a more specific embodiment, the method according to the invention comprises steps (a) and (b),

[0673] wherein step (a) is to provide an ankyrin repeat domain having a percentage of Arg and Lys residues higher than 6.7% among the framework residues comprised in the repeat domain; and

[0674] wherein step (b) is (1) replacing at least one Arg or Lys residue in the framework residues of the repeat domain of step (a) with a neutral or acidic amino acid residue; and

[0675] (2) replacing at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid;

[0676] wherein the modified repeat domain has a percentage of Arg and Lys residues equal to or lower than 6.7% among the framework residues comprised in the repeat domain.

[0677] In another more specific embodiment, the method according to the present invention comprises steps (a) and (b),

[0678] wherein step (a) is to provide an ankyrin repeat domain having a percentage of Arg and Lys residues higher than 6.7% among the framework residues comprised in the repeat domain; and

[0679] wherein step (b) is to replace at least one Arg or Lys residue in the framework residues of the repeat domain of step (a) with a neutral or acidic amino acid residue;

[0680] wherein the modified repeat domain has a percentage of Arg and Lys residues equal to or lower than 6.7% among the framework residues comprised in the repeat domain.

[0681] In other embodiments of the method according to the invention, the repeat domain in step (a) has a KR / DE ratio higher than 0.44, and the modified repeat domain has a KR / DE ratio equal to or lower than 0.44.

[0682] In other embodiments of the method according to the invention, the repeat domain in step (a) has a KR / DE ratio in framework residues higher than 0.36, and the modified repeat domain has a KR / DE ratio equal to or lower than 0.36.

[0683] In other embodiments of the method according to the present invention, the repeat domain in step (a) has a KR / DE ratio higher than 0.66 among all residues of the N-terminal capping module, and the modified repeat domain has a KR / DE ratio equal to or lower than 0.66 among all residues of the N-terminal capping module.

[0684] In other embodiments of the method according to the present invention, the repeat domain in step (a) has a KR / DE ratio higher than 0.44, and the modified repeat domain has a KR / DE ratio equal to or lower than 0.44, and / or the repeat domain in step (a) has a KR / DE ratio higher than 0.36 in framework residues, and the modified repeat domain has a KR / DE ratio equal to or lower than 0.36, and / or the repeat domain in step (a) has a KR / DE ratio higher than 0.66 in all residues of the N-terminal capping module, and the modified repeat domain has a KR / DE ratio equal to or lower than 0.66 in all residues of the N-terminal capping module.

[0685] In some embodiments, at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen, at least fourteen, or at least fifteen residues of the repeat domain in (a) are substituted.

[0686] In some embodiments, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, %, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, or at least 82% of the residues are substituted. Where such percentages are to be converted to corresponding residue numbers, conventional rounding of the calculated residues to the nearest integer may be used.

[0687] In some embodiments, at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, at least 8%, at least 9%, at least 10%, at least 11%, at least 12%, at least 13%, at least 14%, at least 15%, at least 16%, at least 17%, at least 18%, at least 19%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, At least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, or at least 82% of the residues are substituted. In such embodiments, each repeat module wording includes an N-terminal capping module and / or a C-terminal capping module and / or an internal repeat module.

[0688] In one embodiment, the method according to the present invention also includes the step of connecting the drug moiety to the modified repeat domain. In some embodiments, the drug moiety is covalently or non-covalently connected to the modified repeat domain. In a more specific embodiment, the drug moiety is connected to the modified repeat domain through a chelating agent. Suitable chelating agents can be selected according to the application. In a specific embodiment, the chelating agent is DTPA. In a more specific embodiment, the drug moiety is a radionuclide. The selection of the radionuclide can depend on the expected application (e.g., diagnosis and treatment) of the modified repeat domain.

[0689] In one embodiment, the drug moiety is a therapeutic moiety. In one embodiment, the therapeutic moiety is a toxin. In one embodiment, the therapeutic moiety is a radionuclide. In a more specific embodiment, the radionuclide is indium-111.

[0690] In other embodiments, the drug moiety is a diagnostic moiety. In a more specific embodiment, the diagnostic moiety is a fluorophore, a chromophore, an imaging agent, or a radionuclide.

[0691] The residue selected for replacement may be located at a randomized position or a non-randomized position of the repeat domain. Therefore, in some embodiments, the residue replaced is selected from the residue located at the non-randomized position of the repeat domain. In other embodiments, the residue replaced is selected from the residue located at the randomized position of the repeat domain. In some embodiments, the residue replaced is selected from the residue located at the randomized position and the non-randomized position of the repeat domain. In other embodiments, the residue selected for replacement may be a framework residue or a target interaction residue. Therefore, in some embodiments, the residue replaced is selected from the framework residue contained in the repeat domain. In other embodiments, the residue replaced is selected from the target interaction residue contained in the repeat domain. In other embodiments, the residue replaced is selected from all residues contained in the repeat domain. In this sense, all residues should refer to any one of the target interaction residues or framework residues contained in the repeat domain. In some embodiments, the randomized position of the repeat domain corresponds to the position of the potential target interaction residue. In other embodiments, the non-randomized position corresponds to the position of the framework residue. The preferred positions of the framework residues are shown in Table 4. Table 5 shows the preferred positions of the potential target interaction residues.

[0692] Table 5

[0693] Repeating Modules Representative sequences Positions of potential target-interacting residues N-terminal capping module SEQ ID NO:43 4, 8, 11 and 12 Internal repeating module SEQ ID NO:48 3, 4, 6, 11, 14 and 15 C-terminal capping module SEQ ID NO:54 3, 4, 6, 14 and 15

[0694] In certain embodiments, the substitution is performed outside the structural core residues of the ankyrin repeat domain, for example, in the β loop connecting the α-helix. In certain embodiments, the substitution is performed within the structural core residues of the ankyrin repeat domain. For example, the ankyrin repeat domain of step (a) may comprise a sequence disclosed herein, for example, selected from the following consensus sequences: XDXXGXTPLXXAXXXGXLXIXXVLLXAGADVNA (SEQ ID NO: 32), DLGXXLLXAAXXGQLDXVRXLLXAGADVNA (SEQ ID NO: 20) and QDXXGXTPAXXAAXXGXXXIAXVLQXAA (SEQ ID NO: 41), wherein "X" represents any amino acid. In one embodiment, the residue named "X" is substituted. In another embodiment, the substitution is performed outside the residue named "X". In a preferred embodiment, the substituted amino acid is not cysteine, glycine or proline. In some embodiments, the term "any amino acid" as defined for certain positions "X" in the disclosed sequences of the present invention corresponds to "any naturally occurring amino acid".

[0695] The repeat domain of step (a) can be any repeat domain commonly known to those skilled in the art. Preferably, the repeat domain of step (a) is a designed ankyrin repeat domain. In some embodiments, the repeat domain of step (a) comprises an N-terminal end-capping module, one or more internal repeat modules and a C-terminal end-capping module. Preferably, the repeat domain of step (a) comprises 1, 2, 3, 4, 5, 6, 7, 8 or 9 internal repeat modules, and most preferably, the repeat domain comprises 2, 3 or 4 internal repeat modules. In some embodiments, the repeat domain of step (a) may include a sequence selected from SEQ ID NO: 19 to 25, 57 to 60, 65 to 81, 31 to 35, 61 to 62, 92 to 94, 40 to 42, 63 to 64 and 82 to 91. In some embodiments, each internal repeat module included in the repeat domain of step (a) may independently include a sequence selected from SEQ ID NO: 31 to 35, 61 to 62 and 92 to 94. Alternatively or additionally, the N-terminal capping module comprised in the repeat domain of step (a) may comprise a sequence selected from SEQ ID NOs: 19 to 25, 57 to 60, 65 to 81. Alternatively or additionally, the C-terminal capping module comprised in the repeat domain of step (a) may comprise a sequence selected from SEQ ID NOs: 40 to 42, 63 to 64 and 82 to 91.

[0696] In some embodiments, the modified repeat domain produced by the methods of the invention comprises one internal repeat module, two internal repeat modules, three internal repeat modules, or four internal repeat modules.

[0697] In one embodiment, the repeat domain of step (a) specifically binds to the target. In some embodiments, the repeat domain of step (a) is about 10 -5 M or smaller, about 10 -6 M or smaller, about 10 -7 M or smaller, about 10 - 8 M or smaller, about 10 -9 M or smaller, about 10 -10 M or smaller, about 10 -11 M or smaller, about 10 -12 M or smaller, about 10 -13 M or smaller, about 10 -14 M or less dissociation constant (K D ) binds to the target.

[0698] In a preferred embodiment, the modified repeat domain produced by the method of the present invention retains the binding characteristics of the repeat domain of step (a). Therefore, in some embodiments, the modified repeat domain is as follows K D Binds to the target: (1) approximately equal to the K of the repeat domain binding to the target in step (a) D , or (2) the K of the repeat domain of step (a) binding to the target D less than 100 times, less than 10 times, less than 5 times or less than 2 times.

[0699] In some embodiments, the modified repeat domain produced by the method of the present invention has an isoelectric point (pi) that is at least 0.1 pH points lower than the repeat domain of step (a). In some embodiments, the pi is at least 0.1, at least 0.2, at least 0.3, at least 0.4, at least 0.5, at least 0.6, at least 0.7, at least 0.8, at least 0.9, at least 1.0, or at least 1.1 pH points lower than the repeat domain of step (a).

[0700] In one embodiment, the modified repeat domain produced by the method of the invention is used in a medicament, wherein the medicament comprises the modified repeat domain linked to a drug moiety, and wherein the drug moiety is optionally a radionuclide or a cytotoxic agent.

[0701] The properties described with respect to the designed ankyrin repeat domains of the present invention are analogously applicable to the modified repeat domains obtained by the methods of the present invention.

[0702] In some embodiments, the modified repeat domains produced by the methods of the invention have a pi of at or below pH 4.9, at or below pH 4.8, at or below pH 4.7, at or below pH 4.6, at or below pH 4.5, at or below pH 4.4, at or below pH 4.3, at or below pH 4.2, at or below pH 4.1, at or below pH 4.0, at or below pH 3.9, at or below pH 3.8, at or below pH 3.7, at or below pH 3.6, or at or below pH 3.5.

[0703] In some embodiments, the modified repeat domains produced by the methods of the invention have a pH of at or below pH 4.53, at or below pH 4.52, at or below pH 4.50, at or below pH 4.48, at or below pH 4.46, at or below pH 4.44, at or below pH 4.42, at or below pH 4.40, at or below pH 4.38, at or below pH 4.36, at or below pH 4.34, at or below pH 4.32, at or below pH 4.30, at or below pH 4.28, at or below pH 4.26, at or below pH 4.24, at or below pH 4.22, at or below pH 4.20, at or below pH 4.18, at or below pH 4.16, at or below pH 4.14, at or below pH 4.12, at or below pH 4.10, equal to or lower than pH 4.08, equal to or lower than pH 4.06, equal to or lower than pH 4.04, equal to or lower than pH 4.02, equal to or lower than pH 4.00, equal to or lower than pH 3.98, equal to or lower than pH 3.96, equal to or lower than pH 3.94, equal to or lower than pH 3.92, equal to or lower than pH 3.90, equal to or lower than pH 3.88, equal to or lower than pH 3.86, equal to or lower than pH 3.84, equal to or lower than pH 3.82, equal to or lower than pH 3.80, equal to or lower than pH 3.78, equal to or lower than pH 3.76, equal to or lower than pH 3.74, equal to or lower than pH 3.72, equal to or lower than pH 3.70, equal to or lower than pH 3.68, equal to or lower than pH 3.66, equal to or lower than pH 3.64, equal to or lower than pH 3.62, at or below pH 3.60, at or below pH 3.58, at or below pH 3.56, at or below pH 3.54, at or below pH 3.52, or at or below pH 3.50.

[0704] In some embodiments, the modified repeat domains produced by the methods of the invention have a pH between 3.00 and pH 4.60, between 3.10 and pH 4.60, between 3.20 and pH 4.60, between 3.30 and pH 4.60, between 3.40 and pH 4.60, between 3.50 and pH 4.60, between 3.50 and pH 4.60, between 3.50 and pH 4.58, between 3.50 and pH 4.56, between 3.50 and pH 4.54, between 3.50 and pH 4.53, between 3.50 and pH 4.52, between 3.50 and pH 4.50, between 3.50 and pH 4.48, between 3.50 and pH 4.46, between 3.50 and pH 4.57, between 3.50 and pH 4.58, between 3.50 and pH 4.59, between 3.50 and pH 4.60, between 3.50 and pH 4.61, between 3.50 and pH 4.62, between 3.50 and pH 4.63, between 3.50 and pH 4.64, between 3.50 and pH 4.65, between 3.50 and pH 4.66 4.44, between pH 3.50 and pH 4.42, between pH 3.50 and pH 4.40, between pH 3.50 and pH 4.38, between pH 3.50 and pH 4.36, between pH 3.50 and pH 4.34, between pH 3.50 and pH 4.32, between pH 3.50 and pH 4.30, between pH 3.60 and pH 4.60, between pH 3.60 and pH 4.58, between pH 3.60 and pH 4.56, between pH 3.60 and pH 4.54, between pH 3.60 and pH 4.53, between pH 3.60 and pH 4.52, between pH 3.60 and pH 4.50, between pH 3.60 and pH 4.48, between pH 3.60 and pH 4.46, between pH 3.60 and pH 4.44, between pH 3.60 and pH 4.42, between pH 3.60 and pH 4.40, between pH 3.60 and pH 4.38, between pH 3.60 and pH 4.36, between pH 3.60 and pH 4.34, between pH 3.60 and pH 4.32, between pH 3.60 and pH 4.30, between pH 3.70 and pH 4.60, between pH 3.70 and pH 4.58, between pH 3.70 and pH 4.56, between pH 3.70 and pH 4.54, between pH 3.70 and pH 4.53, between pH 3.70 and pH 4.52, between pH between pH 3.70 and pH 4.50, between pH 3.70 and pH 4.48, between pH 3.70 and pH 4.46, between pH 3.70 and pH 4.44, between pH 3.70 and pH 4.42, between pH 3.70 and pH 4.40, between pH 3.70 and pH 4.38, between pH 3.70 and pH 4.36, between pH 3.70 and pH 4.34, between pH 3.70 and pH 4.32, or between pH 3.70 and pH 4.30.

[0705] In some embodiments, the modified repeat domain produced by the methods of the invention has a percentage of basic amino acid residues of equal to or less than 12.0%, equal to or less than 11.5%, equal to or less than 11.0%, equal to or less than 10.5%, equal to or less than 10.0%, equal to or less than 9.5%, or equal to or less than 9.0%.

[0706] In some embodiments, the modified repeat domains produced by the methods of the invention have a molecular weight of 8.87% or less, 8.80% or less, 8.70% or less, 8.60% or less, 8.50% or less, 8.40% or less, 8.30% or less, 8.20% or less, 8.10% or less, 8.00% or less, 7.90% or less, 7.80% or less, 7.70% or less, 7.60% or less, 7.50% or less, 7.40% or less, 7.30% or less, 7.20% or less, 7.10% or less, 7.00% or less, etc. %, equal to or less than 5.90%, equal to or less than 5.80%, equal to or less than 5.70%, equal to or less than 5.60%, equal to or less than 5.50%, equal to or less than 5.40%, equal to or less than 5.30%, equal to or less than 5.20%, equal to or less than 5.10%, equal to or less than 5.00%, equal to or less than 4.90% or equal to or less than 4.80% basic amino acid residues.

[0707] In some embodiments, the modified repeat domains produced by the methods of the invention have a percentage of Arg and Lys residues equal to or less than 8.0%, equal to or less than 7.5%, equal to or less than 7.0%, equal to or less than 6.5%, or equal to or less than 6.0%.

[0708] In some embodiments, the modified repeat domains produced by the methods of the invention have a % affinity for 5.65%, 5.60%, 5.50%, 5.40%, 5.30%, 5.20%, 5.10%, 5.00%, 4.90%, 4.80%, 4.70%, 4.60%, 4.70%, 4.80%, 4.90%, 4.90%, 4.80%, 4.80%, 4.90%, 4.80%, 4.80%, 4.80%, 4.80%, 4.80%, 4.90%, 4.90%, 4.8 ... 4.50%, equal to or less than 4.40%, equal to or less than 4.30%, equal to or less than 4.20%, equal to or less than 4.10%, equal to or less than 4.00%, equal to or less than 3.90%, equal to or less than 3.80%, equal to or less than 3.70%, equal to or less than 3.60%, equal to or less than 3.50%, equal to or less than 3.40%, equal to or less than 3.30%, equal to or less than 3.20%, equal to or less than 3.10%, equal to or less than 3.0 0%, 2.90% or less, 2.80% or less, 2.70% or less, 2.60% or less, 2.50% or less, 2.40% or less, 2.30% or less, 2.20% or less, 2.10% or less, 2.00% or less, 1.90% or less, 1.80% or less, 1.70% or less, 1.60% or less, 1.50% or less, The percentage of Arg and Lys residues of 1.40% or less, 1.30% or less, 1.20% or less, 1.10% or less, 1.00% or less, 0.90% or less, 0.80% or less, 0.70% or less, 0.60% or less, 0.50% or less, 0.40% or less, 0.30% or less, 0.20% or less, or 0.10% or less.

[0709] In some embodiments, the modified repeat domains produced by the methods of the invention have a percentage of basic amino acid residues in the framework residues of 9.6% or less, 9.4% or less, 9.2% or less, 9.0% or less, 8.8% or less, 8.6% or less, 8.4% or less, 8.2% or less, 8.0% or less, 7.8% or less.

[0710] In some embodiments, the modified repeat domains produced by the methods of the invention have a 7.77% or less, 7.70% or less, 7.60% or less, 7.50% or less, 7.40% or less, 7.30% or less, 7.20% or less, 7.10% or less, 7.00% or less, 6.90% or less, 6.80% or less, 6.70% or less, 6.60% or less, 6.50% or less, 6.40% or less, 6.30% or less in the framework residues. %, equal to or less than 6.20%, equal to or less than 6.10%, equal to or less than 6.00%, equal to or less than 5.90%, equal to or less than 5.80%, equal to or less than 5.70%, equal to or less than 5.60%, equal to or less than 5.50%, equal to or less than 5.40%, equal to or less than 5.30%, equal to or less than 5.20%, equal to or less than 5.10%, equal to or less than 5.00%, equal to or less than 4.90%, equal to or less than 4.80%, equal to or less than 4.70%, equal to or less than 4.60% or equal to or less than 4.50% basic amino acid residues.

[0711] In some embodiments, the designed ankyrin repeat domain of the invention has a percentage of Arg and Lys residues among framework residues that is equal to or lower than 6.7%, equal to or lower than 6.6%, equal to or lower than 6.5%, equal to or lower than 6.4%, equal to or lower than 6.3%, equal to or lower than 6.2%, equal to or lower than 6.1%, equal to or lower than 6.0%, or equal to or lower than 5.9%.

[0712] In some embodiments, the modified repeat domains produced by the methods of the invention have a 5.83% or less, 5.80% or less, 5.70% or less, 5.60% or less, 5.50% or less, 5.40% or less, 5.30% or less, 5.20% or less, 5.10% or less, 5.00% or less, 4.90% or less, 4.80% or less, 4.90% or less, 4.10% or less, 4.20% or less, 4.30% or less, 4.40% or less, 4.50% or less, 4.60% or less, 4.70% or less, 4.80% or less, 4.9 ... .70%, 4.60%, 4.50%, 4.40%, 4.30%, 4.20%, 4.10%, 4.00%, 3.90%, 3.80%, 3.70%, 3.60%, 3.50%, 3.40%, 3.30%, 3.20%, 3. 3.10%, 3.00%, 2.90%, 2.80%, 2.70%, 2.60%, 2.50%, 2.40%, 2.30%, 2.20%, 2.10%, 2.00%, 1.90%, 1.80%, 1.70%, 1.60%, 2. The percentage of Arg and Lys residues that are less than 1.50%, equal to or less than 1.40%, equal to or less than 1.30%, equal to or less than 1.20%, equal to or less than 1.10%, equal to or less than 1.00%, equal to or less than 0.90%, equal to or less than 0.80%, equal to or less than 0.70%, equal to or less than 0.60%, equal to or less than 0.50%, equal to or less than 0.40%, equal to or less than 0.30%, equal to or less than 0.20% or equal to or less than 0.10%.

[0713] The modified repeat domain produced by the method of the present invention may comprise an N-terminal capping module, a C-terminal capping module and at least one internal repeat module. Preferably, the modified repeat domain comprises 1, 2, 3, 4, 5, 6, 7, 8 or 9 internal repeat modules. More preferably, the modified repeat domain of the present invention comprises 1, 2, 3 or 4 internal repeat modules.

[0714] In some embodiments, the total number of basic amino acid residues contained in the modified repeat domain generated by the method of the present invention can be expressed as a function based on the number of repeat modules contained in the repeat domain. In one embodiment, the modified repeat domain has a total number of basic amino acid residues equal to or lower than n, wherein n=R, n=1+R, n=2+R, n=3+R, n=4+R, n=5+R, n=6+R, n=7+R, n=8+R, n=9+R, n=2R, n=1+2R, n=2+2R, n=3+2R, n=4+2R, n=5+2R, n=6+2R, n=7+2R, n=8+2R, n=9+2R, n=3R, n=1+3R, n=2+3R, n=3+3R, n=4+3R, n=5+3R, n=6+3R, n=7+3R, n=8+3R, n=9+3 In some embodiments, the invention relates to a modified repeat domain in which the number of internal repeat modules is greater than or equal to 4R, wherein the number of internal repeat modules is greater than or equal to 4R, wherein the number of internal repeat modules is greater than or equal to 4R, wherein the number of internal repeat modules is greater than or equal to 4R, wherein the number of internal repeat modules is greater than or equal to 4R, wherein the number of internal repeat modules is greater than or equal to 4R, wherein the number of internal repeat modules is greater than or equal to 4R, wherein the number of internal repeat modules is greater than or equal to 4R, wherein the number of internal repeat modules is greater than or equal to 4R, wherein the number of internal repeat modules is greater than or equal to 4R, wherein the number of internal repeat modules is greater than or equal to 4R, wherein the number of internal repeat modules is greater than or equal to 4R, wherein the number of internal repeat modules is greater than or equal to 4R, wherein the number of internal repeat modules is greater than or equal to 4R, wherein the number of internal repeat modules is greater than or equal to 4R, wherein the number of internal repeat modules is greater than or equal to 4R, wherein the number of internal repeat modules is greater than or equal to 4R, wherein the number of internal repeat modules is greater than or equal to 4R, wherein the number of internal repeat modules is greater than or equal to 4R, wherein the number of internal repeat modules is greater than or equal to 4R, wherein the number of internal repeat modules is greater than or equal to 4R

[0715] In another embodiment, the total number of Arg and Lys residues contained in the modified repeat domain produced by the method of the present invention is equal to or lower than m, wherein m=R, m=1+R, m=2+R, m=3+R, m=4+R, m=5+R, m=6+R, m=7+R, m=8+R, m=9+R, m=2R, m=1+2R, m=2+2R, m=3+2R, m=4+2R, m=5+2R, m=6+2R, m=7+2R, m=8+2R , m=9+2R, m=3R, m=1+3R, m=2+3R, m=3+3R, m=4+3R, m=5+3R, m=6+3R, m=7+3R, m=8+3R, m=9+3R, m=4R, m=1+4R, m=2+4R, m=3+4R, m=4+4R, m=5+4R, m=6+4R, m=7+4R, m=8+4R or m=9+4R, wherein R is the number of internal repeat modules contained in the modified repeat domain.

[0716] In some embodiments, the modified repeat domain produced by the method of the present invention has a total number of basic amino acid residues in the framework residues that is equal to or lower than x, wherein x=R, x=1+R, x=2+R, x=3+R, x=4+R, x=5+R, x=6+R, x=7+R, x=2R, x=1+2R, x=2+2R, x=3+2R, x=4+2R, x=5+2R, x=6+2R, x=7+2R, x=3R, x=1+3R, x=2+3R, x=3+3R, x=4+3R, x=5+3R, x=6+3R or x=7+3R, wherein R is the number of internal repeat modules contained in the modified repeat domain.

[0717] In other embodiments, the modified repeat domain produced by the method of the present invention has a total number of Arg and Lys residues in the framework residues that is equal to or less than y, wherein y=1+R, y=2+R, y=3+R, y=4+R, y=5+R, y=6+R, y=2R, y=1+2R, y=2+2R, y=3+2R, y=4+2R, y=5+2R or y=6+2R, wherein R is the number of internal repeat modules contained in the modified repeat domain.

[0718] Preferably, the modified repeat domain produced by the method of the present invention specifically binds to the target. In a preferred embodiment, the modified repeat domain is about 10 -5 M or smaller, about 10 -6 M or smaller, about 10 -7 M or smaller, about 10 -8 M or smaller, about 10 -9 M or smaller, about 10 -10 M or smaller, about 10 -11 M or smaller, about 10 -12 M or smaller, about 10 -13 M or smaller, about 10 -14 M or less dissociation constant (K D ) binds to the target.

[0719] In some embodiments, the modified repeat domain produced by the methods of the invention has a KR / DE ratio of 0.44 or less, 0.42 or less, 0.40 or less, 0.38 or less, 0.36 or less, 0.34 or less, 0.32 or less, 0.30 or less, 0.28 or less, 0.26 or less, 0.24 or less, 0.22 or less, 0.20 or less, 0.18 or less, 0.16 or less, 0.14 or less, 0.12 or less, 0.10 or less, or 0.08 or less. Preferably, the KR / DE ratio is equal to or lower than 0.3.

[0720] In some embodiments, the modified repeat domains produced by the methods of the invention have a KR / DE ratio of 0.36 or less, 0.34 or less, 0.32 or less, 0.30 or less, 0.28 or less, 0.26 or less, 0.24 or less, 0.22 or less, 0.20 or less, 0.18 or less, 0.16 or less, 0.14 or less, 0.12 or less, 0.10 or less, or 0.08 or less in framework residues. Preferably, the KR / DE ratio in framework residues is equal to or lower than 0.25.

[0721] In some embodiments, the modified repeat domain produced by the methods of the invention has a 50% to 100% affinity residue in all residues of the N-terminal capping module. In some embodiments, the modified repeat domain produced by the methods of the invention has a 50% to 100% affinity residue in all residues of the N-terminal capping module. Preferably, the KR / DE ratio of all residues in the N-terminal capping module is equal to or lower than 0.5.

[0722] In some embodiments, the modified repeat domains produced by the methods of the invention do not have Arg residues and Lys residues within the framework residue positions.

[0723] In some embodiments, the modified repeat domain produced by the method of the present invention comprises two internal repeat modules. In some embodiments, the modified repeat domain comprising two internal repeat modules may have a total number of basic amino acid residues equal to or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14. Preferably, the total number of basic residues is equal to or less than 14. More preferably, the total number of basic residues is equal to or less than 10. In another specific embodiment, the modified repeat domain comprising two internal repeat modules may have a total number of Arg and Lys residues equal to or less than 1, 2, 3, 4, 5, 6, 7, 8 or 9. Preferably, the total number of Arg and Lys residues is equal to or less than 9. More preferably, the total number of Arg and Lys residues is equal to or less than 5. In another specific embodiment, the modified repeat domain comprising two internal repeat modules may have a total number of Arg and Lys residues in the framework residues equal to or lower than 1, 2, 3, 4, 5 or 6. Preferably, the total number of Arg and Lys residues in the framework residues is equal to or lower than 6. More preferably, the total number of Arg and Lys residues in the framework residues is equal to or lower than 5.

[0724] In some embodiments, the modified repeat domain produced by the methods of the present invention comprises an N-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID NOs: 25 and 115 to 122, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in any one of SEQ ID NOs: 25 and 115 to 122 is substituted by another amino acid.

[0725] In some embodiments, the N-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 115 and 121, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in any of SEQ ID NOs: 115 and 121 are substituted with another amino acid.

[0726] In other embodiments, the N-terminal capping module has a sequence selected from the group consisting of: (1) SEQ ID NO: 115, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in SEQ ID NO: 115 are substituted with another amino acid.

[0727] As an alternative or in addition to the N-terminal capping module defined above, in some embodiments, the modified repeat domain produced by the methods of the present invention comprises a C-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID NOs: 123 to 130, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in any one of SEQ ID NOs: 123 to 130 is substituted by another amino acid.

[0728] In some embodiments, the C-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 123 and 126, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in any of SEQ ID NOs: 123 and 126 is substituted with another amino acid.

[0729] In other embodiments, the C-terminal capping module has a sequence selected from the group consisting of: (1) SEQ ID NO: 123, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in SEQ ID NO: 123 is substituted with another amino acid.

[0730] In a specific embodiment, the modified repeat domain produced by the method of the invention comprises:

[0731] An N-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID NOs: 115 and 121, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in any of SEQ ID NOs: 115 and 121 are substituted with another amino acid, and

[0732] A C-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID NOs: 123 and 126, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in any of SEQ ID NOs: 123 and 126 is substituted by another amino acid.

[0733] In another specific embodiment, the modified repeat domain produced by the method of the present invention comprises:

[0734] An N-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID NO: 115, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in SEQ ID NO: 115 are substituted with another amino acid, and

[0735] A C-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID NO: 123, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in SEQ ID NO: 123 is substituted by another amino acid.

[0736] In other embodiments, the modified repeat domain produced by the method of the present invention comprises one or more internal repeat modules, which independently comprise a sequence selected from the group consisting of: (1) SEQ ID NO:31, and (2) a sequence in which at most 5, 4, 3, 2 or 1 framework residues other than positions 1, 10, 13, 17, 19, 21, 22 and 26 in SEQ ID NO:31 are substituted by another amino acid, and / or the N-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NO:19, and (2) a sequence in which at most 5, 4, 3, 2 or 1 framework residues other than positions 5, 17, 20 and 23 in SEQ ID NO:19 are substituted by another amino acid, and / or the C-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NO:40, and (2) a sequence in which at most 5, 4, 3, 2 or 1 framework residues other than positions 5, 17, 20 and 23 in SEQ ID NO:41 are substituted by another amino acid. A sequence in which at most 5, 4, 3, 2 or 1 framework residues other than positions 10, 11, 17, 18, 19, 22 and 26 in NO:40 are substituted with another amino acid.

[0737] In a specific embodiment, the modified repeat domain of the invention comprises a sequence selected from SEQ ID NO: 19 to 25, 57 to 60, 65 to 81, 31 to 35, 61 to 62, 92 to 94, 40 to 42, 63 to 64 and 82 to 91.

[0738] In a specific embodiment, each internal repeat module contained in the modified repeat domain produced by the method of the present invention may independently comprise a sequence selected from SEQ ID NOs: 31 to 35, 61 to 62, and 92 to 94. Alternatively or additionally, the N-terminal capping module contained in the modified repeat domain produced by the method of the present invention may comprise a sequence selected from SEQ ID NOs: 19 to 25, 57 to 60, and 65 to 81. Alternatively or additionally, the C-terminal capping module contained in the modified repeat domain produced by the method of the present invention may comprise a sequence selected from SEQ ID NOs: 40 to 42, 63 to 64, and 82 to 91.

[0739] In a specific embodiment, each internal repeat module comprised in the modified repeat domain produced by the method of the present invention independently comprises a sequence selected from the group consisting of SEQ ID NOs: 31 to 35, 61 to 62, 92 to 94, and 131 to 142. Alternatively or additionally, the N-terminal capping module comprised in the modified repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of SEQ ID NOs: 19 to 25, 57 to 60, 65 to 81, and 115 to 122. Alternatively or additionally, the C-terminal capping module comprised in the modified repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of SEQ ID NOs: 40 to 42, 63 to 64, 82 to 91, and 123 to 130.

[0740] In other embodiments, each internal repeat module contained in the modified repeat domain produced by the method of the present invention independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 31 to 35, 61 to 62, and 92 to 94, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues, or up to 1 framework residue in any one of SEQ ID NOs: 31 to 35, 61 to 62, and 92 to 94 are substituted by another amino acid. Alternatively or additionally, the N-terminal capping module comprised in the modified repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 19 to 25, 57 to 60 and 65 to 81, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues or up to 1 framework residue in any one of SEQ ID NOs: 19 to 25, 57 to 60 and 65 to 81 are substituted by another amino acid. Alternatively or additionally, the C-terminal capping module comprised in the modified repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 40 to 42, 63 to 64 and 82 to 91, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues or up to 1 framework residue in any one of SEQ ID NOs: 40 to 42, 63 to 64 and 82 to 91 are substituted by another amino acid.

[0741] In a specific embodiment, each internal repeat module contained in the modified repeat domain produced by the method of the present invention independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 31 to 32, and (2) a sequence in which up to 9 framework residues, up to 8 framework residues, up to 7 framework residues, up to 6 framework residues, up to 5 framework residues, up to 4 framework residues, up to 3 framework residues, up to 2 framework residues or up to 1 framework residue in any one of SEQ ID NOs: 31 to 32 is substituted by another amino acid. Alternatively or additionally, the N-terminal capping module comprised in the modified repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 19 to 20, and (2) a sequence in which at most 9 framework residues, at most 8 framework residues, at most 7 framework residues, at most 6 framework residues, at most 5 framework residues, at most 4 framework residues, at most 3 framework residues, at most 2 framework residues or at most 1 framework residue in any one of SEQ ID NOs: 19 to 20 is substituted by another amino acid. Alternatively or additionally, the C-terminal capping module comprised in the modified repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 40 to 41, and (2) a sequence in which at most 9 framework residues, at most 8 framework residues, at most 7 framework residues, at most 6 framework residues, at most 5 framework residues, at most 4 framework residues, at most 3 framework residues, at most 2 framework residues or at most 1 framework residue in any one of SEQ ID NOs: 40 to 41 is substituted by another amino acid.

[0742] In other embodiments, each internal repeat module contained in the modified repeat domain produced by the method of the present invention may independently comprise a sequence selected from the group consisting of: (1) SEQ ID NOs: 26 to 30, and (2) a sequence in which 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids in any one of SEQ ID NOs: 26 to 30 are substituted by another amino acid. Alternatively or additionally, the N-terminal capping module contained in the modified repeat domain produced by the method of the present invention may comprise a sequence selected from the group consisting of: (1) SEQ ID NOs: 15 to 18, and (2) a sequence in which 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids in any one of SEQ ID NOs: 15 to 18 are substituted by another amino acid. Alternatively or additionally, the C-terminal capping module comprised in the modified repeat domain produced by the method of the present invention may comprise a sequence selected from the group consisting of: (1) SEQ ID NOs: 36 to 39, and (2) a sequence in which 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids in any one of SEQ ID NOs: 36 to 39 are substituted by another amino acid.

[0743] In other embodiments, each internal repeat module contained in the modified repeat domain produced by the method of the present invention may also independently contain a sequence selected from the group consisting of: (1) SEQ ID NOs: 46 to 52, and (2) a sequence in which 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids in any one of SEQ ID NOs: 46 to 52 are substituted by another amino acid. Alternatively or additionally, the N-terminal capping module contained in the modified repeat domain produced by the method of the present invention may also contain a sequence selected from the group consisting of: (1) SEQ ID NOs: 43 to 45, and (2) a sequence in which 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids in any one of SEQ ID NOs: 43 to 45 are substituted by another amino acid. Alternatively or additionally, the C-terminal capping module contained in the modified repeat domain produced by the method of the present invention may also contain a sequence selected from the group consisting of: (1) SEQ ID NOs: 53 to 56, and (2) a sequence in which 1, 2, 3, 4, 5, 6, 7, 8 or 9 amino acids in any one of SEQ ID NOs: 53 to 56 are substituted by another amino acid.

[0744] In other embodiments, each internal repeat module contained in the modified repeat domain produced by the method of the present invention independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 26 to 30 and 46 to 52, and (2) a sequence in which up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids, or up to 1 amino acid in any one of SEQ ID NOs: 26 to 30 and 46 to 52 is substituted by another amino acid. Alternatively or additionally, the N-terminal capping module comprised in the modified repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 15 to 18, 43 to 45 and 99 to 101, and (2) a sequence in which at most 9 amino acids, at most 8 amino acids, at most 7 amino acids, at most 6 amino acids, at most 5 amino acids, at most 4 amino acids, at most 3 amino acids, at most 2 amino acids or at most 1 amino acid in any one of SEQ ID NOs: 15 to 18, 43 to 45 and 99 to 101 is substituted by another amino acid. Alternatively or additionally, the C-terminal capping module comprised in the modified repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 36 to 39, 53 to 56 and 102 to 103, and (2) a sequence in which up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids or up to 1 amino acid in any one of SEQ ID NOs: 36 to 39, 53 to 56 and 102 to 103 is substituted by another amino acid.

[0745] In other embodiments, each internal repeat module contained in the modified repeat domain produced by the method of the present invention independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 26 to 30 and 47 to 48, and (2) a sequence in which up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids, or up to 1 amino acid in any one of SEQ ID NOs: 26 to 30 and 47 to 48 is substituted by another amino acid. Alternatively or additionally, the N-terminal capping module comprised in the modified repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 15 to 18, 43 and 99 to 101, and (2) a sequence in which at most 9 amino acids, at most 8 amino acids, at most 7 amino acids, at most 6 amino acids, at most 5 amino acids, at most 4 amino acids, at most 3 amino acids, at most 2 amino acids or at most 1 amino acid in any one of SEQ ID NOs: 15 to 18, 43 and 99 to 101 is substituted by another amino acid. Alternatively or additionally, the C-terminal capping module comprised in the modified repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 36 to 39, 54 and 102 to 103, and (2) a sequence in which up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids or up to 1 amino acid in any one of SEQ ID NOs: 36 to 39, 54 and 102 to 103 is substituted by another amino acid.

[0746] In other embodiments, each internal repeat module contained in the modified repeat domain produced by the method of the present invention independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 47 to 48, and (2) a sequence in which up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids or up to 1 amino acid in any one of SEQ ID NOs: 47 to 48 is substituted by another amino acid. Alternatively or additionally, the N-terminal capping module comprised in the modified repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NO: 43, 100, 99 and 101, and (2) a sequence in which at most 9 amino acids, at most 8 amino acids, at most 7 amino acids, at most 6 amino acids, at most 5 amino acids, at most 4 amino acids, at most 3 amino acids, at most 2 amino acids or at most 1 amino acid in any one of SEQ ID NO: 43, 100, 99 and 101 is substituted by another amino acid. Alternatively or additionally, the C-terminal capping module comprised in the modified repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 54 and 102 to 103, and (2) a sequence in which up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids or up to 1 amino acid in any one of SEQ ID NOs: 54 and 102 to 103 is substituted by another amino acid.

[0747] In other embodiments, each internal repeat module contained in the modified repeat domain produced by the method of the present invention independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 29, 30 and 47 to 50, and (2) a sequence in which up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids or up to 1 amino acid in any one of SEQ ID NOs: 29, 30 and 47 to 50 is substituted by another amino acid. Alternatively or additionally, the N-terminal capping module comprised in the modified repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 18, 43, 44 and 99 to 101, and (2) a sequence in which up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids or up to 1 amino acid in any one of SEQ ID NOs: 18, 43, 44 and 99 to 101 is substituted by another amino acid. Alternatively or additionally, the C-terminal capping module comprised in the modified repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NO: 39, 54, 55, 102 and 103, and (2) a sequence in which up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids or up to 1 amino acid in any one of SEQ ID NO: 39, 54, 55, 102 and 103 is substituted by another amino acid.

[0748] In other embodiments, each internal repeat module contained in the modified repeat domain produced by the method of the present invention independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 26 to 28 and 46, and (2) a sequence in which up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids, or up to 1 amino acid in any one of SEQ ID NOs: 26 to 28 and 46 is substituted by another amino acid. Alternatively or additionally, the N-terminal capping module comprised in the modified repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 15 to 17, and (2) a sequence in which up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids or up to 1 amino acid in any one of SEQ ID NOs: 15 to 17 is substituted by another amino acid. Alternatively or additionally, the C-terminal capping module comprised in the modified repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 36 to 38 and 53, and (2) a sequence in which up to 9 amino acids, up to 8 amino acids, up to 7 amino acids, up to 6 amino acids, up to 5 amino acids, up to 4 amino acids, up to 3 amino acids, up to 2 amino acids or up to 1 amino acid in any one of SEQ ID NOs: 36 to 38 and 53 is substituted by another amino acid.

[0749] In other embodiments, each internal repeat module contained in the modified repeat domain produced by the method of the present invention independently comprises a sequence selected from the group consisting of: (1) SEQ ID NO: 51 to 52, and (2) a sequence in which at most 9 amino acids, at most 8 amino acids, at most 7 amino acids, at most 6 amino acids, at most 5 amino acids, at most 4 amino acids, at most 3 amino acids, at most 2 amino acids, or at most 1 amino acid in any one of SEQ ID NO: 51 to 52 is substituted by another amino acid. Alternatively or additionally, the N-terminal capping module contained in the modified repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NO: 45, and (2) a sequence in which at most 9 amino acids, at most 8 amino acids, at most 7 amino acids, at most 6 amino acids, at most 5 amino acids, at most 4 amino acids, at most 3 amino acids, at most 2 amino acids, or at most 1 amino acid in SEQ ID NO: 45 is substituted by another amino acid. Alternatively or additionally, the C-terminal capping module comprised in the modified repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NO:56, and (2) a sequence in which at most 9 amino acids, at most 8 amino acids, at most 7 amino acids, at most 6 amino acids, at most 5 amino acids, at most 4 amino acids, at most 3 amino acids, at most 2 amino acids or at most 1 amino acid in SEQ ID NO:56 is substituted by another amino acid.

[0750] In some embodiments, each internal repeat module contained in the modified repeat domain produced by the methods of the present invention comprises one or more residues numbered relative to SEQ ID NO:48 selected from the group consisting of: (i) Gln at position 1, and (ii) Glu at position 26.

[0751] In some embodiments, the N-terminal capping module contained in the modified repeat domain produced by the methods of the present invention comprises one or more residues numbered relative to SEQ ID NO:43 selected from the group consisting of: (i) Thr at position 4, (ii) Gln at position 5, (iii) Leu, Val, Ile, Ala or Met at position 15, and (iv) Gln or Ala at position 23.

[0752] In some embodiments, the C-terminal capping module contained in the modified repeat domain produced by the methods of the present invention comprises one or more residues numbered relative to SEQ ID NO:54 selected from the group consisting of: (i) Thr or Asn at position 3, (ii) Ser at position 4, (iii) Thr or Ser at position 6, (iv) Asn at position 17, (v) Glu at position 18, and (vi) Asp at position 19.

[0753] Thus, in an exemplary embodiment, the modified repeat domain produced by the method of the present invention comprises (1) an N-terminal capping module comprising Thr at position 4, Gln at position 5, Leu at position 15, and Ala at position 23, numbered relative to SEQ ID NO:43, (2) two internal repeat modules, each of which comprises Glu at position 26, numbered relative to SEQ ID NO:48, and (3) a C-terminal capping module comprising Thr at position 3, Ser at position 4, Ser at position 6, Asn at position 17, Glu at position 18, and Asp at position 19, numbered relative to SEQ ID NO:54.

[0754] In another exemplary embodiment, the modified repeat domain produced by the method of the present invention comprises (1) an N-terminal capping module comprising Thr at position 4, Gln at position 5, Leu at position 15, and Gln at position 23 relative to the numbering of SEQ ID NO:43, (2) two internal repeat modules, each of which comprises Gln at position 1 and Glu at position 26 relative to the numbering of SEQ ID NO:48, and (3) a C-terminal capping module comprising Asn at position 17, Glu at position 18, and Asp at position 19 relative to the numbering of SEQ ID NO:54.

[0755] In other embodiments, the modified repeat domain produced by the methods of the invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 104 to 114, and (2) a sequence having 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%, or at least 99% amino acid sequence identity in the framework residues of any one of SEQ ID NOs: 104 to 114. In some embodiments, the modified repeat domain produced by the methods of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 104 and 112, and (2) a sequence having 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%, or at least 99% amino acid sequence identity in the framework residues of any of SEQ ID NOs: 104 and 112. In other embodiments, the modified repeat domain produced by the methods of the invention comprises a sequence selected from the group consisting of: (1) SEQ ID NO: 104, and (2) a sequence having 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%, or at least 99% amino acid sequence identity in the framework residues of SEQ ID NO: 104 replaced by another amino acid.

[0756] In a specific embodiment, the modified repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 2 to 5, 8 to 10, 12, and 95 to 98, and (2) a sequence having 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%, or at least 99% amino acid sequence identity to any one of SEQ ID NOs: 2 to 5, 8 to 10, 12, and 95 to 98. Therefore, in a more specific embodiment, the modified designed repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NO: 2 to 5, 8 to 10, 12 and 95 to 98, and (2) a sequence having at least 80% amino acid sequence identity with any one of SEQ ID NO: 2 to 5, 8 to 10, 12 and 95 to 98.

[0757] In a specific embodiment, the modified repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 8 to 10, 12, and 95 to 98, and (2) a sequence having 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%, or at least 99% amino acid sequence identity to any one of SEQ ID NOs: 8 to 10, 12, and 95 to 98. Therefore, in a more specific embodiment, the modified designed repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 8 to 10, 12 and 95 to 98, and (2) a sequence having at least 80% amino acid sequence identity with any one of SEQ ID NOs: 8 to 10, 12 and 95 to 98.

[0758] In a specific embodiment, the modified repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 8 to 10 and 95 to 98, and (2) a sequence having 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% or at least 99% amino acid sequence identity to any one of SEQ ID NOs: 8 to 10 and 95 to 98. Therefore, in a more specific embodiment, the modified designed repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 8 to 10 and 95 to 98, and (2) a sequence having at least 80% amino acid sequence identity with any one of SEQ ID NOs: 8 to 10 and 95 to 98.

[0759] In a specific embodiment, the modified repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 8 to 9 and 95 to 98, and (2) a sequence having 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% or at least 99% amino acid sequence identity to any one of SEQ ID NOs: 8 to 9 and 95 to 98. Thus, in a more specific embodiment, the modified designed repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 8 to 9 and 95 to 98, and (2) a sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 8 to 9 and 95 to 98.

[0760] In a specific embodiment, the modified repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 95 to 98, and (2) a sequence having 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%, or at least 99% amino acid sequence identity to any one of SEQ ID NOs: 95 to 98. Thus, in a more specific embodiment, the modified designed repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 95 to 98, and (2) a sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 95 to 98.

[0761] In a specific embodiment, the modified repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NO: 12, and (2) a sequence having 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% or at least 99% amino acid sequence identity to SEQ ID NO: 12. Thus, in a more specific embodiment, the modified designed repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NO: 12, and (2) a sequence having at least 80% amino acid sequence identity to SEQ ID NO: 12.

[0762] In a specific embodiment, the modified repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 2 to 5, and (2) a sequence having 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% or at least 99% amino acid sequence identity to any one of SEQ ID NOs: 2 to 5. Thus, in a more specific embodiment, the modified designed repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 2 to 5, and (2) a sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 2 to 5.

[0763] In a specific embodiment, the designed ankyrin repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 2, 3 and 5, and (2) a sequence having 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%, or at least 99% amino acid sequence identity to any one of SEQ ID NOs: 2, 3 and 5. Thus, in a more specific embodiment, the modified designed repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 2, 3 and 5, and (2) a sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 2, 3 and 5.

[0764] In other embodiments, the designed ankyrin repeat domain produced by the methods of the invention comprises one or more residues numbered relative to SEQ ID NO:6 selected from the group consisting of: (i) Thr at position 4, (ii) Gln at position 5, (iii) Leu, Val, Ile, Ala or Met at position 15, (iv) Gln or Ala at position 23, (v) Gln at position 31, (vi) Glu at position 56, (vii) Gln at position 64, (viii) Glu at position 89, (ix) Thr or Asn at position 99, (x) Ser at position 100, (xi) Thr or Ser at position 102, (xii) Asn at position 113, (xiii) Glu at position 114, and (xiv) Asp at position 115.

[0765] Thus, in an exemplary embodiment, the designed ankyrin repeat domain produced by the method of the present invention comprises Thr at position 4, Gln at position 5, Leu at position 15, Ala at position 23, Glu at position 56, Glu at position 89, Thr at position 99, Ser at position 100, Ser at position 102, Asn at position 113, Glu at position 114, and Asp at position 115, numbered relative to SEQ ID NO:6.

[0766] In another exemplary embodiment, the designed ankyrin repeat domain produced by the method of the present invention comprises Thr at position 4, Gln at position 5, Leu at position 15, Gln at position 23, Gln at position 31, Glu at position 56, Gln at position 64, Glu at position 89, Asn at position 113, Glu at position 114, and Asp at position 115, numbered relative to SEQ ID NO:6.

[0767] In some embodiments, the modified repeat domain produced by the method of the present invention is connected to a drug moiety. In some embodiments, the drug moiety is covalently or non-covalently connected to the modified repeat domain produced by the method of the present invention. In a more specific embodiment, the drug moiety is connected to the modified repeat domain produced by the method of the present invention through a chelator. Suitable chelators can be selected according to the application of the modified repeat domain of the present invention. In a specific embodiment, the chelator is DTPA. In a more specific embodiment, the drug moiety is a radionuclide. The selection of the radionuclide can depend on the intended application (e.g., diagnosis and treatment) of the modified repeat domain produced by the method of the present invention.

[0768] In a specific embodiment, the drug moiety is a therapeutic moiety. In one embodiment, the therapeutic moiety is a toxin. In one embodiment, the therapeutic moiety is a radionuclide. In a more specific embodiment, the radionuclide is indium-111. In another specific embodiment, the drug moiety is a diagnostic moiety. In a more specific embodiment, the diagnostic moiety is a fluorophore, a chromophore, an imaging agent, or a radionuclide.

[0769] In a preferred embodiment, the modified repeat domain produced by the method of the present invention is a modified designed ankyrin repeat domain.

[0770] Recombinant protein

[0771] In one aspect, the present invention provides a recombinant protein comprising a designed repeat domain of the present invention.

[0772] The designed repeat domains of the present invention can be genetically fused to other components, such as drug moieties, proteins or pharmaceutical agents, and such fusions are also referred to as "recombinant proteins". Linkers known in the art can be used between repeat domains in such repeat proteins (see, for example, WO 2021 / 116469) or between repeat domains and the other components. Such recombinant proteins are particularly contemplated for use in medicine.

[0773] In some embodiments, the recombinant protein of the present invention comprises a repeat domain of the present invention connected to a drug moiety. In some embodiments, additional drug moieties may be connected to such recombinant proteins. In other embodiments, the recombinant protein of the present invention comprises a repeat domain of the present invention that is not connected to a drug moiety. In more specific embodiments, one or more drug moieties may be connected to the recombinant protein.

[0774] In some embodiments, the recombinant proteins of the present invention comprise one or more additional designed ankyrin repeat domains.

[0775] Nucleic acids, vectors and host cells

[0776] In another aspect, the present invention relates to an isolated nucleic acid encoding the amino acid sequence of the designed repeat domain of the present invention or the recombinant protein of the present invention. In one embodiment, the present invention relates to an isolated nucleic acid encoding the amino acid sequence of the recombinant protein of the present invention. In one embodiment, the present invention relates to an isolated nucleic acid encoding the amino acid sequence of the designed repeat domain of the present invention.

[0777] In addition, the present invention relates to a vector comprising any nucleic acid of the present invention.Therefore, on the other hand, the invention provides a recombinant expression vector comprising nucleic acid according to the present invention, wherein the vector optionally comprises an expression control sequence, allows expression of the polypeptide operably connected to the nucleic acid in a prokaryotic host cell or a eukaryotic host cell.Can be by methods well known to those skilled in the art, for example at MOLECULAR CLONING:A LABORATORY MANUAL, Sambrook et al, 4th Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N Y., those methods described in 2001 insert nucleotide sequence into the recombinant vector.

[0778] Nucleic acids are well known to those skilled in the art. Nucleic acids are used to produce the designer ankyrin repeat domains or recombinant binding proteins of the invention in E. coli, for example, as further described in US Pat. No. 7,417,130.

[0779] On the other hand, the invention provides a host cell comprising a recombinant expression vector according to the present invention. The host cell can be, for example, a bacterial cell (such as Escherichia coli or Streptomyces), a fungal cell (such as Aspergillus (Aspergillus)) and a yeast (such as Saccharomyces (Saccharomyces)), an insect cell, a mammalian cell (such as Chinese hamster ovary (CHO) cell, CI 27 mouse cell line, BHK cell line of Syrian hamster cell, human embryonic kidney 293 (HEK293) cell). In some embodiments, the host cell is a CHO cell or a HEK 293 cell. The host cell can be used, for example, to express a recombinant protein of the present invention.

[0780] Composition

[0781] The present invention also relates to pharmaceutical compositions comprising one or more of the designed ankyrin repeat domains, recombinant proteins, nucleic acids and / or recombinant expression vectors described herein and a pharmaceutically acceptable carrier or diluent. The present invention also relates to uses and therapeutic and diagnostic methods using the pharmaceutical compositions disclosed herein. The methods and uses included in the present invention are described in more detail below.

[0782] The pharmaceutical compositions described herein can be prepared using methods known in the art.

[0783] The pharmaceutical composition optionally comprises a pharmaceutically acceptable carrier or excipient or diluent. Standard pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions such as oil / water or water / oil emulsions and various types of wetting agents.

[0784] The pharmaceutical composition of the present invention may contain any other pharmaceutically acceptable ingredients, including, for example, acidifiers, additives, adsorbents, aerosol propellants, air displacers, alkalizers, anti-caking agents, anticoagulants, antimicrobial preservatives, antioxidants, antibacterial agents, bases, binders, buffers, chelating agents, coating agents, colorants, desiccants, detergents, diluents, disinfectants, disintegrants, dispersants, solubility enhancers, dyes, emollients, emulsifiers, emulsion stabilizers, fillers, film formers, Flavor enhancers, flavoring agents, flow enhancers, gelling agents, granulating agents, humectants, lubricants, mucoadhesives, cream bases, ointments, oily vehicles, organic bases, pastille bases, pigments, plasticizers, polishing agents, preservatives, chelating agents, skin penetrants, solubilizers, solvents, stabilizers, suppository bases, surfactants, suspending agents, sweeteners, therapeutic agents, thickeners, tonicity agents, toxicants, viscosity enhancers, water absorbents, water miscible co-solvents, water softeners or wetting agents. See, e.g., Handbook of Pharmaceutical Excipients, 3rd edition, AH Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated by reference in its entirety. Remington's Pharmaceutical Sciences, 16th edition, EW Martin (Maccadk Publishing Co., Easton, Pa., 1980), which is incorporated by reference in its entirety.

[0785] In one embodiment, the present invention provides a pharmaceutical composition comprising one or more of the following: (i) a designed ankyrin repeat domain according to the present invention, (ii) a recombinant protein according to the present invention, (iii) a nucleic acid according to the present invention, and / or (iv) a recombinant expression vector according to the present invention, and optionally at least one pharmaceutically acceptable carrier or diluent.

[0786] Therapeutic and diagnostic uses and methods according to the invention

[0787] In the context of the present invention, the terms "medical condition", "disease" and "illness" are used interchangeably and include, but are not limited to, cancer. In a preferred embodiment, the medical condition is cancer.

[0788] In one aspect, the present invention provides a method for treating and / or diagnosing a medical condition, the method comprising the step of administering to a patient in need thereof a therapeutically and / or diagnostically effective amount of a designed repeat domain of the present invention, a recombinant protein of the present invention, a nucleic acid of the present invention, or a pharmaceutical composition of the present invention.

[0789] Also provided are the designer repeat domains, recombinant proteins, nucleic acids or pharmaceutical compositions of the invention for use in methods of treating and / or diagnosing a medical disorder.

[0790] In one embodiment, the present invention relates to the use of the designed repeat domains, recombinant proteins, nucleic acids or pharmaceutical compositions according to the present invention for treating and / or diagnosing diseases. For this purpose, a therapeutic and / or diagnostic effective amount of the designed repeat domains, recombinant proteins, nucleic acids or pharmaceutical compositions according to the present invention is administered to a patient in need.

[0791] In one embodiment, the present invention relates to a method for treating and / or diagnosing a medical condition, comprising the step of administering to a patient in need thereof a therapeutically and / or diagnostically effective amount of a designed repeat domain, recombinant protein, nucleic acid or pharmaceutical composition of the present invention.

[0792] In one embodiment, the present invention relates to the use of a designer repeat domain, a recombinant protein, a nucleic acid or a pharmaceutical composition of the present invention for the treatment and / or diagnosis of a medical disorder.

[0793] In one embodiment, the present invention relates to a designed repeat domain, a recombinant protein, a nucleic acid or a pharmaceutical composition of the present invention for use in the treatment and / or diagnosis of a medical condition. In one embodiment, the present invention relates to the use of a designed repeat domain, a recombinant protein, a nucleic acid or a pharmaceutical composition of the present invention as a medicament for the treatment and / or diagnosis of a medical condition.

[0794] In one embodiment, the present invention relates to a method for treating and / or diagnosing a medical condition using the designed repeat domains, recombinant proteins, nucleic acids or pharmaceutical compositions of the present invention. In one embodiment, the present invention relates to a method for manufacturing a medicament for treating and / or diagnosing a medical condition, wherein the designed repeat domains, recombinant proteins, nucleic acids or pharmaceutical compositions of the present invention are the active ingredients of the medicament.

[0795] In one embodiment, the present invention relates to the use of the designed repeat domain, recombinant protein, nucleic acid or pharmaceutical composition of the present invention for the manufacture of a medicament.

[0796] In one embodiment, the present invention relates to the use of a designer repeat domain, a recombinant protein, a nucleic acid or a pharmaceutical composition of the present invention for the manufacture of a medicament for the treatment and / or diagnosis of a medical disorder.

[0797] In another embodiment, the present invention relates to the use of the designed repeat domains, recombinant proteins, nucleic acids or pharmaceutical compositions of the present invention for the manufacture of a medicament for the treatment and / or diagnosis of a medical disorder, preferably a neoplastic disease, more preferably cancer.

[0798] In some preferred embodiments, the medical condition is cancer.

[0799] In some embodiments, the patient is a mammal. In preferred embodiments, the patient is a human.

[0800] In some embodiments, a single administration of the designed ankyrin repeat domain, recombinant protein, nucleic acid or pharmaceutical composition of the invention may be sufficient. In other embodiments, repeated administration may be necessary. Various factors will affect the number and frequency of administration, such as the age and general health of the subject, as well as the properties and typical dosage regimens of any drug moiety included in such designed ankyrin repeat domain, recombinant protein, nucleic acid or pharmaceutical composition of the invention.

[0801] The designed repeat domains, recombinant proteins, nucleic acids or pharmaceutical compositions described herein can be used in combination with another therapeutic agent and / or diagnostic agent. Each therapeutic agent and / or diagnostic agent can be administered simultaneously (e.g., in the same drug or administered simultaneously), administered in parallel (i.e., administered one by one in any order in a separate drug) or administered sequentially in any order. When the therapeutic agent and / or diagnostic agent in the combination therapy and / or diagnosis is in different dosage forms (e.g., one medicament is a tablet or capsule, and the other medicament is a sterile liquid) and / or is administered with different dosing regimens, sequential administration may be useful, for example, an analgesic administered at least daily and a less frequently administered biological therapeutic agent, such as once a week or once every two weeks.

[0802] Administration may include topical administration, oral administration and parenteral administration. The typical administration route is parenteral administration.

[0803] In specific embodiments, oral administration is excluded from any of the above methods or uses according to the present invention. Therefore, in some specific embodiments, the designed repeat domains of the present invention, the recombinant proteins of the present invention, the nucleic acids of the present invention or the pharmaceutical compositions of the present invention for use in the methods for treating and / or diagnosing medical conditions according to the present invention are not administered orally. In some embodiments, the present invention provides a method for treating and / or diagnosing a medical condition, the method comprising administering to a patient in need thereof a therapeutically and / or diagnostically effective amount of the designed repeat domains of the present invention, the recombinant proteins of the present invention, the nucleic acids of the present invention or the pharmaceutical compositions of the present invention, and wherein the administration is not oral administration.

[0804] In some other embodiments, the designed repeat domains, recombinant proteins, or pharmaceutical compositions described herein can be used for radiopharmaceutical therapy or diagnosis. Exemplary methods and instructions are disclosed, for example, in Sgouros, George et al., "Radiopharmaceutical therapy in cancer: clinical advances and challenges." Nature Reviews Drug Discovery 19.9 (2020): 589-608.

[0805] In some alternative embodiments, the designed repeat domains, recombinant proteins, or pharmaceutical compositions described herein can be used in therapeutic and / or diagnostic methods that can also use antibody-drug conjugates. Such methods and instructions are disclosed, for example, in Drago, Joshua Z., Shanu Modi, and Sarat Chandarlapaty. Nature Reviews Clinical Oncology 18.6 (2021) and Tarantino, Paolo et al., CA: a cancer journal for clinicians 72.2 (2022): 165-182.

[0806] The present invention is not limited to the specific embodiments described in the examples.

[0807] Example

[0808] Material

[0809] Chemicals were purchased from Sigma-Aldrich (USA). Oligonucleotides were from Microsynth (Switzerland). Unless otherwise stated, DNA polymerases, restriction enzymes, and buffers were from New England Biolabs (USA) or Fermentas / Thermo Fisher Scientific (USA). Inducible E. coli expression strains were used for cloning and protein production, such as E. coli XL1-blue (Stratagene, USA) or BL21 (Novagen, USA). TEV protease was from Sigma-Aldrich (USA). Double-stranded gene fragments (eBlock) were obtained from IDT (US). Maleimide DTPA was purchased from Chematech, metal-free PBS was purchased from VWR, and Chelex 100 chelating resin was purchased from BioRad.

[0810] Molecular Biology

[0811] Unless otherwise stated, methods were performed according to known protocols (see, e.g., Sambrook J., Fritsch E. F., and Maniatis T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory 1989, New York).

[0812] Designed Ankyrin Repeat Protein Library

[0813] Methods for generating designed ankyrin repeat protein libraries have been described in the following: for example, U.S. Patent No. 7,417,130; Binz et al., 2003, supra; Binz et al., 2004, supra. By such methods, designed ankyrin repeat protein libraries with randomized ankyrin repeat modules and / or randomized end-capping modules can be constructed. For example, such libraries can be assembled based on fixed N-terminal end-capping modules or randomized N-terminal end-capping modules, one or more randomized repeat modules, and fixed C-terminal end-capping modules or randomized C-terminal end-capping modules, respectively (see, for example, N-terminal end-capping modules and C-terminal end-capping modules provided in WO2021116462 and WO2021116469). Preferably, such libraries are assembled to have no amino acid C, G, M, N (in front of the G residue) and P at the randomized position of the repeat or end-capping module.

[0814] In addition, such randomization modules in such libraries may include additional polypeptide loop insertions with randomized amino acid positions. Examples of such polypeptide loop insertions are complementary determining region (CDR) loop libraries of antibodies or peptide libraries generated from scratch. For example, the structure of the N-terminal ankyrin repeat domain of human ribonuclease L (Tanaka, N., Nakanishi, M, Kusakabe, Y, Goto, Y., Kitade, Y, Nakamura, KT, EMBO J. 23 (30), 3929-3938, 2004) can be used as a guide to design such loop insertions. Similar to the ankyrin repeat domain in which ten amino acids are inserted into the β-turn present near the boundary of two ankyrin repeats, the ankyrin repeat protein library can include randomized loops (with fixed positions and randomized positions) of variable length (e.g., 1 to 20 amino acids) inserted into one or more β-turns of the ankyrin repeat domain.

[0815] The N-terminal capping module of the ankyrin repeat protein library preferably has a RILLAA, RILLKA or RELLKA motif, and any such C-terminal capping module of the ankyrin repeat protein library preferably has a KLN, KLA or KAA motif.

[0816] The design of such ankyrin repeat protein libraries can be guided by the known structures of ankyrin repeat domains that interact with the target. Examples of such structures identified by their protein data bank (PDB) unique accession numbers or identification codes (PDB-ID) are 1WDY, 3V31, 3V30, 3V2X, 3V2O, 3UXG, 3TWQ-3TWX, 1N11, 1S70 and 2ZGD.

[0817] Examples of designed ankyrin repeat protein libraries, such as the N2C and N3C designed ankyrin repeat protein libraries, have been described (U.S. Pat. No. 7,417,130; Binz et al., 2003, supra; Binz et al., 2004, supra). The numbers in N2C and N3C describe the number of randomized repeat modules present between the N-terminal and C-terminal capping modules.

[0818] The nomenclature used to define the positions within the repeat unit and the module is based on Binz et al., 2004 (supra), with the modification that the boundaries of the ankyrin repeat module and the ankyrin repeat unit are shifted by one amino acid position. For example, position 1 of the ankyrin repeat module of Binz et al. 2004 (supra) corresponds to position 2 of the ankyrin repeat module of the present disclosure, and thus position 33 of the ankyrin repeat module of Binz et al. 2004 (supra) corresponds to position 1 of the following ankyrin repeat module of the present disclosure.

[0819] Some exemplary experimental conditions are further described in WO2012069654, WO2016156596 and WO2021116462.

[0820] Example 1: Construction, expression and purification of DARPin variants

[0821] DARPins with defined amino acid sequences can be produced by gene synthesis of the corresponding reverse-translated nucleic acid sequence, subcloning into an appropriate expression vector of an expression system (e.g., an E. coli expression system), and protein expression and purification. Such methods are known to those skilled in the art.

[0822] Charge engineered DARPin variants are constructed and studied by evaluating which amino acid positions will allow charge-reducing substitutions of the parent amino acid residue while still retaining favorable biophysical properties and / or biological activity (binding to the target). Certain amino acid substitutions have been studied in Simeon, RA et al., Protein Engineering, Design and Selection, 34, (2021) with the goal of increasing the protease stability of bacterial exotoxin-specific DARPins.

[0823] In general, a panel of nine DARPin variants with varying numbers of positively and negatively charged amino acid residues were designed. These variants and their parent DARPins were subsequently generated and characterized as described in the following paragraphs.

[0824] The three different parent DARPins selected as starting points for engineering DARPin charge variants were DARPin01 (SEQ ID NO: 1), DARPin06 (SEQ ID NO: 6), and DARPin11 (SEQ ID NO: 11). Parent DARPin06 and DARPin11 were previously described in WO2018054971 and parent DARPin01 was described in WO2020245746 and WO2020245175. DARPin07 was designed to be used as a negative control subsequently; DARPin07 has a higher pI and a higher percentage of basic amino acids in the framework residues compared to its parent DARPin06. Details on the generated DARPin variants and parent DARPins are shown in Table 6 and Figure 1.

[0825] Table 6

[0826]

[0827]

[0828] The DNA encoding each of the designed ankyrin repeat domains of SEQ ID NO: 1 to 12 was cloned into an expression vector based on pQE (QIAgen, Germany), providing an N-terminal 6xHis tag to facilitate simple protein purification. Proteins comprising one o...

Claims

1. A designed ankyrin repeat domain, comprising an N-terminal capping module, at least one internal repeating module and a C-terminal capping module, wherein the repeat domain has at least one feature selected from the following list of features: (a) an isoelectric point (pI) equal to or lower than pH 4.07; (b) a percentage of basic amino acid residues of 7.0% or less among all amino acid residues contained in the repeat domain; and (c) Among the framework residues contained in the repeat domain, the percentage of basic amino acid residues is equal to or lower than 6.1%.

2. The repeat domain of claim 1, wherein the repeat domain has at least two features selected from features (a), (b) and (c).

3. The repeat domain according to any one of claims 1 to 2, wherein the repeat domain has at least features (a) and (b), (a) and (c), or (b) and (c).

4. The repeat domain according to any one of claims 1 to 3, wherein the repeat domain has features (a), (b) and (c).

5. The repeat domain according to any one of claims 1 to 4, wherein the repeat domain specifically binds to a target.

6. The repeat domain of claim 5, wherein the repeat domain has a length of less than 10 - 7 The dissociation constant (K D ) binds to the target.

7. The repeat domain of any one of claims 1 to 6, wherein the repeat domain comprises one internal repeat module, two internal repeat modules, three internal repeat modules or four internal repeat modules.

8. The repeat domain according to any one of claims 1 to 7, wherein the repeat domain has a KR / DE ratio equal to or lower than 0.44, and / or wherein the repeat domain has a KR / DE ratio equal to or lower than 0.36 in the framework residues, and / or wherein the repeat domain has a KR / DE ratio equal to or lower than 0.66 in all residues of the N-terminal capping module.

9. The repeat domain of any one of claims 1 to 8, wherein each internal repeat module independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 131 to 142, and (2) a sequence in which up to 9 framework residues in any one of SEQ ID NOs: 131 to 142 are substituted by another amino acid, and / or the N-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 25 and 115 to 122, and (2) a sequence in which up to 9 framework residues in any one of SEQ ID NOs: 25 and 115 to 122 are substituted by another amino acid, and / or the C-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 123 to 130, and (2) a sequence in which up to 9 framework residues in any one of SEQ ID NOs: 123 to 130 are substituted by another amino acid.

10. The repeat domain of any one of claims 1 to 9, wherein each internal repeat module independently comprises a sequence selected from the group consisting of: (1) SEQ ID NO: 31, and (2) a sequence in which up to 5 framework residues other than positions 1, 10, 13, 17, 19, 21, 22 and 26 in SEQ ID NO: 31 are substituted by another amino acid, and / or the N-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NO: 19, and (2) a sequence in which up to 5 framework residues other than positions 5, 17, 20 and 23 in SEQ ID NO: 19 are substituted by another amino acid, and / or the C-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NO: 40, and (2) a sequence in which up to 5 framework residues other than positions 10, 11, 17, 18, 19, 22 and 26 in SEQ ID NO: 40 are substituted by another amino acid.

11. The repeat domain of any one of claims 1 to 10, wherein each internal repeat module independently comprises a sequence selected from SEQ ID NOs: 31 to 35, 61 to 62, 92 to 94 and 131 to 142, and / or the N-terminal capping module comprises a sequence selected from SEQ ID NOs: 19 to 25, 57 to 60, 65 to 81 and 115 to 122, and / or the C-terminal capping module comprises a sequence selected from SEQ ID NOs: 40 to 42, 63 to 64, 82 to 91 and 123 to 130.

12. The repeat domain of any one of claims 1 to 11, wherein each internal repeat module independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 26 to 30 and 47 to 48, and (2) a sequence in which up to 9 amino acids in any one of SEQ ID NOs: 26 to 30 and 47 to 48 are substituted by another amino acid, and / or the N-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 15 to 18, 43 and 99 to 101, and (2) a sequence in which up to 9 amino acids in any one of SEQ ID NOs: 15 to 18, 43 and 99 to 101 are substituted by another amino acid, and / or the C-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 36 to 39, 54 and 102 to 103, and (2) a sequence in which up to 9 amino acids in any one of SEQ ID NOs: 15 to 18, 43 and 99 to 101 are substituted by another amino acid. A sequence in which up to 9 amino acids in any one of NO: 36 to 39, 54, and 102 to 103 are substituted with another amino acid.

13. The repeat domain of any one of claims 1 to 12, wherein the repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 104, 108, 109, and 112 to 114, and (2) a sequence having at least 80% amino acid sequence identity in the framework residues of any one of SEQ ID NOs: 104, 108, 109, and 112 to 114.

14. The repeat domain of any one of claims 1 to 13, wherein the repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 8 to 9 and 95 to 98, and (2) a sequence having at least 80% amino acid sequence identity with any one of SEQ ID NOs: 8 to 9 and 95 to 98.

15. A method for producing a modified ankyrin repeat domain, the method comprising steps (a) and (b), wherein step (a) is to provide an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.07, and / or a percentage of basic amino acid residues higher than 7.0% among all amino acid residues contained in the repeat domain, and / or a percentage of basic amino acid residues higher than 6.1% among the framework residues contained in the repeat domain; and wherein step (b) is (1) replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; and / or (2) replacing at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; wherein the modified ankyrin repeat domain has at least one feature selected from the following list of features: (i) an isoelectric point (pI) equal to or lower than pH 4.07; (ii) a percentage of basic amino acid residues of equal to or lower than 7.0% among all amino acid residues contained in the repeat domain; and (iii) Among the framework residues contained in the repeat domain, the percentage of basic amino acid residues is equal to or lower than 6.1%.

16. The method according to claim 15, wherein the method comprises steps (a) and (b), wherein step (a) is to provide an ankyrin repeat domain, wherein the ankyrin repeat domain has an isoelectric point (pI) higher than pH 4.07; and wherein step (b) is (1) replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; and / or (2) replacing at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; wherein the modified ankyrin repeat domain has an isoelectric point (pi) equal to or lower than pH 4.

07.

17. The method according to claim 15, wherein the method comprises steps (a) and (b), wherein step (a) is to provide an ankyrin repeat domain, said ankyrin repeat domain having a percentage of basic amino acid residues higher than 7.0% among all amino acid residues contained in said repeat domain; and wherein step (b) comprises replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; wherein the modified ankyrin repeat domain has a percentage of basic amino acid residues equal to or lower than 7.0% among all amino acid residues comprised in the repeat domain.

18. The method according to claim 15, wherein the method comprises steps (a) and (b), wherein step (a) is to provide an ankyrin repeat domain having a percentage of basic amino acid residues higher than 6.1% among the framework residues contained in the repeat domain; and wherein step (b) comprises replacing at least one basic amino acid residue in the framework residues of the repeat domain of step (a) with a neutral or acidic amino acid residue; wherein the modified ankyrin repeat domain has a percentage of basic amino acid residues among the framework residues comprised in the repeat domain that is equal to or lower than 6.1%.

19. The method according to any one of claims 15 to 18, wherein the repeat domain in step (a) has a KR / DE ratio higher than 0.44 and the modified repeat domain has a KR / DE ratio equal to or lower than 0.44, and / or wherein the repeat domain in step (a) has a KR / DE ratio higher than 0.36 in the framework residues and the modified repeat domain has a KR / DE ratio equal to or lower than 0.36, and / or wherein the repeat domain in step (a) has a KR / DE ratio higher than 0.66 in all residues of the N-terminal capping module and the modified repeat domain has a KR / DE ratio equal to or lower than 0.66 in all residues of the N-terminal capping module.

20. The method according to any one of claims 15 to 19, wherein the repeat domain of step (a) is optionally present in an amount less than 10 -7 The dissociation constant (K D ) specifically binds to the target.

21. The method of claim 20, wherein the modified repeat domain is less than 10 -7 M of K D Specifically binds to the target, preferably wherein the modified repeat domain is expressed as follows: D Binds to the target: (1) approximately equal to the K of the repeat domain binding to the target in step (a) D , or (2) is the K of the repeat domain of step (a) binding to the target D less than 100 times, less than 10 times, less than 5 times or less than 2 times.

22. The method of any one of claims 15 to 21, wherein the modified repeat domain comprises one internal repeat module, two internal repeat modules, three internal repeat modules, or four internal repeat modules.

23. A designed ankyrin repeat domain, comprising an N-terminal capping module, at least one internal repeat module and a C-terminal capping module, wherein the repeat domain is linked to a drug moiety, and wherein the repeat domain has at least one feature selected from the following list of features: (a) an isoelectric point (pI) equal to or lower than pH 4.6; (b) a percentage of basic amino acid residues equal to or lower than 12.0% among all amino acid residues contained in the repeat domain; (c) a percentage of Arg and Lys residues of 8.0% or less among all the amino acid residues contained in the repeat domain; (d) among the framework residues contained in the repeat domain, the percentage of basic amino acid residues is equal to or lower than 9.6%; and (e) Among the framework residues contained in the repeat domain, the percentage of Arg and Lys residues is equal to or lower than 6.7%.

24. The repeat domain of claim 23, wherein the repeat domain has at least feature (a), and wherein the isoelectric point (pi) is optionally in the range between pH 3.0 and pH 4.6, preferably between pH 3.3 and pH 4.6, and more preferably between pH 3.50 and pH 4.

53.

25. The repeat domain according to any one of claims 23 to 24, wherein the repeat domain has at least feature (b), and wherein the percentage in (b) is optionally equal to or lower than 8.7%.

26. The repeat domain according to any one of claims 23 to 25, wherein the repeat domain has at least feature (c), and wherein the percentage in (c) is optionally equal to or lower than 4.7%.

27. The repeat domain according to any one of claims 23 to 26, wherein the repeat domain has at least feature (d), and wherein the percentage in (d) is optionally equal to or lower than 6.7%.

28. The repeat domain according to any one of claims 23 to 27, wherein the repeat domain has at least feature (e), and wherein the percentage in (e) is optionally equal to or lower than 5.7%.

29. The repeat domain of any one of claims 23 to 28, wherein the repeat domain has a KR / DE ratio equal to or lower than 0.44, and / or wherein the repeat domain has a KR / DE ratio equal to or lower than 0.36 in the framework residues, and / or wherein the repeat domain has a KR / DE ratio equal to or lower than 0.66 in all residues of the N-terminal capping module.

30. The repeat domain according to any one of claims 23 to 29, wherein the repeat domain has a total number of basic amino acid residues equal to or lower than n, wherein n=4+5R, wherein R is the number of internal repeat modules contained in the repeat domain.

31. A repeat domain according to any one of claims 23 to 30, wherein the repeat domain has a total number of Arg and Lys residues equal to or lower than m, wherein m=5+2R, wherein R is the number of internal repeat modules contained in the repeat domain.

32. The repeat domain according to any one of claims 23 to 31, wherein the repeat domain does not have Arg residues and Lys residues within the framework residue positions.

33. The repeat domain of any one of claims 23 to 32, wherein the repeat domain comprises one internal repeat module, two internal repeat modules, three internal repeat modules, or four internal repeat modules.

34. The repeat domain of any one of claims 23 to 33, wherein the repeat domain specifically binds to a target.

35. The repeat domain of claim 34, wherein the repeat domain is present in a frequency range of less than 10 - 7 The dissociation constant (K D ) binds to the target.

36. The repeat domain of any one of claims 23 to 35, wherein the drug moiety is a therapeutic moiety or a diagnostic moiety.

37. The repeat domain of any one of claims 23 to 36, wherein the drug moiety is a toxin.

38. The repeat domain of claim 37, wherein the toxin is a radionuclide.

39. The repeat domain of claim 37, wherein the toxin is a cytotoxin.

40. The repeat domain of any one of claims 23 to 39, wherein the repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 104 to 114, and (2) a sequence having at least 80% amino acid sequence identity in the framework residues of any one of SEQ ID NOs: 104 to 114.

41. A repeat domain according to any one of claims 23 to 40, wherein the repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 104 and 112, and (2) a sequence having at least 80% amino acid sequence identity in the framework residues of either SEQ ID NOs: 104 and 112.

42. A repeat domain according to any one of claims 23 to 41, wherein the repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 8 to 10, 12 and 95 to 98, and (2) a sequence having at least 80% amino acid sequence identity with any one of SEQ ID NOs: 8 to 10, 12 and 95 to 98.

43. A repeat domain linked to a drug moiety according to any one of claims 23 to 42 for use in a method of treating and / or diagnosing a medical condition, the method comprising the step of administering a therapeutically and / or diagnostically effective amount of the repeat domain to a patient in need thereof, and wherein optionally the administration is not oral administration.

44. A designer ankyrin repeat domain linked to a drug moiety for use in a method of treating and / or diagnosing a medical condition according to claim 43, wherein the medical condition is cancer.

45. A method of producing a modified ankyrin repeat domain linked to a drug moiety, the method comprising steps (a) and (b), wherein step (a) is to provide an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.6, and / or a percentage of basic amino acid residues higher than 12.0% among all amino acid residues contained in the repeat domain, and / or a percentage of Arg and Lys residues higher than 8.0% among all amino acid residues contained in the repeat domain, and / or a percentage of basic amino acid residues higher than 9.6% among the framework residues contained in the repeat domain, and / or a percentage of Arg and Lys residues higher than 6.7% among the framework residues contained in the repeat domain; and wherein step (b) is (1) replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; and / or (2) replacing at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; Wherein the modified repeat domain has at least one feature selected from the following list of features: (i) an isoelectric point (pI) equal to or lower than pH 4.6; (ii) a percentage of basic amino acid residues equal to or lower than 12.0% among all amino acid residues contained in the repeat domain; (iii) a percentage of Arg and Lys residues equal to or lower than 8.0% among all the amino acid residues contained in the repeat domain; (iv) among the framework residues contained in the repeat domain, the percentage of basic amino acid residues is equal to or lower than 9.6%; and (v) Among the framework residues contained in the repeat domain, the percentage of Arg and Lys residues is equal to or lower than 6.7%.

46. ​​The method of claim 45, wherein the method comprises steps (a) and (b), wherein step (a) is to provide an ankyrin repeat domain, wherein the ankyrin repeat domain has an isoelectric point (pI) higher than pH 4.6; and wherein step (b) is (1) replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; and / or (2) replacing at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; wherein the modified repeat domain has an isoelectric point (pi) equal to or lower than pH 4.

6.

47. The method of claim 45, wherein the method comprises steps (a) and (b), wherein step (a) is to provide an ankyrin repeat domain having a percentage of basic amino acid residues higher than 12.0% among all amino acid residues contained in the repeat domain; and wherein step (b) comprises replacing at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; wherein the modified repeat domain has a percentage of basic amino acid residues equal to or lower than 12.0% among all amino acid residues contained in the repeat domain.

48. The method of claim 45, wherein the method comprises steps (a) and (b), wherein step (a) is to provide an ankyrin repeat domain having a percentage of Arg and Lys residues higher than 8.0% among all amino acid residues contained in the repeat domain; and wherein step (b) comprises replacing at least one Arg or Lys residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; wherein the modified repeat domain has a percentage of Arg and Lys residues equal to or lower than 8.0% among all amino acid residues comprised in the repeat domain.

49. The method of claim 45, wherein the method comprises steps (a) and (b), wherein step (a) is to provide an ankyrin repeat domain having a percentage of basic amino acid residues higher than 9.6% among the framework residues contained in the repeat domain; and wherein step (b) comprises replacing at least one basic amino acid residue in the framework residues of the repeat domain of step (a) with a neutral or acidic amino acid residue; wherein the modified repeat domain has a percentage of basic amino acid residues equal to or lower than 9.6% among the framework residues comprised in the repeat domain.

50. The method of claim 45, wherein the method comprises steps (a) and (b), wherein step (a) is to provide an ankyrin repeat domain having a percentage of Arg and Lys residues higher than 6.7% among the framework residues comprised in the repeat domain; and wherein step (b) comprises replacing at least one Arg or Lys residue in the framework residues of the repeat domain of step (a) with a neutral or acidic amino acid residue; wherein the modified repeat domain has a percentage of Arg and Lys residues equal to or lower than 6.7% among the framework residues comprised in the repeat domain.

51. The method of any one of claims 45 to 50, wherein the repeat domain in step (a) has a KR / DE ratio higher than 0.44 and the modified repeat domain has a KR / DE ratio equal to or lower than 0.44, and / or wherein the repeat domain in step (a) has a KR / DE ratio higher than 0.36 in the framework residues and the modified repeat domain has a KR / DE ratio equal to or lower than 0.36, and / or wherein the repeat domain in step (a) has a KR / DE ratio higher than 0.66 in all residues of the N-terminal capping module and the modified repeat domain has a KR / DE ratio equal to or lower than 0.66 in all residues of the N-terminal capping module.

52. The method of any one of claims 45 to 51, wherein the method further comprises the step of linking a drug moiety to the modified repeat domain.

53. The method according to any one of claims 45 to 52, wherein the repeat domain of step (a) is optionally present in an amount less than 10 -7 The dissociation constant (K D ) specifically binds to the target.

54. The method of claim 53, wherein the modified repeat domain is present at a concentration of less than 10 -7 M of K D Specifically binds to the target, preferably wherein the modified repeat domain is expressed as follows: D Binds to the target: (1) approximately equal to the K of the repeat domain binding to the target in step (a) D , or (2) the K of the repeat domain of step (a) binding to the target D less than 100 times, less than 10 times, less than 5 times or less than 2 times.

55. The method of any one of claims 45 to 54, wherein the modified repeat domain comprises one internal repeat module, two internal repeat modules, three internal repeat modules, or four internal repeat modules.

56. A designed ankyrin repeat domain obtainable by or by the method according to any one of claims 15 to 22 or 45 to 55.

57. A recombinant protein comprising the designed repeat domain according to any one of claims 1 to 14, 23 to 42 and 56.

58. An isolated nucleic acid encoding the designed repeat domain of any one of claims 1 to 14, 23 to 42 and 56 or the recombinant protein of claim 57.

59. A recombinant expression vector comprising the nucleic acid according to claim 58.

60. A host cell comprising the recombinant expression vector according to claim 59.

61. A pharmaceutical composition comprising one or more of the following: (i) a repeat domain according to any one of claims 1 to 14, 23 to 42 and 56, (ii) the recombinant protein according to claim 57, (iii) the nucleic acid according to claim 58, and / or (iv) the recombinant expression vector according to claim 59, and optionally at least one pharmaceutically acceptable carrier or diluent.

62. A method for treating and / or diagnosing a medical condition, the method comprising the step of administering to a patient in need thereof a therapeutically and / or diagnostically effective amount of a repeat domain according to any one of claims 1 to 14, 23 to 42 and 56, a recombinant protein according to claim 57, a nucleic acid according to claim 58 or a pharmaceutical composition according to claim 61.

63. The method of claim 62, wherein the medical condition is cancer.

64. The repeat domain according to any one of claims 1 to 14, 23 to 42 and 56, the recombinant protein according to claim 57, the nucleic acid according to claim 58 or the pharmaceutical composition according to claim 61 for use in a method of treating and / or diagnosing a medical condition.

65. The repeat domain, recombinant protein, nucleic acid or pharmaceutical composition for use according to claim 64, wherein the medical condition is cancer.

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