Charge-engineered engineered repeat domains and their uses

Abstract

The present invention relates to designed ankyrin repeat domains with a reduced isoelectric point (pI) and / or a reduced number of basic amino acid residues. The present invention further provides such repeat domains linked to a drug moiety (e.g., a radionuclide or a cytotoxic drug). The present invention further provides methods for producing such repeat domains, and the use of such repeat domains in therapeutic and / or diagnostic methods. Furthermore, the present invention further 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.

Description

[Technical Field]

[0001] (CROSS-REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of priority to European Patent Application No. 22188160, filed August 1, 2022, European Patent Application No. 23151023, filed January 10, 2023, and European Patent Application No. 23168056, filed April 14, 2023, the disclosures of which are incorporated herein by reference in their entirety for all purposes.

[0002] FIELD OF THE INVENTION The present invention relates to designed ankyrin repeat domains with a reduced isoelectric point (pI) and / or a reduced number of basic amino acid residues. The present invention further provides such repeat domains linked to a drug moiety (e.g., a radionuclide or a cytotoxic drug). The present invention further provides methods for producing such repeat domains, and the use of such repeat domains in therapeutic and / or diagnostic methods. Furthermore, the present invention further 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 technology]

[0003] Targeted radiopharmaceuticals have emerged as promising tools in cancer diagnosis and treatment. Such radiopharmaceuticals typically consist of a radiopharmaceutical (e.g., a radionuclide) linked to a binding molecule (e.g., an antibody or fragment thereof, an engineered protein scaffold, a peptide, or a small molecule). By combining specificity for a biological target with an ionizing radiation source, radiopharmaceuticals can focus radiation emission in the vicinity of a biomarker of interest. High target selectivity and tumor retention, low uptake in non-tumorous organs and tissues, and rapid clearance are desirable characteristics for accurate cancer diagnosis (e.g., the radiopharmaceutical reveals the location of the tumor) and / or treatment (e.g., the radiopharmaceutical damages the tumor) with radiopharmaceuticals.

[0004] Early studies in radioimmunotherapy highlighted significant drawbacks of using radiolabeled antibodies, such as their slow extravasation and clearance from the blood due to the size of intact antibodies (150 kDa). With antibody-based radiopharmaceuticals, optimal tumor-to-background ratios can typically be reached only after several days, thus inducing indirect damage to radiosensitive organs and tissues, such as bone marrow. Subsequently, alternative lower molecular weight binders were developed to improve pharmacokinetics and increase tumor-to-normal tissue dose ratios. Drug clearance in the context of radiolabeled peptides containing small molecule fragments is primarily driven by renal excretion, which includes the physiological processes of glomerular filtration, active tubular secretion, and tubular reabsorption. Glomerular filtration ensures that circulating cells and valuable macromolecular components of plasma are selectively retained based on molecular size. Molecules with a molecular weight greater than 70 kDa or a radius greater than 4.2 nm, and molecules bound to plasma proteins (such as albumin), undergo negligible glomerular filtration (Parihar, A.S. et al., Translational Oncology 15.1(2022):101295).

[0005] Due to their inherent properties, some radiopharmaceuticals are retained in the kidney, thereby contributing to an increased radiation absorption dose to the kidney. Small-format binding molecules with lower molecular weights can offer the combined advantages of rapid targeting and rapid clearance with minimal uptake in normal tissues or organs early after injection, but their use also induces undesirable high renal accumulation of radioactivity, preventing their wider clinical application. Both the choice of radionuclide and the properties of the binding molecule can affect the severity of nephrotoxicity resulting from such high radioactivity accumulation in the kidney (Chigoho, DM et al., Current Opinion in Chemical Biology 63(2021):219-228).

[0006] A major contributor to nephrotoxicity is the process of renal reabsorption. Low- to medium-molecular-weight radiolabeled molecules are readily filtered through the glomerulus and subsequently reabsorbed via charge attraction to the negatively charged phospholipid bilayer of proximal tubular cells, followed by intracellular catabolism. After proteolysis in the lysosome, radiolabeled catabolic products are released and, depending on their physical properties, are either freely washed out of the cell (non-residualized radionuclides) or retained intracellularly (residualized radionuclides). Residualized radionuclides are typically advantageous in terms of tumor cytotoxicity, but may increase the toxicity profile in normal tissues due to off-target localization.

[0007] Pharmacological and / or physicochemical approaches have been attempted to mitigate such renal retention, for example, by co-injecting positively charged amino acids that inhibit reabsorption of the radiotracer from the negatively charged proximal tubule membrane and / or by adjusting the net charge of the binding protein used in the conjugate via acylation, respectively. 99mIt has been previously reported that the renal clearance and tumor targeting of Tc-labeled humanized anti-Tac monoclonal antibody Fab can be enhanced by neutralizing the positive charge of the Fab through acylation. Furthermore, simultaneous injection of lysine further improved the blockade of renal uptake (Kim, MK et al., Nuclear medicine and biology, 29.2 (2002): 139-146). Furthermore, the effect of a single amino acid substitution on radioactivity accumulation in the kidney was investigated by Akizawa et al. using a very short 8-amino acid peptide ( 111 It has been studied in the context of In-DT PA conjugates (octreotide derivatives), but not with larger binding domains or proteins (Akizawa, H. et al., Nuclear medicine and biology 28.7(2001):761-768).

[0008] Pharmacological approaches to reduce renal uptake of peptide- and protein-based radiopharmaceuticals can help circumvent the effort required to tailor the structural characteristics of radiolabeled compounds. However, it has been shown that administration of such mitigation compounds, which act on various parts of the renal reabsorption system, is not effective for all radiopharmaceuticals. 99m A study on preventing the renal uptake of Tc-labeled engineered ankyrin repeat proteins (DARPins) showed that common clinical strategies were not effective in reducing the renal uptake of these radiolabeled DARPins in mice. More specifically, co-injection of lysine or gelofuscin did not reduce renal uptake. Pre-administration of high doses of maleate or fructose, which inhibit ATP-mediated endocytosis, resulted in a reduction in the renal uptake of these protein scaffolds, but the doses required were not suitable for clinical application. No other compounds were effective. Therefore, according to the authors, this study: 99mThese findings suggest that renal uptake of Tc-labeled DARPins proceeds via a mechanism independent of the DARPin structure and binding site composition (Altai, M. et al., EJNMMI Research 10.1(2020):1-8). Thus, despite attempts to reduce renal uptake and mitigate nephrotoxicity using pharmacological and / or physicochemical approaches, this has not consistently been achieved, and nephrotoxicity remains a hurdle for the application of radiopharmaceuticals in radiopharmaceutical therapy or diagnosis. Similar considerations apply to pharmaceuticals in which a small-format target-specific binding agent is linked to a cytotoxic agent instead of a radionuclide, for example, in antibody mimics or protein conjugates using small binding moieties (Richards, DADrug Discovery Today: Technologies 30(2018):35-46).

[0009] In summary, there remains a need for improved target-specific binding agents that have beneficial properties that result in reduced renal accumulation, particularly of the linked drug moiety (e.g., a radionuclide or cytotoxic agent), when administered to a mammal. Such improved target-specific binding agents can be used in therapeutic or diagnostic applications, such as with radiopharmaceutical or cytotoxic drug conjugates. Summary of the Invention

[0010] The present invention provides engineered DARPins that have a reduced number of basic amino acids (positive charge) and / or a reduced isoelectric point (pI) compared to conventional DARPins. These properties have surprisingly been found to reduce kidney accumulation of the linked drug moiety (e.g., a radionuclide) following in vivo administration of a drug moiety-linked DARPin of the invention.

[0011] DARPins are small, genetically engineered scaffold proteins (approximately 14 kDa for a single, designed repeat domain) that can be selected to bind to a given target protein with high affinity and specificity. Their applications in the context of radiopharmaceutical therapy or diagnostics include, for example, the use of DARPins for breast cancer imaging. 99m Phase I clinical trials involving Tc-labeled DARPins have begun (Bragina, O., et al., Journal of Nuclear Medicine 63.4(2022):528-535). However, as with other radiopharmaceuticals based on low molecular weight binders, solutions to address nephrotoxicity are needed to fully exploit the potential of DARPin-based radiopharmaceuticals. While modification of the net charge of DARPins has been described, for example, in International Patent Application Publication No. WO 2016 / 023898 (in which DARPins were modified to reduce their negative charge in an approach to creating DARPins capable of crossing the lipophilic membrane barrier of cells), modification of the charge properties or pI of DARPins has not been evaluated in relation to kidney accumulation.

[0012] The present invention relates to designed ankyrin repeat proteins (DARPins) with structural modifications that improve pharmacokinetic properties, in particular reducing renal accumulation. More specifically, the present invention provides designed ankyrin repeat domains that have a reduced number of basic amino acid residues and / or a lowered isoelectric point (pI) compared to commonly described designed ankyrin repeat domains. The present invention also provides improved repeat domains as described above linked to a drug moiety (e.g., a radionuclide or a cytotoxic drug). The present invention also provides methods for producing such repeat domains and the use of such repeat domains in therapeutic and / or diagnostic methods. Furthermore, the present invention also 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, such as humans.

[0013] The DARPins of the present invention, when linked to radioactive or cytotoxic drugs, show reduced drug accumulation in the kidney upon in vivo administration. Thus, the improved repeat domains and recombinant proteins provided herein open the door to new therapeutic and diagnostic applications, for example in the field of nuclear medicine, such as radiopharmaceutical therapy, or in cancer therapy using cytotoxic drug-conjugated proteins.

[0014] In one aspect, the invention provides an engineered 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 comprises the following list of features: (a) isoelectric point (pI) of pH 4.07 or less; (b) the percentage of basic amino acid residues among all amino acid residues contained in the repeat domain is 7.0% or less; and (c) the percentage of basic amino acid residues among the framework residues contained in the repeat domain is 6.1% or less.

[0015] In another aspect, the present invention provides a method for producing a modified ankyrin repeat domain, the method comprising steps (a) and (b), step (a) providing an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.07 and / or a percentage of basic amino acid residues among all amino acid residues contained in the repeat domain higher than 7.0% and / or a percentage of basic amino acid residues among framework residues contained in the repeat domain higher than 6.1%; and Step (b) comprises: (1) substituting 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) substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; The engineered ankyrin repeat domain has the following list of features: (i) an isoelectric point (pI) of pH 4.07 or less; (ii) the percentage of basic amino acid residues among all amino acid residues contained in the repeat domain is 7.0% or less; and (iii) the percentage of basic amino acid residues among the framework residues contained in the repeat domain is 6.1% or less.

[0016] In another aspect, the invention provides an engineered 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 exhibits the following list of features: (a) isoelectric point (pI) of pH 4.6 or less; (b) the percentage of basic amino acid residues among all amino acid residues contained in the repeat domain is 12.0% or less; (c) a percentage of Arg and Lys residues of 8.0% or less among all amino acid residues contained in the repeat domain; (d) a percentage of basic amino acid residues of 9.6% or less among framework residues contained in the repeat domain; and (e) Among the framework residues contained in the repeat domain, the percentage of Arg residues and Lys residues is 6.7% or less.

[0017] 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): step (a) providing an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.6, and / or a percentage of basic amino acid residues among all amino acid residues contained in the repeat domain higher than 12.0%, and / or a percentage of Arg and Lys residues among all amino acid residues contained in the repeat domain higher than 8.0%, and / or a percentage of basic amino acid residues among framework residues contained in the repeat domain higher than 9.6%, and / or a percentage of Arg and Lys residues among framework residues contained in the repeat domain higher than 6.7%, and Step (b) comprises: (1) substituting 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) substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; The modified repeat domain has the following list of characteristics: (i) an isoelectric point (pI) of pH 4.6 or less; (ii) the percentage of basic amino acid residues among all amino acid residues contained in the repeat domain is 12.0% or less; (iii) the percentage of Arg residues and Lys residues among all amino acid residues contained in the repeat domain is 8.0% or less; (iv) the percentage of basic amino acid residues among the framework residues contained in the repeat domain is 9.6% or less; and (v) the percentage of Arg residues and Lys residues among the framework residues contained in the repeat domain is 6.7% or less.

[0018] In another aspect, the present invention provides a designed ankyrin repeat domain obtainable or obtained by the method of the present invention.

[0019] In another aspect, the present invention provides recombinant proteins comprising such engineered repeat domains of the invention.

[0020] In another aspect, the present invention provides isolated nucleic acids encoding designed repeat domains of the invention or encoding recombinant proteins of the 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 invention, and optionally at least one pharmaceutically acceptable carrier or diluent.

[0021] In another aspect, the present invention provides a method for treating and / or diagnosing a medical condition, comprising 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 one particular aspect, the medical condition is cancer.

[0022] In another aspect, the present invention provides an engineered repeat domain, recombinant protein, nucleic acid, or pharmaceutical composition of the invention for use in a method for treating a medical condition. In one particular aspect, the medical condition is cancer.

[0023] Based on the disclosure provided herein, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by embodiment (E) below. E1. 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 has the following list of features: (a) isoelectric point (pI) of pH 4.07 or less; (b) the percentage of basic amino acid residues among all amino acid residues contained in the repeat domain is 7.0% or less; and (c) A repeat domain having at least one characteristic selected from the following: the percentage of basic amino acid residues among the framework residues contained in the repeat domain is 6.1% or less. E2. The repeat domain of E1, wherein the repeat domain has at least two features selected from features (a), (b), and (c). E3. The repeat domain of E1 or E2, wherein the repeat domain has at least features (a) and (b), (a) and (c), or (b) and (c). E4. The repeat domain of any one of E1-E3, wherein the repeat domain has features (a), (b), and (c). E5. The repeat domain of any one of E1-E4, wherein the repeat domain specifically binds to a target. E6. Repeat domains are 10 -7 A dissociation constant (K D ) the repeat domain described in E5 that binds to the target. 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. E8. The repeat domain of any one of E1-E7, wherein the repeat domain has a KR / DE ratio of 0.44 or less, and / or a KR / DE ratio among framework residues of 0.36 or less, and / or a KR / DE ratio among all residues of the N-terminal capping module of 0.66 or less. E9. The repeat domain of any one of E1-E8, wherein each internal repeat module independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 131-142, and (2) sequences in which up to nine framework residues in any of SEQ ID NOs: 131-142 are replaced by another amino acid; and / or wherein the N-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 25, and 115-122, and (2) sequences in which up to nine framework residues in any of SEQ ID NOs: 25, and 115-122 are replaced by another amino acid; and / or wherein the C-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 123-130, and (2) sequences in which up to nine framework residues in any of SEQ ID NOs: 123-130 are replaced by another amino acid. E10. The repeat domain of any one of E1-E9, 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 five 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 wherein 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 five framework residues other than positions 5, 17, 20, and 23 in SEQ ID NO: 19 are substituted by another amino acid; and / or wherein the C-terminal capping module comprises: (1) SEQ ID NO: 40; and (2) a sequence in which up to five framework residues other than positions 10, 11, 17, 18, 19, 22, and 26 in SEQ ID NO: 40 are substituted by another amino acid. E11. The repeat domain of any one of E1 to E10, wherein each internal repeat module independently comprises a sequence selected from SEQ ID NOs: 31-35, 61, 62, 92-94, and 131-142, and / or wherein the N-terminal capping module comprises a sequence selected from SEQ ID NOs: 19-25, 57-60, 65-81, and 115-122, and / or wherein the C-terminal capping module comprises a sequence selected from SEQ ID NOs: 40-42, 63, 64, 82-91, and 123-130. The repeat domain of any one of E1-E11, wherein each internal repeat module independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 26-30, 47, and 48; and (2) sequences in which up to 9 amino acids in any of SEQ ID NOs: 26-30, 47, and 48 are substituted by another amino acid; and / or wherein the N-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 15-18, 43, and 99-101; and (2) sequences in which up to 9 amino acids in any of SEQ ID NOs: 15-18, 43, and 99-101 are substituted by another amino acid; and / or wherein the C-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 36-39, 54, 102, and 103; and (2) sequences in which up to 9 amino acids in any of SEQ ID NOs: 36-39, 54, 102, and 103 are substituted by another amino acid. In certain embodiments, the substitutions are according to Table 3. In a further embodiment, the substitution is a conservative substitution according to Table 3. E12a. each internal repeat module independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 26-30, 47, and 48; and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 26-30, 47, and 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: 15-18, 43, and 99-101; and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, or up to 1 amino acid in any of SEQ ID NOs: 15-18, 43, and 99-101 is substituted with another amino acid. The repeat domain of any one of E1 to E12, wherein the repeat domain comprises a sequence selected from the group consisting of: sequences in which up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid is substituted by another amino acid; and / or wherein the C-terminal capping module comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 36-39, 54, 102, and 103; and (2) sequences in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 36-39, 54, 102, and 103 is substituted by another amino acid. In a particular embodiment, the substitution is a substitution according to Table 3. In a further embodiment, the substitution is a conservative substitution according to Table 3. E12b. The repeat domain of any one of E1-E12a, wherein each internal repeat module independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 26-30, 47, and 48; and (2) sequences in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 26-30, 47, and 48 is substituted by another amino acid. The repeat domain of any one of E1 to E12b, wherein the N-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 15-18, 43, and 99-101; and (2) sequences in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 15-18, 43, and 99-101 is substituted by another amino acid. The repeat domain of any one of E1 to E12c, wherein the C-terminal capping module comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 36 to 39, 54, 102, and 103; and (2) sequences in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 36 to 39, 54, 102, and 103 is substituted with another amino acid. E13. The repeat domain of any one of E1-E12, wherein the repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 104, 108, 109, and 112-114; and (2) a sequence having at least 80% amino acid sequence identity among framework residues of any one of SEQ ID NOs: 104, 108, 109, and 112-114. E14. The repeat domain of any one of E1 to E13, wherein the repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 8, 9, and 95-98, and (2) a sequence having at least 80% amino acid sequence identity to any one of the amino acid sequences of SEQ ID NOs: 8, 9, and 95-98. E15. A method for generating a modified ankyrin repeat domain, the method comprising steps (a) and (b), step (a) providing an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.07 and / or a percentage of basic amino acid residues among all amino acid residues contained in the repeat domain higher than 7.0% and / or a percentage of basic amino acid residues among framework residues contained in the repeat domain higher than 6.1%; and Step (b) comprises: (1) substituting 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) substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; The engineered ankyrin repeat domain has the following list of features: (i) an isoelectric point (pI) of pH 4.07 or less; (ii) the percentage of basic amino acid residues among all amino acid residues contained in the repeat domain is 7.0% or less; and (iii) the percentage of basic amino acid residues among the framework residues contained in the repeat domain is 6.1% or less. E16. A method comprising steps (a) and (b), step (a) providing an ankyrin repeat domain with an isoelectric point (pI) higher than pH 4.07; and Step (b) comprises: (1) substituting 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) substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; The method of E15, wherein the modified ankyrin repeat domain has an isoelectric point (pI) of pH 4.07 or less. E17. comprising step (a) and step (b), Step (a) is a step of providing an ankyrin repeat domain having a percentage of basic amino acid residues of more than 7.0% among all amino acid residues contained in the repeat domain; and step (b) comprising substituting at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; The method according to E15, wherein the modified ankyrin repeat domain has a percentage of basic amino acid residues of 7.0% or less among all amino acid residues contained in the repeat domain. E18. comprising step (a) and step (b); Step (a) is a step of providing 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 step (b) comprising substituting at least one basic amino acid residue with a neutral or acidic amino acid residue among the framework residues of the repeat domain of step (a); The method according to E15, wherein the modified ankyrin repeat domain has a percentage of basic amino acid residues of 6.1% or less among the framework residues contained in the repeat domain. E19. The method of 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 of 0.44 or less, and / or the repeat domain in step (a) has a KR / DE ratio among framework residues higher than 0.36 and the modified repeat domain has a KR / DE ratio of 0.36 or less, and / or the repeat domain in step (a) has a KR / DE ratio among all residues of the N-terminal capping module higher than 0.66 and the modified repeat domain has a KR / DE ratio among all residues of the N-terminal capping module of 0.66 or less. E20. The repeat domain of step (a) specifically binds to a target and optionally -7 A dissociation constant (K DThe method according to any one of E15 to E19, wherein the binding is performed at E21. The modified repeat domain is 10 -7 K less than M D and preferably the modified repeat domain specifically binds to the target at (1) the K D K is approximately equal to D or (2) the repeat domain of step (a) binds to the target K D K less than 100 times, less than 10 times, less than 5 times, or less than 2 times higher than D and binding to the target. E22. The method of 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. 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 the following list of features: (a) isoelectric point (pI) of pH 4.6 or less; (b) the percentage of basic amino acid residues among all amino acid residues contained in the repeat domain is 12.0% or less; (c) a percentage of Arg and Lys residues of 8.0% or less among all amino acid residues contained in the repeat domain; (d) a percentage of basic amino acid residues of 9.6% or less among framework residues contained in the repeat domain; and (e) A repeat domain having at least one characteristic selected from the following: the percentage of Arg residues and Lys residues among the framework residues contained in the repeat domain is 6.7% or less. E24. The repeat domain according to E23, wherein the isoelectric point (pI) is within the range of pH 3.0 to pH 5.0, preferably within the range of pH 3.0 to pH 4.6, more preferably within the range of pH 3.50 to pH 4.53. E25. The repeat domain of E23 or E24, wherein the repeat domain has at least characteristic (b), and optionally the percentage in characteristic (b) is 8.7% or less. E26. The repeat domain of any one of E23-E25, wherein the repeat domain has at least characteristic (c), and optionally the percentage in characteristic (c) is 4.7% or less. E27. The repeat domain of any one of E23-E26, wherein the repeat domain has at least characteristic (d), and optionally the percentage in characteristic (d) is 6.7% or less. E28. The repeat domain of any one of E23-E27, wherein the repeat domain has at least feature (e), and optionally the percentage in feature (e) is 5.7% or less. E29. The repeat domain of any one of E23 to E28, wherein the repeat domain has a KR / DE ratio of 0.44 or less, and / or a KR / DE ratio among framework residues of 0.36 or less, and / or a KR / DE ratio among all residues of the N-terminal capping module of 0.66 or less. E30. The repeat domain of any one of E23 to E29, wherein the repeat domain has a total number of basic amino acid residues equal to or less than n, where n=4+5R, where R is the number of internal repeat modules comprised in the repeat domain. E31. The repeat domain of any one of E23 to E30, wherein the repeat domain has a total number of Arg and Lys residues equal to or less than m, where m=5+2R, and R is the number of internal repeat modules comprised in the repeat domain. E32. The repeat domain of any one of E23-E31, wherein the repeat domain has no Arg or Lys residues in any framework residue position. E33. The repeat domain of any one of E23 to E26, wherein the repeat domain comprises one internal repeat module, two internal repeat modules, three internal repeat modules, or four internal repeat modules. E34. The repeat domain of any one of E23-E33, wherein the repeat domain specifically binds to a target. E35. Repeat domain is 10 -7 A dissociation constant (K D ) a repeat domain as described in E34 that binds to a target at E36. The repeat domain of any one of E23-E35, wherein the drug moiety is a therapeutic moiety or a diagnostic moiety. E37. The repeat domain of any one of E23-E36, wherein the drug moiety is a toxin. E38. The repeat domain of E37, wherein the toxin is a radionuclide. E39. The repeat domain of E37, wherein the toxin is a cytotoxin. E40. The repeat domain of any one of E23 to E39, wherein the repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 104-114; (2) a sequence having at least 80% amino acid sequence identity within the framework residues of any one of SEQ ID NOs: 104-114. E41. The repeat domain of any one of E23 to E40, wherein the repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 104-112, (2) a sequence having at least 80% amino acid sequence identity between framework residues of any one of SEQ ID NOs: 104-112. E42. The repeat domain of any one of E23 to E41, 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; (2) a sequence having at least 80% amino acid sequence identity with SEQ ID NOs: 8 to 10, 12, and 95 to 98. E43. The repeat domain of any one of E23-E42, wherein the repeat domain is linked to a drug moiety for use in a method for treating and / or diagnosing a medical condition, the method comprising administering to a patient in need of treatment and / or diagnosis a therapeutically and / or diagnostically effective amount of the repeat domain, optionally wherein the administration is not oral. E44. A designed ankyrin repeat domain according to E43 linked to a drug moiety for use in a method for treating and / or diagnosing a medical condition, wherein the medical condition is cancer. E45. A method for producing a modified ankyrin repeat domain linked to a drug moiety, the method comprising steps (a) and (b): step (a) providing an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.6, and / or a percentage of basic amino acid residues among all amino acid residues contained in the repeat domain higher than 12.0%, and / or a percentage of Arg and Lys residues among all amino acid residues contained in the repeat domain higher than 8.0%, and / or a percentage of basic amino acid residues among framework residues contained in the repeat domain higher than 9.6%, and / or a percentage of Arg and Lys residues among framework residues contained in the repeat domain higher than 6.7%, and Step (b) comprises: (1) substituting 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) substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; The modified repeat domain has the following list of characteristics: (i) an isoelectric point (pI) of pH 4.6 or less; (ii) the percentage of basic amino acid residues among all amino acid residues contained in the repeat domain is 12.0% or less; (iii) the percentage of Arg residues and Lys residues among all amino acid residues contained in the repeat domain is 8.0% or less; (iv) the percentage of basic amino acid residues among the framework residues contained in the repeat domain is 9.6% or less; and (v) the percentage of Arg residues and Lys residues among the framework residues contained in the repeat domain is 6.7% or less. E46. comprising step (a) and step (b), step (a) providing an ankyrin repeat domain with an isoelectric point (pI) higher than pH 4.6; and Step (b) comprises: (1) substituting 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) substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; The method of E45, wherein the modified repeat domain has an isoelectric point (pI) of pH 4.6 or less. E47. Comprising step (a) and step (b), Step (a) is a step of providing 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 step (b) comprising substituting at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; The method according to E45, wherein the modified repeat domain has a percentage of basic amino acid residues of 12.0% or less among all amino acid residues contained in the repeat domain. E48. Comprising step (a) and step (b), Step (a) is a step of providing an ankyrin repeat domain having a percentage of Arg and Lys residues of more than 8.0% among all amino acid residues contained in the repeat domain; and step (b) comprising substituting at least one Arg and Lys residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; The method according to E45, wherein the modified repeat domain has a proportion of Arg residues and Lys residues of 8.0% or less among all amino acid residues contained in the repeat domain. E49. comprising step (a) and step (b); Step (a) is a step of providing 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 step (b) comprising substituting at least one basic amino acid residue with a neutral or acidic amino acid residue among the framework residues of the repeat domain of step (a); The method according to E45, wherein the modified repeat domain has a percentage of basic amino acid residues of 9.6% or less among the framework residues contained in the repeat domain. E50. comprising step (a) and step (b); Step (a) is to provide an ankyrin repeat domain having a percentage of Arg and Lys residues among the framework residues contained in the repeat domain that is higher than 6.7%; and step (b) comprising substituting at least one Arg or Lys residue among the framework residues of the repeat domain of step (a) with a neutral or acidic amino acid residue; The method according to E45, wherein the modified repeat domain has a percentage of Arg and Lys residues of 6.7% or less among the framework residues contained in the repeat domain. E51. The method of 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 of 0.44 or less, and / or the repeat domain in step (a) has a KR / DE ratio among framework residues higher than 0.36 and the modified repeat domain has a KR / DE ratio of 0.36 or less, and / or the repeat domain in step (a) has a KR / DE ratio among all residues of the N-terminal capping module higher than 0.66 and the modified repeat domain has a KR / DE ratio among all residues of the N-terminal capping module of 0.66 or less. E52. The method of any one of E45-E51, further comprising linking a drug moiety to the modified repeat domain. E53. The repeat domain of step (a) specifically binds to a target and optionally -7 A dissociation constant (K D The method according to any one of E45 to E52, wherein the binding is performed at E54. The modified repeat domain is 10 -7 K less than M D and preferably the modified repeat domain specifically binds to the target at (1) the K D K is approximately equal to D or (2) the repeat domain of step (a) binds to the target K D K less than 100 times, less than 10 times, less than 5 times, or less than 2 times higher than D and binding to the target. E55. The method of 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. E56. A designed ankyrin repeat domain obtainable or obtained by the method according to any one of E15 to E22, or E45 to E55. E57. A recombinant protein comprising the repeat domain according to any one of E1 to E14, E23 to E42, and E56. E58. An isolated nucleic acid encoding the repeat domain of any one of E1 to E14, E23 to E42, and E56, or the recombinant protein of E57. E59. A recombinant expression vector comprising a nucleic acid according to E58. E60. A host cell comprising a recombinant expression vector according to E59. E61. A pharmaceutical composition comprising one or more of: (i) a repeat domain described in any one of E1-E14, E23-E42, and E56; (ii) a recombinant protein described in E57; (iii) a nucleic acid described in E58; and / or (iv) a recombinant expression vector described in E59, and optionally a pharmaceutically acceptable carrier or diluent. E62. A method for treating and / or diagnosing a medical condition, comprising administering to a patient in need thereof a therapeutically and / or diagnostically effective amount of a repeat domain described in any one of E1-E14, E23-E42, and E56, a recombinant protein described in E57, a nucleic acid described in E58, or a pharmaceutical composition described in E61. E63. The method of E62, wherein the medical condition is cancer. E64. The repeat domain of any one of E1-E14, E23-E42, and E56, the recombinant protein of E57, the nucleic acid of E58, or the pharmaceutical composition of E61 for use in a method for treating and / or diagnosing a medical condition. E65. The repeat domain, recombinant protein, nucleic acid, or pharmaceutical composition for use according to E64, wherein the medical condition is cancer. [Brief explanation of the drawings]

[0024] [Figure 1A]The sequences and properties of a representative DARPin mutant according to the invention, a parent DARPin, a negative control DARPin mutant (DARPin07), and a consensus DARPin are shown. The underlined and bolded sequence names represent the parent DARPin used as the basis for the engineered mutants. Figure 1A shows some characteristics of DARPins calculated for the entire repeat domain (i.e., by considering all residues contained in the repeat domain) or for framework positions only (i.e., by considering all residues contained in the repeat domain excluding potential target interaction residues). The symbol "#", as used, for example, in #(Arg+Lys), refers to the total number of Arg and Lys residues. The symbol "%", as used, for example, in %(Arg+Lys), refers to the percentage of Arg and Lys residues contained in the indicated repeat domain or framework position. [Figure 1B] The sequences and properties of a representative DARPin mutant according to the invention, a parent DARPin, a negative control DARPin mutant (DARPin07), and a consensus DARPin are shown. The underlined and bolded sequence names represent the parent DARPin used as the basis for the engineered mutants. As shown in FIG. 1B, the exemplary DARPin mutant comprises an N-terminal capping module, a C-terminal capping module, and two internal repeat modules. FIG. 1B shows an alignment of all DARPin mutant sequences, with randomized positions marked by an underlined "X" in the consensus column (the complete sequence is divided into different boxes for practical reasons only). Some positions have a strong preference for specific acidic / basic residues based on the consensus sequence (such as position 10 in internal repeat module 1), but have been found to be different for the mutants shown (e.g., due to affinity-increasing mutations). [Figure 2]Figures 2-1, 2-2, and 2-3 show size-exclusion chromatography (SEC) profiles for the parent DARPin and DARPin variants tested. Plots 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). All SEC profiles show a dimer peak before the main monomer peak due to partial formation of disulfide-bonded dimers (C-terminal Cys). [Figure 3] Schematic overview of the process for producing radiolabeled DARPins according to the invention. DARPin variants were expressed in Escherichia coli and purified by immobilized metal affinity chromatography (IMAC) and gel filtration (GF). The constructs were cleaved with recombinant TEV protease to cleave the His-tag. Subsequently, uncleaved DARPin variants as well as the His-tagged TEV protease were removed by reverse IMAC, and the flow-through was collected and loaded onto an SEC column. The purified DARPin was reduced and coupled with the chelator DTPA. The chelated DARPin was then loaded with the radionuclide indium-111 (also referred to as 111In). [Figure 4]Figures 4-1, 4-2, and 4-3 show single-trace SPR profiles of DARPins coupled with DTPA (also referred to as constructs, see Table 7) against biotinylated full-length HER2. For reference, plots 1 and 2 show the profiles of the HER2-binding parent DARPin06 (SEQ ID NO: 6) and parent DARPin11 (SEQ ID NO: 11), respectively. Plots 3, 4, 5, 6, and 7 show the profiles 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), respectively. Plots 8 and 9 show construct 11 (DARPin11-GSGSC-DTPA) and construct 12 (DARPin12-GSGSC-DTPA), respectively. A GSGSC tag (SEQ ID NO: 14) was fused to the C-terminus of the DARPin. All analytes (500 nM) were injected sequentially for 120 seconds, and dissociation was recorded for 180 seconds (25 μL / min PBS-Tween 20 (0.005%)). Each injection was followed by a 60-second regeneration step with glycine pH 2.0. Data were double-referenced (control spot and buffer injection) and fitted to a 1:1 Langmuir model. [Figure 5] Figure 1 shows the renal uptake of indium-labeled DARPins. Radiolabeled DARPin01 to DARPin12 were injected into wild-type mice. Data are presented as the mean % injected activity / gram tissue mass (%IA / g). Measurements were performed 4 hours after injection. Error bars indicate standard deviation. Bars shown in black correspond to the parent DARPin. DARPin07 is a negative control with a higher pI and a higher percentage of basic amino acids among the framework residues compared to its parent DARPin06. [Figure 6A]Figure 6 shows a 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 DARPin (1 mg / kg, approximately 150 KBq) 2 weeks (n = 6 per group) or 3 weeks (n = 6 per group) after SKOV3ip tumor cell implantation. Measurements were performed 4 hours after injection. Data are presented as the mean % injected activity / gram tissue mass (% IA / g, pooled n = 12 / group). Figure 6A shows the kidney accumulation of In-radiolabeled pI engineered DARPins compared to the In-labeled parent DARPin. A strong reduction in kidney accumulation is observed for the engineered variants (DARPin02 and DARPin08) compared to the respective parent DARPins (DARPin01 and DARPin06, respectively). More specifically, a 90% (DARPin02 vs. DARPin01) and 78% (DARPin08 vs. DARPin06) reduction occurs as a result of pI engineering of the DARPin variants. In Figures 6A and 6B, the results corresponding to the engineered variants are represented by open bars, and the results corresponding to the parent DARPins are represented by closed bars. [Figure 6B]Figure 6B shows a 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 DARPin (1 mg / kg, approximately 150 KBq) 2 weeks (n = 6 per group) or 3 weeks (n = 6 per group) after SKOV3ip tumor cell implantation. Measurements were performed 4 hours after injection. Data are presented as the mean % injected activity per gram of tissue mass (% IA / g, pooled n = 12 per group). Figure 6B shows DARPin uptake in tumors. Significant accumulation of HER2-specific DARPins was observed in tumors, and accumulation of DARPin08 in tumors was comparable to that of its parent DARPin06. Only negligible non-specific accumulation of unbound DARPin01 and DARPin02 was observed in the tumors. In Figures 6A and 6B, the results corresponding to the engineered mutants are represented by open bars, and the results corresponding to the parent DARPin are represented by closed bars. [Figure 6C]Figure 6 shows a 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 DARPin (1 mg / kg, approximately 150 KBq) 2 weeks (n = 6 per group) or 3 weeks (n = 6 per group) after SKOV3ip tumor cell implantation. Measurements were performed 4 hours after injection. Data are presented as the mean % injected activity / gram tissue mass (% IA / g, pooled n = 12 / group). Figure 6C: Measurement of DARPin accumulation in additional tissues or organs (blood, heart, lung, spleen, liver, small intestine, large intestine, muscle, bone, and tail). Overall, the accumulation of the engineered variants (DARPin02 and DARPin08) was similar to that of the respective parent DARPins (DARPin01 and DARPin06, respectively). Error bars indicate standard deviation. [Figure 7] Figure 1 shows a time course biodistribution study of In-labeled DARPins (DARPin06 and DARPin08) in mice bearing HER2-expressing SKOV3ip tumors. Mice were injected with radiolabeled DARPin (1 mg / kg, approximately 150 KBq) at a tumor volume of approximately 350 mm. Radioactivity development in isolated kidney, tumor, and blood samples was measured 1, 4, 24, 48, and 96 hours after injection. Data are presented as mean % injected activity / gram tissue mass (% IA / g, n=4 / group). The area under the curve (AUC) in the kidney of charge-modified DARPin08 is reduced by 76% compared to parent DARPin06. The AUC in the tumor and blood remains comparable for the two DARPins. [Figure 8A]Figure 8 shows the sequences and properties of further exemplary DARPin variants according to the invention, derived from the parent DARPin06. Figure 8A shows the characteristics of the DARPin calculated for the entire repeat domain (i.e., by considering all residues contained in the repeat domain) or for framework positions only (i.e., by considering all residues contained in the repeat domain excluding potential target interaction residues). The definitions and legend in Figure 8 correspond to those in Figure 1. [Figure 8B] 8B shows the sequences and properties of further exemplary DARPin variants according to the invention, derived from the parent DARPin06. As shown in FIG. 8B, these further exemplary DARPins comprise an N-terminal capping module, a C-terminal capping module, and two internal repeat modules. Randomized positions are marked by underlined "X"s in the consensus column (the complete sequences are divided into different boxes for practical reasons only). The definitions and legend in FIG. 8 correspond to those in FIG. 1. [Figure 9] Figures 9A, 9B, 9C, 9D, 9E, and 9F show the multitrace SPR profiles of DARPins coupled with DTPA (also referred to as constructs, see Tables 7 and 16) against biotinylated full-length HER2. Figures 9A, 9B, 9C, 9D, 9E, and 9F show 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. A GSGSC tag (SEQ ID NO: 14) was fused to the C-terminus of the DARPin. Three-fold dilutions of analyte (50, 16.667, 5.556, 1.852, and 0.617 nM) were injected for 300 seconds, and dissociation was recorded for 1500 seconds (25 μL / min). Each injection was followed by a 60-second regeneration step with glycine pH 2.0. Dissociation constants (KD) were calculated from the globally fitted on- and off-rates using a standard 1:1-Langmuir model (see Table 17). [Figure 10A]Figure 10A shows a biodistribution study of In-labeled DARPins in mice bearing HER2-expressing SKOV3ip tumors. All tested DARPins have binding specificity for HER2. DARPin 06 is the parent DARPin, and DARPins 08, 13, 14, 15, and 16 are charge-engineered variants according to the present invention. Mice were injected with radiolabeled DARPin (1 mg / kg, approximately 150 KBq) at a tumor volume of approximately 350 mm. Measurements were performed 4 hours after injection. Data are presented as the mean % injected activity per gram of tissue mass (% IA / g, n=4 / group). Figure 10B shows the kidney accumulation of In-radiolabeled charge-engineered DARPins 08, 13, 14, 15, and 16 compared to In-labeled parent DARPin 06. A strong decrease in kidney accumulation is observed for the engineered variants compared to the parent DARPin. More specifically, a reduction of 82% (DARPin08 vs. DARPin06), 90% (DARPin13 vs. DARPin06), 85% (DARPin14 vs. DARPin06), 93% (DARPin15 vs. DARPin06), and 87% (DARPin16 vs. DARPin06) occurs as a result of charge engineering of the DARPin mutants. In Figures 10A and 10B, the results corresponding to the engineered mutants are represented by open bars, and the results corresponding to the parent DARPin are represented by closed bars. Error bars indicate the standard deviation. [Figure 10B]Figure 10B shows a biodistribution study of In-labeled DARPins in mice bearing HER2-expressing SKOV3ip tumors. All tested DARPins have binding specificity for HER2. DARPin 06 is the parent DARPin, and DARPins 08, 13, 14, 15, and 16 are charge-engineered variants according to the invention. Mice were injected with radiolabeled DARPin (1 mg / kg, approximately 150 KBq) at a tumor volume of approximately 350 mm. Measurements were performed 4 hours after injection. Data are presented as the mean % injected activity per gram of tissue mass (% IA / g, n=4 / group). Figure 10B shows DARPin uptake in tumors. Accumulation of the HER2-specific DARPin was observed in the tumor, and the accumulation of the charge-engineered DARPin variants in the tumor was comparable to that of the parent DARPin 06. In Figures 10A and 10B, the results corresponding to the engineered variants are represented by open bars, while the results corresponding to the parent DARPin are represented by closed bars. Error bars indicate standard deviation. DETAILED DESCRIPTION OF THE INVENTION

[0025] Designed ankyrin repeat domains are the structural units of designed ankyrin repeat proteins. Designed repeat protein libraries, including designed ankyrin repeat protein libraries (WO 2002 / 020565, Binz et al., Nat. Biotechnol. 22, 575-582, 2004; Stumpp et al., Drug Discov. Today 13, 695-701, 2008), can be used to select target-specific designed repeat domains that bind to targets with high affinity. Such target-specific designed repeat domains can then be used as valuable components of recombinant binding proteins for the treatment and / or diagnosis of diseases.

[0026] Designed ankyrin repeat proteins are a class of binding molecules that have the potential to overcome the limitations of monoclonal antibodies and thus enable novel therapeutic and / or diagnostic approaches. Such ankyrin repeat proteins may contain a single designed ankyrin repeat domain or a combination of two, three, four, five, or more 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 containing only a single designed ankyrin repeat domain are small proteins (14 kDa) that can be selected to bind to a given target protein with high affinity and specificity. These features, along with the possibility of combining two, three, four, five, or more designed ankyrin repeat domains in one protein, make designed ankyrin repeat proteins ideal candidates for agonistic, antagonistic, and / or inhibitory drugs. Furthermore, such ankyrin repeat proteins can be engineered to carry various effector functions, e.g., cytotoxic agents or half-life extenders, enabling entirely new drug formats. Taken together, designed ankyrin repeat proteins represent the next generation of protein therapeutics with the potential to go beyond existing antibody drugs.

[0027] The present inventors surprisingly discovered 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 upon in vivo administration. Furthermore, when designed repeat domains of the present invention are linked to drug moieties, the drug moieties exhibit reduced accumulation in the kidney upon in vivo administration. For example, when designed repeat domains of the present invention are linked to radioactive drugs, the accumulation of radioactivity in the kidney upon in vivo administration is reduced. This property is highly desirable because kidney accumulation of toxic drug moieties in the kidney leads to nephrotoxicity. For the above reasons, kidney accumulation of radioactivity limits the use of radiolabeled DARPins in therapy or diagnosis, and similarly, kidney accumulation of cytotoxic agents limits the use of cytotoxic agent-conjugated DARPins. The biophysical properties and target-specific accumulation of designed ankyrin repeat proteins of the present invention in target-expressing tumors are not significantly affected by charge engineering. The designed ankyrin repeat domains of the present invention therefore provide an improved tumor-to-kidney ratio. The improved repeat domains and recombinant proteins provided herein therefore open the door to new therapeutic and / or diagnostic applications, for example in the field of nuclear medicine, such as radiopharmaceutical therapy, or in the field of cancer therapy using cytotoxic drug-conjugated proteins.

[0028] definition Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclature used in connection with the techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry described herein is that well known and commonly used in the art.

[0029] The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms unless otherwise specified. When aspects of the invention describe a feature as "comprising," in embodiments, the feature can also be construed as "consisting of" or "consisting essentially of." Any and all examples provided herein, or the use of illustrative language (e.g., "such as"), are intended merely to better explain the disclosure and do not impose limitations on the scope of the disclosure unless specifically stated otherwise. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure. As used herein, the term "about" corresponds to ±10% of a given numerical value, unless otherwise specified.

[0030] The recitation of ranges of values herein, unless otherwise indicated herein, is merely intended to serve as a shorthand method of referring individually to each separate value and each endpoint falling within the range, and each separate value and endpoint is incorporated herein as if it were individually listed herein.

[0031] The term "nucleic acid" refers to a polynucleotide molecule that can be a ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) molecule, either single-stranded or double-stranded, and includes modified and artificial forms of DNA or RNA. The nucleic acid can be present in either isolated form or contained in a recombinant nucleic acid molecule or vector.

[0032] In the context of the present invention, the term "protein" refers to a molecule comprising a polypeptide, at least a portion of which has or can acquire a defined three-dimensional sequence by forming secondary, tertiary, or quaternary structures within a single polypeptide chain and / or between multiple polypeptide chains. When a protein comprises two or more polypeptide chains, the individual polypeptide chains can be linked by non-covalent or covalent bonds, for example, by disulfide bonds between two polypeptides. The portions of a protein that individually have or can acquire a defined three-dimensional sequence by forming secondary and / or tertiary structures are referred to as "protein domains." Such protein domains are well known to those skilled in the art.

[0033] The term "recombinant," as used in reference to recombinant protein and recombinant polypeptide, means that the protein or polypeptide is produced by use of recombinant DNA technology, as is well known to those skilled in the art. For example, a recombinant DNA molecule (e.g., produced by gene synthesis) encoding a polypeptide can be cloned into a bacterial expression plasmid (e.g., pQE30, QIAgen), yeast expression plasmid, mammalian expression plasmid, or plant expression plasmid, or into DNA enabling in vitro expression. For example, when such a recombinant bacterial expression plasmid is inserted into suitable bacteria (e.g., E. coli), these bacteria can produce the polypeptide encoded by the recombinant DNA. The polypeptide or protein produced accordingly is referred to as a recombinant polypeptide or recombinant protein.

[0034] In the context of the present invention, the term "polypeptide" relates to a molecule consisting of a chain of multiple, i.e., two or more, amino acids linked by peptide bonds. Preferably, a polypeptide consists of more than eight amino acids linked by peptide bonds. The term "polypeptide" also includes multiple chains of amino acids linked by cysteine S-S bridges. Polypeptides are well known to those skilled in the art.

[0035] The term "target" refers to an individual molecule, such as a nucleic acid, polypeptide or protein, carbohydrate, or any other natural or non-natural molecule or moiety, including any portion of such an individual molecule, or a complex of two or more such molecules. A target can be a whole cell sample or a tissue sample. Preferably, a target is a naturally occurring or non-natural polypeptide, or a polypeptide that contains a chemical modification, e.g., modified by natural or non-natural phosphorylation, acetylation, or methylation.

[0036] WO 2002 / 020565 and Forrer et al., 2003 (Forrer, P., Stumpp, MT, Binz, HK, Pluckthun, A., 2003. FEBS Letters 539, 2-6) contain a general description of the characteristics of repeat proteins and of the characteristics, techniques and applications of repeat domains. The term "repeat protein" refers to a protein comprising one or more repeat domains. Preferably, a repeat protein comprises one, two, three, four, five or six repeat domains. Furthermore, a repeat protein may comprise additional non-repeat protein domains, polypeptide tags and / or peptide linkers.

[0037] The term "repeat domain" refers to a protein domain comprising two or more consecutive repeat modules as structural units, wherein the repeat modules have structural and sequence homology. Preferably, the repeat domain further comprises an N-terminal and / or a C-terminal capping module. For clarity, the capping module may be a repeat module. Such repeat domains, repeat modules and capping modules, sequence motifs and structural and sequence homologies are well known to those skilled in the art from the examples of ankyrin repeat domains (Binz et al., J. Mol. Biol. 332, 489-503, 2003; Binz et al., 2004, cited above, WO 2002 / 020565, WO 2012 / 069655), leucine-rich repeat domains (WO 2002 / 020565), tetratricopeptide repeat domains (Main, E. R., Xiong, Y., Cocco, M. J., D'Andrea, L., Regan, L., Structure 11(5), 497-508, 2003) and armadillo repeat domains (WO 2009 / 040338). It is further known to those skilled in the art that such repeat domains are distinct from proteins containing repeated amino acid sequences, all of which can form individual domains (e.g., the FN3 domain of fibronectin). The repeat domains can be binding domains.

[0038] The term "ankyrin repeat domain" refers to a repeat domain that comprises two or more consecutive ankyrin repeat modules as structural units, wherein the ankyrin repeat modules have structural and sequence homology.

[0039] The term "designed" as used in designed repeat proteins, designed repeat domains etc. refers to the property that such repeat proteins and repeat domains, respectively, are artificial and do not occur in nature. The binding 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.

[0040] The term "repeat module" refers to the repeated amino acid sequence and structural unit of a designed repeat domain, which is originally derived from the repeat unit of a naturally occurring repeat protein. Each repeat module comprised in a repeat domain is derived from one or more repeat units of a family or subfamily of naturally occurring repeat proteins, preferably the ankyrin repeat protein family. Furthermore, each repeat module comprised in a repeat domain may contain a "repeat sequence motif" deduced from homologous repeat modules with the same target specificity obtained from the selected repeat domain on a target. Repeat modules as used in the present invention encompass internal repeat modules and capping modules, such as N- and C-terminal capping modules. An "internal repeat module" refers to a repeat module flanked by two repeat modules. In other words, an internal repeat module is flanked at the N-terminus by one repeat module and at the C-terminus by another repeat module.

[0041] Therefore, the term "ankyrin repeat module" refers to a repeat module derived from the repeat unit of an ankyrin repeat protein that originally occurs in nature. Ankyrin repeat proteins are well known to those skilled in the art. Designed ankyrin repeat proteins have been previously disclosed; see, for example, International Patent Applications Nos. 2002 / 020565, 2010 / 060748, 2011 / 135067, 2012 / 069654, 2012 / 069655, 2014 / 001442, 2014 / 191574, 2014 / 083208, 2016 / 156596, and 2018 / 054971, all of which are incorporated by reference in their entirety. Typically, ankyrin repeat modules contain approximately 31-33 amino acid residues that form two alpha helices separated by a loop.

[0042] A repeat module may comprise positions with amino acid residues that are not randomized in the library for the purpose of selecting target-specific repeat domains ("non-randomized positions" or "fixed positions", used interchangeably herein) and positions with amino acid residues that are randomized in the library for the purpose of selecting target-specific repeat domains ("randomized positions"). Non-randomized positions include framework residues and may also include target-interaction residues. Randomized positions include target-interaction residues. "Randomized" means, for example, that more than one amino acid is allowed at an amino acid position of a repeat module, and that any of the 20 common naturally occurring amino acids is allowed, or that amino acids other than cysteine, or most of the 20 naturally occurring amino acids are allowed, such as amino acids other than glycine, cysteine, and proline. For the purposes of this patent application, amino acid residues 3, 4, 6, 11, 14, and 15 of SEQ ID NOs: 26-35, 46-52, 61, 62, 92-94, and 131-142, and amino acid residues 4, 8, 11, and 12 of SEQ ID NOs: 15-25, 43-45, 57-60, 65-81, 99-101, and 115-122, and amino acid residues 3, 4, 6, 14, and 15 of SEQ ID NOs: 36-42, 53-56, 63, 64, 82-91, 102, 103, and 123-130 are randomized positions of the ankyrin repeat module of the invention.

[0043] The term "repeat sequence motif" refers to an amino acid sequence deduced from one or more repeat modules. Preferably, the repeat modules are derived from repeat domains with binding specificity for the same target. Such a repeat sequence motif comprises framework residue positions and target interaction residue positions. The framework residue positions correspond to the framework residue positions of a repeat module. Similarly, the target interaction residue positions correspond to the target interaction residue positions of a repeat module. The repeat sequence motif comprises non-randomized positions and randomized positions.

[0044] The term "repeat unit" refers to an amino acid sequence that contains one or more naturally occurring protein sequence motifs, where the "repeat unit" is found in multiple copies and exhibits a defined folding topology common to all motifs that determines protein folding. Examples of such repeat units include leucine-rich repeat units, ankyrin repeat units, armadillo repeat units, tetratricopeptide repeat units, HEAT repeat units, and leucine-rich variant repeat units.

[0045] Residue or amino acid residue refers to an amino acid comprised in the peptide chain. The term "target interaction residue" refers to an amino acid residue of a repeat module that contributes to direct interaction with a target. Such contribution of a residue can be tested, for example, in binding assays, e.g., in mutagenesis studies performed to identify sufficient and / or necessary residues required for the repeat domain to bind to the target with its original binding affinity or amount (i.e., its binding affinity or amount in the absence of any mutations). Target interaction residues can further be determined by structural analysis of the repeat domain bound to a target.

[0046] The term "framework residues" refers to amino acid residues of a repeat module that contribute to the folding topology, i.e., contribute to the folding of the repeat module or contribute to interactions with neighboring modules. Such contributions may be interactions with other residues within the repeat module, or influence on the polypeptide backbone structure found in α-helices or β-sheets, or participation in amino acid stretches that form linear polypeptides or loops. Specifically, for the purposes of calculating (1) the percentage of basic amino acid residues among the framework residues comprised in a designed ankyrin repeat domain of the invention, or (2) the combined percentage of Arg and Lys residues among the framework residues comprised in a designed ankyrin repeat domain of the invention, the term "framework residue" includes amino acid residues present at positions in a designed ankyrin repeat domain corresponding to the positions listed in Table 4 (i.e., positions 1-3, 5-7, 9, 10, and 13-30 of SEQ ID NO: 43) for representative N-terminal capping modules, amino acid residues present at positions in a designed ankyrin repeat domain corresponding to the positions listed in Table 4 (i.e., positions 1, 2, 5, 7-10, 12, 13, and 16-33 of SEQ ID NO: 48) for representative internal repeat modules, and amino acid residues present at positions in a designed ankyrin repeat domain corresponding to the positions listed in Table 4 (i.e., positions 1, 2, 5, 7-13, and 16-28 of SEQ ID NO: 54) for representative C-terminal capping modules.The term "framework residues" does not include amino acid residues present at positions in designed ankyrin repeat domains corresponding to the positions listed in Table 5 for representative N-terminal capping modules (i.e., positions 4, 8, 11 and 12 of SEQ ID NO: 43), amino acid residues present at positions in designed ankyrin repeat domains corresponding to the positions listed in Table 5 for representative internal repeat modules (i.e., positions 3, 4, 6, 11, 14 and 15 of SEQ ID NO: 48), and amino acid residues present at positions in designed ankyrin repeat domains corresponding to the positions listed in Table 5 for representative C-terminal capping modules (i.e., positions 3, 4, 6, 14 and 15 of SEQ ID NO: 54).

[0047] Such framework and target interaction residues can be identified by analysis of structural data obtained by physicochemical methods such as X-ray crystallography, NMR and / or CD spectroscopy, or by comparison with known relevant structural information well known to those skilled in the art in structural biology and / or bioinformatics.

[0048] The terms "binding specificity," "having binding specificity for a target," "specifically binds to a target," "binds with high specificity to a target," "specific for a target," or "target specificity," etc., mean that a binding protein or binding domain binds to a target with a lower dissociation constant (i.e., binds with higher affinity) than it binds to an unrelated protein, such as E. coli maltose binding protein (MBP). Preferably, the dissociation constant for the target ("K D ") is at least 10 times larger than the corresponding dissociation constant for MBP. 2 times, more preferably at least 10 3 times, more preferably at least 10 4 times, or more preferably at least 10 5Methods for measuring the dissociation constant of a protein-protein interaction, such as surface plasmon resonance (SPR)-based techniques (e.g., SPR equilibrium analysis) or isothermal titration calorimetry (ITC), are well known to those skilled in the art. The K of a particular protein-protein interaction D Measurements of K may vary when measured under different conditions (e.g., salt concentration, pH). D The determination of the values is preferably carried out using a standardized solution of the protein and a standardized buffer solution such as PBS.

[0049] The binding of any molecule to another molecule is determined by two forces: the association rate (k on ) and dissociation rate (k off ) is then governed by the equilibrium dissociation constant K D This can be expressed as k off / k on is the quotient of.

[0050]

number

[0051] k on is in units of M -1 s -1 is the second order rate constant for the binding reaction with k, and for the dissociation reaction with k off is in units of s -1 This reveals that while the association reaction depends on the reactant concentrations, the dissociation reaction is independent of the concentrations and follows a simple exponential decay function.

[0052] Various methods for measuring binding affinity are well known in the art, any of which can 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 can be determined by the K D It can be expressed as a value, K, which refers to the dissociation constant of the binding moiety and the drug molecule target. D is the "off rate (k off) and the association rate or "on rate (k on )" Therefore, K D is K off / K on is expressed as a molar concentration (M), and K D The smaller the value, the stronger the binding affinity.

[0053] K D The value can be determined using any suitable method. D One exemplary method for measuring K is surface plasmon resonance (SPR) (see, e.g., Nguyen et al., Sensors (Basel). 2015 May 5;15(5):10481-510). D The K value may be measured by SPR using a biosensor system such as the BIACORE® system. BIAcore kinetic analysis involves, for example, analyzing the binding and dissociation of antigens from chips having immobilized molecules (e.g., molecules containing epitope-binding domains) on their surfaces. D Another method for determining K is to use Bio-Layer Interferometry (e.g., Shah et al., J Vis Exp. 2014;(84):51383). D The K value can be measured using OCTET® technology (Octet QKe system, ForteBio). Alternatively or additionally, the KinExA® (kinetic exclusion assay) assay available from Sapidyne Instruments (Boise, Id.) can be used. Any method suitable for assessing binding affinity between two binding partners is encompassed herein. Surface plasmon resonance (SPR) is particularly preferred. Most preferably, the K D Values are determined in PBS and by SPR.

[0054] The term "isoelectric point" or "pI" refers to the pH value at which a macromolecule such as a protein has no net charge. In a protein, there may be many charged groups, but at the isoelectric point, the sum of all these charges is zero. At pH values above the isoelectric point, the total net charge of a polypeptide is negative, while at pH values below the isoelectric point, the total net charge of a polypeptide is positive. The isoelectric point can be determined experimentally or calculated for a polypeptide based on its primary sequence. Those skilled in the art are aware of how to determine 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 are available online that allow for calculating the isoelectric point of a protein. One such preferred tool is "ExPASy Compute pI / Mw" (https: / / web.expasy.org / compute_pi / ); Protein Identification and Analysis Tools on the ExPASy Server; see 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 for determining the pI of ankyrin repeat domains of the present invention. Any N- or C-terminal tags comprising one or more amino acids that may be fused to the repeat domain for production or other purposes, as well as any N- or C-terminal peptide linkers, are not taken into account for calculating the pI of the repeat domains of the present invention. Such tags or linkers are well known in the art and include, for example, the His6-TEV tag of SEQ ID NO: 13 (N-terminus), a GS residue (N-terminus), and the GSGSC tag of SEQ ID NO: 14 (C-terminus), as shown in Figure 3.

[0055] The term "basic amino acid" refers to a hydrophilic amino acid with a positively charged side chain at physiological pH. Among 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. Among the 20 common amino acids, Asp (D) and Glu (E) are acidic amino acids (see Table 1). Basic and acidic amino acids can also be collectively referred to as charged amino acids because their side chains are ionized at physiological pH. A "neutral amino acid" refers to an amino acid that is neither basic nor acidic and therefore does not effectively ionize under physiological conditions. Fifteen of the 20 common amino acids are neutral amino acids (see Table 1). Such considerations are well known to those skilled in the art.

[0056] The term "drug moiety" refers to a chemical moiety that is attached or suitable for attachment to a protein, in particular a designed repeat domain or a designed repeat protein, and includes any therapeutic or diagnostic agent having desired therapeutic or diagnostic properties, respectively. As used herein, drug moiety is intended to encompass the terms "therapeutic moiety" and "diagnostic moiety". Such moieties can be attached to the repeat domains or repeat proteins of the invention using methods available in the art or as described, for example, in Example 3.

[0057] The term "therapeutic moiety" refers to a chemical moiety that can function as a therapeutic agent (or perform a therapeutic function) when administered or otherwise provided to a patient or subject, such as for the treatment of a disease or disorder.

[0058] The term "diagnostic moiety" refers to a chemical moiety that, when administered or otherwise provided to a patient or subject, can function as a diagnostic agent (or perform a diagnostic function), such as for diagnosing a disease or disorder.

[0059] The term "linked" or "linkage" refers to any covalent or non-covalent bond between a chemical moiety and a protein, such as a designed repeat domain or a designed repeat protein.

[0060] The term "toxin" refers to any agent that is detrimental to the growth, proliferation, and / or survival of a cell and can act to reduce, inhibit, kill, and / or destroy a cell or malignancy. This term encompasses, for example, radionuclides (which can be toxic because of their radioactivity) and cytotoxic agents.

[0061] The term "cytotoxic agent" or "cytotoxin" refers to a substance that causes cell death or toxicity primarily by interfering with cellular life processes (e.g., 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 nucleases, antibiotics, toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, auristatins, calicheamicins, maytansinoids, and camptothecin analogs. Further non-limiting examples of cytotoxins are cytotoxins that can be used in antibody-drug conjugates, such as those described in Drago, Joshua Z., Shanu Modi, and Sarat Chandarlapaty. Nature Reviews Clinical Oncology 18.6 (2021).

[0062] The term "radionuclide" or "radioisotope" refers to an isotope, of natural or artificial origin, that has an unstable neutron-to-proton ratio and decays with the emission of particles (i.e., protons (alpha radiation) or electrons (beta radiation)) or electromagnetic radiation (gamma radiation). In other words, a radionuclide undergoes radioactive decay. Such radionuclides include, but are not limited to, the following: 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 Dy. The selection of an appropriate radionuclide may depend on the chemical structure and chelating ability of the chelator, as well as the intended use of the resulting drug (e.g., diagnostic or therapeutic).

[0063] The term "chelator" or "chelating agent" refers to a multidentate (multiple-bonded) ligand that can form two or more separate coordinate bonds ("coordinate") with a central (metal) ion. Specifically, such molecules, or molecules that share one electron pair, are sometimes referred to as "Lewis bases." The central (metal) ion is usually coordinated to the chelating agent by two or more electron pairs. Typically, the electron pairs of the chelating agent form coordinate bonds with a single central (metal) ion, but in certain instances, the chelating agent may form coordinate bonds with two or more metal ions, and various bonding modes are possible. The terms "coordinate" and "coordination" refer to an interaction in which one multi-electron pair donor coordinates ("coordinates") with one central (metal) ion, i.e., shares two or more unshared electron pairs with one central (metal) ion. The chelating agent is preferably selected based on its ability to coordinate a desired central (metal) ion, usually a radionuclide as identified herein.

[0064] The term "percentage", e.g., as used in the percentage of basic amino acid residues, refers to 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- or C-terminal tags comprising one or more amino acids that may be fused to the repeat domain for production or other purposes, as well as any N- or C-terminal peptide linkers, are not taken into account for calculating such percentages. Such tags or linkers are well known in the art and include, for example, the His6-TEV tag of SEQ ID NO: 13 (N-terminus), the GS residue (N-terminus), and the GSGSC tag of SEQ ID NO: 14 (C-terminus), as shown in Figure 3.

[0065] The term "physiological conditions" refers to conditions normally present in a mammalian body. Thus, for example, in humans, physiological conditions mean a pH of 7.35 to 7.45, with an average of 7.40, and a temperature of 36.1°C to 37.2°C, with an average of 37°C.

[0066] Repeat domains of the present invention DARPins (designed ankyrin repeat proteins) are repeat proteins containing designed ankyrin repeat domains, which typically contain one or more internal repeat modules that are identical or similar except for their randomized positions and are flanked by N- and C-terminal capping repeat modules (e.g., Binz et al., 2003, cited above). As used herein, the term "repeat module" encompasses internal repeat modules and terminal repeat modules (C-cap and N-cap modules). While 27 of the 33 amino acid positions in a typical internal repeat module are highly conserved, the other six amino acid positions are less conserved and are mostly involved in the specific interaction of the ankyrin repeat domain with its target (Binz et al., 2003, cited above). The paratope of an ankyrin repeat domain is formed by a continuous surface primarily formed by these variable positions in the internal repeat modules and sometimes the capping repeat modules. DARPins also encompass proteins containing multiple designed ankyrin repeat domains linked together by a suitable linker. Such linkers are known to those skilled in the art.

[0067] Representative designed ankyrin repeat domains according to the invention have been generated and characterized as described in Examples 1-8 and are illustrated in FIGS. 1 and 8.

[0068] Thus, in one aspect, the present invention provides designed ankyrin repeat domains which have a reduced number of basic amino acid residues and / or a reduced pI compared to conventional designed ankyrin repeat domains. As described in more detail below, the designed ankyrin repeat domains of the invention can be obtained by substitution of basic amino acids; methods for making such substitutions are well known in the art and include mutagenesis of cDNA encoding the described repeat domains.

[0069] The ankyrin repeat domains described herein generally comprise a core scaffold that provides structure and target-interacting residues that bind to the target. The structural core comprises conserved amino acid residues (also called framework residues), and the target-binding surface comprises target-interacting residues that vary depending on the target.

[0070] Table 1 shows typical properties of common amino acids considered herein.

[0071] [Table 1]

[0072] The categories of charge-modifying amino acid residue substitutions considered herein and their effect on charge are shown in Table 2.

[0073] [Table 2]

[0074] 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.

[0075] [Table 3]

[0076] In some embodiments, the designed ankyrin repeat domains of the invention have a pI of pH 4.9 or less, pH 4.8 or less, pH 4.7 or less, pH 4.6 or less, pH 4.5 or less, pH 4.4 or less, pH 4.3 or less, pH 4.2 or less, pH 4.1 or less, pH 4.0 or less, pH 3.9 or less, pH 3.8 or less, pH 3.7 or less, pH 3.6 or less, or pH 3.5 or less.

[0077] In some embodiments, the designed ankyrin repeat domains of the invention have a pI of pH 4.53 or less, pH 4.52 or less, pH 4.50 or less, pH 4.48 or less, pH 4.46 or less, pH 4.44 or less, pH 4.42 or less, pH 4.40 or less, pH 4.38 or less, pH 4.36 or less, pH 4.34 or less, pH 4.32 or less, pH 4.30 or less, pH 4.28 or less, pH 4.26 or less, pH 4.24 or less, pH 4.22 or less, pH 4.20 or less, pH 4.18 or less, pH 4.16 or less, pH 4.14 or less, pH 4.12 or less, pH 4.10 or less, pH 4.08 or less, pH 4.0 6 or less, pH 4.04 or less, pH 4.02 or less, pH 4.00 or less, pH 3.98 or less, pH 3.96 or less, pH 3.94 or less, pH 3.92 or less, pH 3.90 or less, pH 3.88 or less, pH 3.86 or less, pH 3.84 or less, pH 3.82 or less, pH 3.80 or less, pH 3.78 or less, pH 3.76 or less, pH 3.74 or less, pH 3.72 or less, pH 3.70 or less, pH 3.68 or less, pH 3.66 or less, pH 3.64 or less, pH 3.62 or less, pH 3.60 or less, pH 3.58 or less, pH 3.56 or less, pH 3.54 or less, pH 3.52 or less, pH 3.50 or less.

[0078] In some embodiments, the designed ankyrin repeat domain of the present invention has a pH in the range of 3.00 to 4.60, 3.10 to 4.60, 3.20 to 4.60, 3.30 to 4.60, 3.40 to 4.60, 3.50 to 4.60, 3.50 to 4.60, 3.50 to 4.58, 3.50 to 4.56, 3.50 to 4.54, 3.50 to 4.53, 3.50 to 4.52, 3.50 to 4.60. 50 range, pH 3.50 - pH 4.48 range, pH 3.50 - pH 4.46 range, pH 3.50 - pH 4.44 range, pH 3.50 - pH 4.42 range, pH 3.50 - pH 4.40 range, pH 3.50 - pH 4.38 range, pH 3.50 - pH 4.36 range, pH 3.50 - pH 4.34 range, pH 3.50 - pH 4.32 range, pH 3.50 - pH 4.30 range, pH 3.60 - pH 4.60 range, pH 3.60 - pH 4.58 range, pH 3.60 - pH 4.56 range, pH 3.60 - pH pH 4.54 range, pH 3.60 to pH 4.53 range, pH 3.60 to pH 4.52 range, pH 3.60 to pH 4.50 range, pH 3.60 to pH 4.48 range, pH 3.60 to pH 4.46 range, pH 3.60 to pH 4.44 range, pH 3.60 to pH 4.42 range, pH 3.60 to pH 4.40 range, pH 3.60 to pH 4.38 range, pH 3.60 to pH 4.36 range, pH 3.60 to pH 4.34 range, pH 3.60 to pH 4.32 range, pH 3.60 to pH 4.30 range, pH 3.70 Within pH 4.60, within pH 3.70 to pH 4.58, within pH 3.70 to pH 4.56, within pH 3.70 to pH 4.54, within pH 3.70 to pH 4.53, within pH 3.70 to pH 4.52, within pH 3.70 to pH 4.50, within pH 3.70 to pH 4.48, within pH 3.70 to pH 4.46, within pH 3.70 to pH 4.44, within pH 3.70 to pH 4.42, within pH 3.70 to pH 4.40, within pH 3.70 to pH 4.38, within pH 3.70 to pH 4.36, pH 3.The pH range is 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.

[0079] In some embodiments, the designed ankyrin repeat domain of the invention has a percentage of basic amino acid residues of 12.0% or less, 11.5% or less, 11.0% or less, 10.5% or less, 10.0% or less, 9.5% or less, or 9.0% or less.

[0080] In some embodiments, the designed ankyrin repeat domain of the invention has a percentage of basic amino acid residues 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, 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, 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, or 4.80% or less.

[0081] Histidine residues under physiological conditions typically have a partial positive charge. The effect of mutating His to a neutral or acidic residue on charge reduction is less pronounced than mutating Lys or Arg to a neutral or acidic residue. Unless otherwise specified, basic amino acid residues include Arg, Lys, and His.

[0082] In some embodiments, the designed ankyrin repeat domain of the invention has a percentage of Arg and Lys residues of 8.0% or less, 7.5% or less, 7.0% or less, 6.5% or less, or 6.0% or less.

[0083] For clarity, the term "percentage of Arg and Lys residues" as used in this disclosure is intended to mean the combined percentage of Arg and Lys residues.

[0084] In some embodiments, the designed ankyrin repeat domain of the invention has a percentage of Arg and Lys residues of 5.65%, 5.60%, 5.50%, 5.40%, 5.30%, 5.20%, 5.10%, 5.00%, 4.90%, 4.80%, 4.70%, 4.60%, 4.50%, 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% 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, 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, 0.10% or less.

[0085] Alternatively, the number of basic amino acid residues in a designed ankyrin repeat domain of the invention may be defined in terms of the position of the basic amino acid residue within the repeat domain. In some embodiments, the number of basic amino acids is considered only among the framework residues of the repeat domain. The position of the framework residues can be determined by one skilled in the art. Preferably, the framework residues shall correspond to residues occupying specific positions within the repeat modules listed in Table 4.

[0086] [Table 4]

[0087] In some embodiments, the designed ankyrin repeat domain of the invention has a percentage of basic amino acid residues among its 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, or 8.0% or less, 7.8% or less.

[0088] In some embodiments, the designed ankyrin repeat domain of the invention has a percentage of basic amino acid residues among its framework residues of 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, 6.70% or less, 6.80% or less, 6.9 ... 0.50% or less, 6.40% or less, 6.30% or less, 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.

[0089] In some embodiments, the designed ankyrin repeat domain of the invention has a percentage of Arg and Lys residues among its framework residues of 6.7% or less, 6.6% or less, 6.5% or less, 6.4% or less, 6.3% or less, 6.2% or less, 6.1% or less, 6.0% or less, or 5.9% or less.

[0090] In some embodiments, the designed ankyrin repeat domain of the invention has a percentage of Arg and Lys residues in its framework residues of 5.83%, 5.80%, 5.70%, 5.60%, 5.50%, 5.40%, 5.30%, 5.20%, 5.10%, 5.00%, 4.90%, 4.80%, 4.70%, 4.60%, 4.50%, 4.40%, 4.30%, 4.20%, 4.10%, 4.00%, 3.90%, 3.80%, 3.70%, 3.60%, 3.50%, 3.4 0% 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, 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 0.10% or less.

[0091] The designed ankyrin repeat domain may comprise an N-terminal capping module, a C-terminal capping module, and at least one internal repeat module. Preferably, the repeat domain of the invention comprises 1, 2, 3, 4, 5, 6, 7, 8 or 9 internal repeat modules. More preferably, the repeat domain of the invention comprises 1, 2, 3 or 4 internal repeat modules.

[0092] In some embodiments, the total number of basic amino acid residues contained in a designed ankyrin repeat domain of the invention can be expressed as a function of the number of repeat modules contained in the repeat domain. In one embodiment, an ankyrin repeat domain of the invention has a total number of basic amino acid residues equal to or less than n, where 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+3R , 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, where R is the number of internal repeat modules contained in the repeat domain. In such embodiments, "xR" means x multiplied by R.

[0093] In another embodiment, the total number of Arg and Lys residues contained in a designed ankyrin repeat domain of the invention is m or less, and 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, where R is the number of internal repeat modules contained in the repeat domain.

[0094] In some embodiments, the designed ankyrin repeat domain of the invention has a total number of basic amino acid residues in the framework residues that is less than or equal to x, where 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, where R is the number of internal repeat modules comprised in the repeat domain.

[0095] In other embodiments, the designed ankyrin repeat domain of the invention has a total number of Arg and Lys residues in the framework residues that is less than or equal to 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 comprised in the repeat domain.

[0096] Preferably, the designed ankyrin repeat domain of the present invention specifically binds to a target. In a preferred embodiment, the repeat domain of the present invention has a specific binding affinity of about 10 -5M or less, about 10 -6 M or less, about 10 -7 M or less, about 10 -8 M or less, about 10 -9 M or less, about 10 -10 M or less, about 10 -11 M or less, about 10 -12 M or less, about 10 -13 M or less, about 10 -14 The dissociation constant (K D ) to bind to the target.

[0097] In some embodiments, the target is HER2. Thus, in certain embodiments, the designed ankyrin repeat domain of the present invention specifically binds to HER2. As used herein, HER2 refers to human epidermal growth factor receptor 2, also known as Neu, ErbB-2, CD340 (cluster of differentiation 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 the UniProtKB / Swiss-Prot number P04626.

[0098] In one embodiment, the invention provides an engineered 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 exhibits the following list of features: (a) isoelectric point (pI) of pH 4.6 or less; (b) the percentage of basic amino acid residues among all amino acid residues contained in the repeat domain is 12.0% or less; (c) the percentage of Arg residues and Lys residues among all amino acid residues contained in the repeat domain is 8.0% or less; (d) a percentage of basic amino acid residues of 9.6% or less among framework residues contained in the repeat domain; and (e) Among the framework residues contained in the repeat domain, the percentage of Arg and Lys residues is 6.7% or less.

[0099] In a preferred embodiment, the repeat domain is linked to a drug moiety.

[0100] The present invention encompasses embodiments in which the repeat domain has any combination of features (a)-(e) above. 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 characteristics (a), (b), (c), (d), and (e). In some preferred embodiments, the repeat domain has an isoelectric point (pI) within the range of pH 3.0 to pH 4.6, preferably pH 3.3 to pH 4.6, more preferably pH 3.50 to pH 4.53. In some preferred embodiments, the percentage in (b) is 8.7% or less. In some most preferred embodiments, the percentage in (b) is 8.06% or less. In some preferred embodiments, the percentage in (c) is 4.7% or less. In some most preferred embodiments, the percentage in (c) is 4.03% or less. In some preferred embodiments, the percentage in (d) is 6.7% or less. In some most preferred embodiments, the percentage in (d) is 6.1% or less. In some preferred embodiments, the percentage in (e) is 5.7% or less. In some most preferred embodiments, the percentage in (e) is 4.85% or less.

[0101] In some embodiments, the repeat domain has a total number of basic amino acid residues equal to or less than n, where 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+3R, n=4 R, 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, where R is the number of internal repeat modules contained in the repeat domain. In some more particular embodiments, n=4+5R. In such embodiments, "xR" means x multiplied by R.

[0102] In some embodiments the repeat domain has a total number of Arg and Lys residues that is m or less, where 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= 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, where R is the number of internal repeat modules contained in the repeat domain. In some more particular embodiments, m=5+2R.

[0103] In some embodiments, the repeat domain has a total number of basic amino acid residues in the framework residues that is less than or equal to x, where 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, where R is the number of internal repeat modules comprised in the repeat domain.

[0104] In some embodiments the repeat domain has a total number of Arg and Lys residues in the framework residues that is y or less, 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 comprised in the repeat domain.

[0105] In some embodiments, the repeat domain has no Arg or Lys residues in the framework residue positions.

[0106] In some embodiments, the repeat domain comprises two internal repeat modules. In some embodiments, a repeat domain comprising two internal repeat modules may have a total number of basic amino acid residues of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 or less. Preferably, the total number of basic residues is 14 or less. More preferably, the total number of basic residues is 10 or less. In another particular embodiment, a repeat domain comprising two internal repeat modules may have a total number of Arg and Lys residues of 1, 2, 3, 4, 5, 6, 7, 8, or 9 or less. Preferably, the total number of Arg and Lys residues is 9 or less. More preferably, the total number of Arg and Lys residues is 5 or less. In another particular embodiment, a repeat domain comprising two internal repeat modules may have a total number of Arg and Lys residues in the framework residues of 1, 2, 3, 4, 5, or 6 or less. Preferably, the total number of Arg and Lys residues in the framework residues is 6 or less. More preferably, the total number of Arg and Lys residues in the framework residues is 5 or less.

[0107] In another embodiment, the invention provides an engineered 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 exhibits the following list of features: (a) isoelectric point (pI) of pH 4.07 or less; (b) the percentage of basic amino acid residues among all amino acid residues contained in the repeat domain is 7.0% or less; and (c) the percentage of basic amino acid residues among the framework residues contained in the repeat domain is 6.1% or less.

[0108] The present invention encompasses embodiments in which the repeat domain has any combination of features (a)-(c) above. Thus, in some embodiments, the repeat domain has at least features (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).

[0109] In some embodiments, the repeat domain comprises about 10 -5 M or less, about 10 -6 M or less, about 10 -7 M or less, about 10 -8 M or less, about 10 -9 M or less, about 10 -10 M or less, about 10 -11 M or less, about 10 -12 M or less, about 10 -13 M or less, or about 10 -14 The dissociation constant (K D ) binds to the target. In a preferred embodiment, the repeat domain has 10 -7 A dissociation constant (K D ) to bind to the target.

[0110] Alternatively or additionally, the charge characteristics of the designed ankyrin repeat domains according to the present invention can be defined by the ratio of the sum of Arg (R) and Lys (K) residues to 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 can be calculated based on the amino acid sequence of the designed ankyrin repeat domain of the present invention.

[0111] Thus, in some embodiments, a designed ankyrin repeat domain 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 0.08 or less. Preferably, the KR / DE ratio is 0.3 or less.

[0112] Unless otherwise specified, the KR / DE ratio is determined by considering all residues contained in the repeat domain. In other embodiments, the KR / DE ratio is calculated only among the framework residues of the repeat domain. Framework residues are defined in Table 4 as residues in a repeat module that correspond to a specific position within the repeat module of a reference sequence. Thus, in some embodiments, a designed ankyrin repeat domain of the invention has a KR / DE ratio among framework residues 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. Preferably, the KR / DE ratio among framework residues is 0.25 or less.

[0113] In a further embodiment, the KR / DE ratio is calculated among all residues contained in the N-terminal capping module only. Thus, in some embodiments, a designed ankyrin repeat domain of the invention has a KR / DE ratio between all residues of the N-terminal capping module of 0.66 or less, 0.64 or less, 0.62 or less, 0.60 or less, 0.58 or less, 0.56 or less, 0.54 or less, 0.52 or less, 0.50 or less, 0.48 or less, 0.46 or less, 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, 0.08 or less, 0.06 or less, 0.04 or less, or 0.02 or less. Preferably, the KR / DE ratio among all residues of the N-terminal capping module is 0.5 or less.

[0114] In some embodiments, a designed ankyrin repeat domain of the invention comprises one or more internal repeat modules, each internal repeat module independently comprising a sequence selected from the group consisting of: (1) SEQ ID NOs: 131-142, and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue in any of SEQ ID NOs: 131-142 is substituted by another amino acid.

[0115] In one embodiment, a designed ankyrin repeat domain of the invention comprises one or more internal repeat modules, each internal repeat module independently comprising a sequence selected from the group consisting of: (1) SEQ ID NOs: 131, 135, and 139; and (2) sequences in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue in any of SEQ ID NOs: 131, 135, and 139 is substituted by another amino acid.

[0116] In another embodiment, a designed ankyrin repeat domain of the invention comprises one or more internal repeat modules, each internal repeat module independently comprising a sequence selected from the group consisting of: (1) SEQ ID NO: 131, and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue in SEQ ID NO: 131 is substituted by another amino acid.

[0117] Instead of or in addition to the internal repeat module defined above, in some embodiments the designed ankyrin repeat domain of the invention comprises an N-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID NOs: 25 and 115-122, and (2) sequences in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue in any of SEQ ID NOs: 25 and 115-122 is replaced by another amino acid.

[0118] 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, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in either of SEQ ID NOs: 115 and 121 is substituted by another amino acid.

[0119] 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, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in SEQ ID NO: 115 is substituted by another amino acid.

[0120] Alternatively or in addition to the internal repeat module and / or the N-terminal capping module defined above, in some embodiments the designed ankyrin repeat domain of the invention comprises a C-terminal capping module, which comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 123-130, and (2) a sequence in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in any of SEQ ID NOs: 123-130 is replaced by another amino acid.

[0121] 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, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in either of SEQ ID NOs: 123 and 126 is substituted by another amino acid.

[0122] 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, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in SEQ ID NO: 123 is substituted by another amino acid.

[0123] In one particular embodiment, the designed ankyrin repeat domain of the invention comprises: (a) 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, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in either of SEQ ID NOs: 115 and 121 is substituted by another amino acid; (b) 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, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in either of SEQ ID NOs: 123 and 126 is replaced by another amino acid.

[0124] In another embodiment, the designed ankyrin repeat domain of the invention comprises: (a) 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, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in SEQ ID NO: 115 is substituted by another amino acid; (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, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in SEQ ID NO: 123 is substituted by another amino acid.

[0125] In one particular embodiment, the designed ankyrin repeat domain of the invention comprises: (a) 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, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in either of SEQ ID NOs: 115 and 121 is substituted by another amino acid; (b) one or more internal repeat modules, each internal repeat module independently comprising 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, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue in any of SEQ ID NOs: 131, 135, and 139 is substituted by another amino acid.

[0126] In another embodiment, the designed ankyrin repeat domain of the invention comprises: (a) 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, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in SEQ ID NO: 115 is substituted by another amino acid; (b) one or more internal repeat modules, each internal repeat module independently having a sequence selected from the group consisting of: (1) SEQ ID NO: 131; and (2) a sequence in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in SEQ ID NO: 131 is substituted by another amino acid.

[0127] In one particular embodiment, the designed ankyrin repeat domain of the invention comprises: (a) 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, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in either of SEQ ID NOs: 123 and 126 is substituted by another amino acid; (b) one or more internal repeat modules, each internal repeat module independently comprising 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, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue in any of SEQ ID NOs: 131, 135, and 139 is substituted by another amino acid.

[0128] In another embodiment, the designed ankyrin repeat domain of the invention comprises: (a) 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, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in SEQ ID NO: 123 is substituted by another amino acid; (b) one or more internal repeat modules, each internal repeat module independently having a sequence selected from the group consisting of: (1) SEQ ID NO: 131; and (2) a sequence in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in SEQ ID NO: 131 is substituted by another amino acid.

[0129] In one particular embodiment, the designed ankyrin repeat domain of the invention comprises: (a) 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, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in either of SEQ ID NOs: 115 and 121 is substituted by another amino acid; (b) one or more internal repeat modules, each internal repeat module independently comprising 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, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue in any of SEQ ID NOs: 131, 135, and 139 is substituted by another amino acid. (c) 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, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in either of SEQ ID NOs: 123 and 126 is replaced by another amino acid.

[0130] In one embodiment, the designed ankyrin repeat domain of the invention comprises: (a) 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, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in SEQ ID NO: 115 is substituted by another amino acid; (b) one or more internal repeat modules, each internal repeat module having a sequence independently selected from the group consisting of: (1) SEQ ID NO: 131, and (2) a sequence in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in SEQ ID NO: 131 is substituted by another amino acid; and (c) 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, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in SEQ ID NO: 123 is substituted by another amino acid.

[0131] In one embodiment, the designed ankyrin repeat domain of the invention comprises: (a) 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 nine framework residues in SEQ ID NO: 115 are replaced by another amino acid; (b) one or more internal repeat modules, each internal repeat module independently having a sequence selected from the group consisting of: (1) SEQ ID NO: 131; and (2) SEQ ID NO: 131, in which up to nine framework residues are substituted by another amino acid. (c) 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, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in SEQ ID NO: 123 is substituted by another amino acid.

[0132] In one embodiment, the designed ankyrin repeat domain of the invention comprises: (a) 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 five framework residues in SEQ ID NO: 115 are replaced by another amino acid; (b) one or more internal repeat modules, each internal repeat module independently having a sequence selected from the group consisting of: (1) SEQ ID NO: 131, and (2) SEQ ID NO: 131 in which up to five framework residues are substituted by another amino acid; and (c) 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, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in SEQ ID NO: 123 is substituted by another amino acid.

[0133] In one embodiment, the designed ankyrin repeat domain of the invention comprises: (a) 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 three framework residues in SEQ ID NO: 115 are replaced by another amino acid; (b) one or more internal repeat modules, each internal repeat module independently having a sequence selected from the group consisting of: (1) SEQ ID NO: 131, and (2) a sequence in which up to three framework residues in SEQ ID NO: 131 are substituted by another amino acid; and (c) 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, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in SEQ ID NO: 123 is substituted by another amino acid.

[0134] In one embodiment, the designed ankyrin repeat domain of the invention comprises: (a) 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 nine framework residues in SEQ ID NO: 115 are replaced by another amino acid; (b) one or more internal repeat modules, each internal repeat module having a sequence independently selected from the group consisting of: (1) SEQ ID NO: 131, and (2) a sequence in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in SEQ ID NO: 131 is substituted by another amino acid; and (c) 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 nine framework residues in SEQ ID NO: 123 are replaced by another amino acid.

[0135] In one embodiment, the designed ankyrin repeat domain of the invention comprises: (a) 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 five framework residues in SEQ ID NO: 115 are replaced by another amino acid; (b) one or more internal repeat modules, each internal repeat module having a sequence independently selected from the group consisting of: (1) SEQ ID NO: 131, and (2) a sequence in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in SEQ ID NO: 131 is substituted by another amino acid; and (c) 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 five framework residues in SEQ ID NO: 123 are replaced by another amino acid.

[0136] In one embodiment, the designed ankyrin repeat domain of the invention comprises: (a) 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 three framework residues in SEQ ID NO: 115 are replaced by another amino acid; (b) one or more internal repeat modules, each internal repeat module having a sequence independently selected from the group consisting of: (1) SEQ ID NO: 131, and (2) a sequence in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in SEQ ID NO: 131 is substituted by another amino acid; and (c) 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 three framework residues in SEQ ID NO: 123 are replaced by another amino acid.

[0137] In one embodiment, the designed ankyrin repeat domain of the invention comprises: (a) 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 two framework residues in SEQ ID NO: 115 are replaced by another amino acid; (b) one or more internal repeat modules, each internal repeat module having a sequence independently selected from the group consisting of: (1) SEQ ID NO: 131, and (2) a sequence in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in SEQ ID NO: 131 is substituted by another amino acid; and (c) 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 two framework residues in SEQ ID NO: 123 are replaced by another amino acid.

[0138] In one embodiment, the designed ankyrin repeat domain of the invention comprises: (a) 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 one framework residue in SEQ ID NO: 115 is replaced by another amino acid; (b) one or more internal repeat modules, each internal repeat module having a sequence independently selected from the group consisting of: (1) SEQ ID NO: 131, and (2) a sequence in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in SEQ ID NO: 131 is substituted by another amino acid; and (c) 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 one amino acid of SEQ ID NO: 123 is replaced by another amino acid.

[0139] In one embodiment, the designed ankyrin repeat domain of the invention comprises: (a) an N-terminal capping module having the sequence of SEQ ID NO: 115; (b) one or more internal repeat modules, each internal repeat module having a sequence independently selected from the group consisting of: (1) SEQ ID NO: 131, and (2) a sequence in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in SEQ ID NO: 131 is substituted by another amino acid; and (c) a C-terminal capping module having the sequence of SEQ ID NO: 123.

[0140] In one embodiment, the designed ankyrin repeat domain of the invention comprises: (a) 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, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in SEQ ID NO: 115 is substituted by another amino acid; (b) one or more internal repeat modules, each internal repeat module independently having a sequence selected from the group consisting of: (1) SEQ ID NO: 131; and (2) SEQ ID NO: 131, in which up to nine framework residues are substituted by another amino acid. (c) 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 nine framework residues in SEQ ID NO: 123 are replaced by another amino acid.

[0141] In one embodiment, the designed ankyrin repeat domain of the invention comprises: (a) 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, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in SEQ ID NO: 115 is substituted by another amino acid; (b) one or more internal repeat modules, each internal repeat module independently having a sequence selected from the group consisting of: (1) SEQ ID NO: 131, and (2) SEQ ID NO: 131 in which up to five framework residues are substituted by another amino acid; and (c) 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 five framework residues in SEQ ID NO: 123 are replaced by another amino acid.

[0142] In one embodiment, the designed ankyrin repeat domain of the invention comprises: (a) 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, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in SEQ ID NO: 115 is substituted by another amino acid; (b) one or more internal repeat modules, each internal repeat module independently having a sequence selected from the group consisting of: (1) SEQ ID NO: 131, and (2) a sequence in which up to three framework residues in SEQ ID NO: 131 are substituted by another amino acid; and (c) 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 three framework residues in SEQ ID NO: 123 are replaced by another amino acid.

[0143] In a further embodiment, the designed ankyrin repeat domain comprises: one or more internal repeat modules, independently selected from the group consisting of: (1) SEQ ID NO: 31; and (2) sequences in which up to 5, 4, 3, 2, or 1 framework residue other than positions 1, 10, 13, 17, 19, 21, 22, and 26 of SEQ ID NO: 31 is substituted with another amino acid; and / or an N-terminal capping module, in which up to 5, 4, 3, 2, or 1 framework residue other than positions 1, 10, 13, 17, 19, 21, 22, and 26 of SEQ ID NO: 31 is substituted with another amino acid; and (2) a C-terminal capping module comprising a sequence selected from the group consisting of sequences in which up to 5, 4, 3, 2, or 1 framework residues other than positions 10, 11, 17, 18, 19, 22, and 26 of SEQ ID NO: 40 are substituted with another amino acid.

[0144] In certain embodiments, the designed ankyrin repeat domain of the invention comprises a sequence selected from SEQ ID NOs: 19-25, 57-60, 65-81, 31-35, 61, 62, 92-94, 40-42, 63, 64, and 82-91.

[0145] In certain embodiments, each internal repeat module comprised in a designed ankyrin repeat domain of the invention may independently comprise a sequence selected from SEQ ID NOs: 31-35, 61-62, and 92-94. Alternatively or additionally, an N-terminal capping module comprised in a designed ankyrin repeat domain of the invention may comprise a sequence selected from SEQ ID NOs: 19-25, 57-60, and 65-81. Alternatively or additionally, a C-terminal capping module comprised in an ankyrin repeat domain of the invention may comprise a sequence selected from SEQ ID NOs: 40-42, 63-64, and 82-91.

[0146] In certain embodiments, each internal repeat module comprised in a designed ankyrin repeat domain of the invention independently comprises a sequence selected from SEQ ID NOs: 31-35, 61-62, 92-94, and 131-142. Alternatively or additionally, an N-terminal capping module comprised in a designed ankyrin repeat domain of the invention comprises a sequence selected from SEQ ID NOs: 19-25, 57-60, 65-81, and 115-122. Alternatively or additionally, a C-terminal capping module comprised in an ankyrin repeat domain of the invention comprises a sequence selected from SEQ ID NOs: 40-42, 63, 64, 82-91, and 123-130.

[0147] In further embodiments, each internal repeat module comprised in a designed ankyrin repeat domain of the invention independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 31-35, 61, 62, and 92-94; and (2) sequences in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue in any of SEQ ID NOs: 31-35, 61, 62, and 92-94 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: 19-25, 57-60, and 65-81; and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue in any of SEQ ID NOs: 19-25, 57-60, and 65-81 is substituted with 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: 40-42, 63, 64, and 82-91; and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue in any of SEQ ID NOs: 40-42, 63, 64, and 82-91 is replaced by another amino acid.

[0148] In certain embodiments, each internal repeat module comprised in a designed ankyrin repeat domain of the invention independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 31 and 32; and (2) a sequence in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in any of SEQ ID NOs: 31 and 32 is substituted by another amino acid. Alternatively or additionally, an N-terminal capping module comprised in a designed ankyrin repeat domain of the invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 19 and 20; and (2) a sequence in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in any of SEQ ID NOs: 19 and 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 and 41, and (2) a sequence in which up to 9, or up to 8, or up to 7, or up to 6, or up to 5, or up to 4, or up to 3, or up to 2, or up to 1 framework residue in either SEQ ID NOs: 40 and 41 is substituted by another amino acid.

[0149] In further embodiments, each internal repeat module comprised in a 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) sequences in which 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids in any of SEQ ID NOs: 26 to 30 are substituted by other amino acids. Alternatively or additionally, an N-terminal capping module comprised in a 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) sequences in which 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids in any of SEQ ID NOs: 15 to 18 are substituted by other amino acids. Alternatively or additionally, a C-terminal capping module comprised in a 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) sequences in which 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids in any of SEQ ID NOs: 36 to 39 are substituted by other amino acids.

[0150] In further embodiments, each internal repeat module comprised in a designed ankyrin repeat domain of the present invention may further independently comprise a sequence selected from the group consisting of: (1) SEQ ID NOs: 46 to 52; and (2) sequences in which 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids in any of SEQ ID NOs: 46 to 52 are substituted by other amino acids. Alternatively or additionally, an N-terminal capping module comprised in a designed ankyrin repeat domain of the present invention may further comprise a sequence selected from the group consisting of: (1) SEQ ID NOs: 43 to 45; and (2) sequences in which 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids in any of SEQ ID NOs: 43 to 45 are substituted by other amino acids. Alternatively or additionally, the C-terminal capping module comprised in the ankyrin repeat domain of the present invention may further comprise a sequence selected from the group consisting of: (1) SEQ ID NOs: 53 to 56, and (2) sequences in which 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids in any of SEQ ID NOs: 53 to 56 are substituted with other amino acids.

[0151] In a further embodiment, each internal repeat module comprised in a designed ankyrin repeat domain of the invention independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 26-30, and 46-52, and (2) sequences in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 26-30, and 46-52 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: 15-18, 43-45, and 99-101; and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 15-18, 43-45, and 99-101 is substituted with 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: 36-39, 53-56, 102, and 103; and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 36-39, 53-56, 102, and 103 is substituted with another amino acid.

[0152] In a further 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: (1) SEQ ID NOs: 26-30, 47, and 48; and (2) sequences in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 26-30, 47, and 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: 15-18, 43, and 99-101; and (2) sequences in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 15-18, 43, and 99-101 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 an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 36 to 39, 54, 102, and 103; and (2) sequences in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 36 to 39, 54, 102, and 103 is substituted with another amino acid.

[0153] In a further embodiment, each internal repeat module comprised in a designed ankyrin repeat domain of the invention independently comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 47 and 48; and (2) sequences in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 47 and 48 is substituted by another amino acid. Alternatively or additionally, an N-terminal capping module comprised in a designed ankyrin repeat domain of the invention comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 43, 99, 100, and 101; and (2) sequences in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 43, 99, 100, 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, 102, and 103; and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 54, 102, and 103 is substituted by another amino acid.

[0154] In some embodiments, each internal repeat module comprised in a designed ankyrin repeat domain of the invention comprises one or more residues selected from the group consisting of: (i) Gln at position 1 and (ii) Glu at position 26, numbered relative to SEQ ID NO: 48.

[0155] In some embodiments, the N-terminal capping module comprised in the designed ankyrin repeat domain of the invention comprises one or more residues 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, numbered relative to SEQ ID NO: 43.

[0156] In some embodiments, the C-terminal capping module comprised in a designed ankyrin repeat domain of the invention comprises one or more residues 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, numbered relative to SEQ ID NO: 54.

[0157] In some embodiments, the designed ankyrin repeat domain of the invention comprises (1) an N-terminal capping module comprising one or more residues 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, numbered relative to SEQ ID NO: 43; and / or (2) one or more internal repeat modules, each internal repeat module independently comprising a repeat sequence selected from the group consisting of SEQ ID NO: 48. and / or (3) a C-terminal capping module numbered relative to SEQ ID NO: 54 and comprising one or more residues 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.

[0158] Thus, in one exemplary embodiment, the designed ankyrin repeat domain of the invention comprises: (1) an N-terminal capping module, numbered relative to SEQ ID NO: 43, comprising Thr at position 4, Gln at position 5, Leu at position 15, and Ala at position 23; (2) two internal repeat modules, each comprising Glu at position 26, numbered relative to SEQ ID NO: 48; and (3) a C-terminal capping module, numbered relative to SEQ ID NO: 54, 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.

[0159] In another 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 Cln at position 23, numbered relative to SEQ ID NO: 43; (2) two internal repeat modules, each comprising 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.

[0160] In further embodiments, the designed ankyrin repeat domain of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 104-114, 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 to any of SEQ ID NOs: 104-114. In one embodiment, preferably, the designed ankyrin repeat domain 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 to any of SEQ ID NOs: 104 and 112. In one embodiment, the designed ankyrin repeat domain 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 to SEQ ID NO: 104.

[0161] In a particular embodiment, the designed ankyrin repeat domain of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 2-5, 8-10, 12, and 95-98; 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: 2-5, 8-10, 12, and 95-98. 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: 2-5, 8-10, 12, and 95-98, and (2) a sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 2-5, 8-10, 12, and 95-98.

[0162] 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: 8-10, 12, and 95-98; 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: 8-10, 12, and 95-98. 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: 8-10, 12, and 95-98; and (2) a sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 8-10, 12, and 95-98.

[0163] 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: 8-10 and 95-98, 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: 8-10 and 95-98. 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: 8-10 and 95-98, and (2) a sequence having at least 80% amino acid identity to any one of SEQ ID NOs: 8-10 and 95-98.

[0164] 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, 9, and 95-98; 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, 9, and 95-98. 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: 8-9, and 95-98; and (2) a sequence having at least 80% amino acid identity to any one of SEQ ID NOs: 8-9, and 95-98.

[0165] 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: 95-98, 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: 95-98. 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: 95-98, and (2) a sequence having at least 80% amino acid identity to any one of SEQ ID NOs: 95-98.

[0166] In a particular embodiment, the designed ankyrin repeat domain of the 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 particular 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 about 80% amino acid sequence identity to SEQ ID NO: 12.

[0167] 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 identity to any one of SEQ ID NOs: 2 to 5.

[0168] In a particular embodiment, the designed ankyrin repeat domain of the invention comprises a sequence selected from (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 particular 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 identity to any one of SEQ ID NOs: 2, 3, and 5.

[0169] In a further embodiment, the designed ankyrin repeat domain of the invention comprises one or more residues 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, numbered relative to SEQ ID NO: 6.

[0170] Thus, in an exemplary embodiment, a 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.

[0171] In another exemplary embodiment, a designed ankyrin repeat domain of the 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, numbering relative to SEQ ID NO: 6.

[0172] Furthermore, any repeat domain sequence of the present invention may optionally comprise at its N-terminus G, S or GS. Furthermore, any repeat domain sequence of the present invention may optionally have an A in the penultimate position substituted with an L and / or an A in the last position substituted with an N.

[0173] In some embodiments, the designed ankyrin repeat domain of the present invention is linked to a drug moiety. In some embodiments, the drug moiety is covalently or non-covalently bound to the designed ankyrin repeat domain of the present invention. In a more specific embodiment, the drug moiety is linked to the designed ankyrin repeat domain of the present invention by a chelator. The appropriate chelator can be selected depending on the use of the repeat domain of the present invention. In a particular embodiment, the chelator is DTPA. In a more specific embodiment, the drug moiety is a radionuclide. The choice of radionuclide can depend on the intended use (e.g., diagnostic or therapeutic) of the designed ankyrin repeat domain of the present invention.

[0174] In one particular 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 one more particular embodiment, the radionuclide is indium-111.

[0175] In another particular embodiment, the drug moiety is a diagnostic moiety, hi a more particular embodiment, the diagnostic moiety is a fluorophore, chromophore, imaging agent, or radionuclide.

[0176] In another aspect, the present invention provides a designed ankyrin repeat domain obtainable or obtained by a method according to the invention as described below.

[0177] Manufacturing method according to the present invention Designed ankyrin repeat domains with reduced pI and / or reduced 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 at or below the thresholds described herein and / or a number of basic amino acids at or below the thresholds described herein.

[0178] The substitution of basic amino acid residues with neutral or acidic amino acid residues is of primary importance for generating engineered ankyrin repeat domains of the present invention with improved pharmacokinetic properties. However, considering that the substitution of neutral amino acid residues in proteins with acidic amino acid residues also typically induces a decrease in the isoelectric point (pI), such substitutions can, where appropriate, be made in a complementary manner to the substitution of basic amino acid residues with neutral or acidic amino acid residues.

[0179] Thus, in one aspect, the present invention provides a method for producing a modified ankyrin repeat domain, the method comprising steps (a) and (b), step (a) providing an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.07 and / or a percentage of basic amino acid residues among all amino acid residues contained in the repeat domain higher than 7.0% and / or a percentage of basic amino acid residues among framework residues contained in the repeat domain higher than 6.1%; and Step (b) comprises: (1) substituting 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) substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; The engineered ankyrin repeat domain has the following list of features: (i) an isoelectric point (pI) of pH 4.07 or less; (ii) the percentage of basic amino acid residues among all amino acid residues contained in the repeat domain is 7.0% or less; and (iii) the percentage of basic amino acid residues among the framework residues contained in the repeat domain is 6.1% or less.

[0180] For clarity, the pI and percentage values in step (a) of the method preferably correlate with the pI and percentage values in properties (i)-(iii), so that in some embodiments, if a different value is selected in one of properties (i)-(iii), the value can be applied to the corresponding feature of the repeat domain of step (a). Thus, if one of properties (i)-(iii) includes a feature that is "less than or equal to" X, the corresponding feature in step (a) must be "higher than" X.

[0181] For further clarity, at least one of the properties listed for the ankyrin repeat domain provided in step (a) is modified by step (b) such that the corresponding characteristics of (i) to (iii) are achieved.

[0182] In one particular embodiment, the method according to the invention comprises steps (a) and (b), step (a) providing an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.07 and / or a percentage of basic amino acid residues among all amino acid residues contained in the repeat domain higher than 7.0% and / or a percentage of basic amino acid residues among framework residues contained in the repeat domain higher than 6.1%; and Step (b) comprises: (1) substituting at least one basic amino acid residue in the repeat domain of step (a) with a neutral or acidic amino acid residue; and (2) substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; The engineered ankyrin repeat domain has the following list of features: (i) an isoelectric point (pI) of pH 4.07 or less; (ii) the percentage of basic amino acid residues among all amino acid residues contained in the repeat domain is 7.0% or less; and (iii) the percentage of basic amino acid residues among the framework residues contained in the repeat domain is 6.1% or less.

[0183] In another embodiment, the method according to the present invention comprises steps (a) and (b), step (a) providing an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.07 and / or a percentage of basic amino acid residues among all amino acid residues contained in the repeat domain higher than 7.0% and / or a percentage of basic amino acid residues among framework residues contained in the repeat domain higher than 6.1%; and step (b) is a step of substituting at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; The engineered ankyrin repeat domain has the following list of features: (i) an isoelectric point (pI) of pH 4.07 or less; (ii) the percentage of basic amino acid residues among all amino acid residues contained in the repeat domain is 7.0% or less; and (iii) the percentage of basic amino acid residues among the framework residues contained in the repeat domain is 6.1% or less.

[0184] In another embodiment, the method according to the present invention comprises steps (a) and (b), step (a) providing an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.07 and / or a percentage of basic amino acid residues among all amino acid residues contained in the repeat domain higher than 7.0% and / or a percentage of basic amino acid residues among framework residues contained in the repeat domain higher than 6.1%; and (2) step (b) is a step of substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; The engineered ankyrin repeat domain has the following list of features: (i) an isoelectric point (pI) of pH 4.07 or less; (ii) the percentage of basic amino acid residues among all amino acid residues contained in the repeat domain is 7.0% or less; and (iii) the percentage of basic amino acid residues among the framework residues contained in the repeat domain is 6.1% or less.

[0185] The present invention encompasses embodiments in which the modified repeat domain in the methods of the invention has any combination of features (i)-(iii) above. Thus, in some embodiments, the modified repeat domain has at least features (i), (ii), or (iii). In other embodiments, the modified repeat domain has at least features (i) and (ii), (i) and (iii), or (ii) and (iii). In other embodiments, the modified repeat domain has features (i), (ii), and (iii).

[0186] In some preferred embodiments, the modified repeat domain has an isoelectric point (pI) within the range of pH 3.0 to pH 4.07, preferably pH 3.3 to pH 4.07, more preferably pH 3.50 to pH 4.07.

[0187] In one embodiment, the method according to the present invention comprises steps (a) and (b), step (a) providing an ankyrin repeat domain with an isoelectric point (pI) higher than pH 4.07; and Step (b) comprises: (1) substituting 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) substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; The modified ankyrin repeat domain has an isoelectric point (pI) of pH 4.07 or less.

[0188] In a more particular embodiment, the method according to the invention comprises steps (a) and (b), step (a) providing an ankyrin repeat domain with an isoelectric point (pI) higher than pH 4.07; and Step (b) comprises: (1) substituting at least one basic amino acid residue in the repeat domain of step (a) with a neutral or acidic amino acid residue; and (2) substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; The modified ankyrin repeat domain has an isoelectric point (pI) of pH 4.07 or less.

[0189] In another particular embodiment, the method according to the invention comprises steps (a) and (b), step (a) providing an ankyrin repeat domain with an isoelectric point (pI) higher than pH 4.07; and step (b) is a step of substituting at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; The modified ankyrin repeat domain has an isoelectric point (pI) of pH 4.07 or less.

[0190] In another particular embodiment, the method according to the invention comprises steps (a) and (b), step (a) providing an ankyrin repeat domain with an isoelectric point (pI) higher than pH 4.07; and (2) step (b) is a step of substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; The modified ankyrin repeat domain has an isoelectric point (pI) of pH 4.07 or less.

[0191] In one embodiment, the method according to the present invention comprises steps (a) and (b), Step (a) is a step of providing an ankyrin repeat domain having a percentage of basic amino acid residues of more than 7.0% among all amino acid residues contained in the repeat domain; and step (b) comprising substituting at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; The modified ankyrin repeat domain has a percentage of basic amino acid residues of 7.0% or less among all amino acid residues contained in the repeat domain.

[0192] In a more particular embodiment, the method according to the invention comprises steps (a) and (b), Step (a) is a step of providing an ankyrin repeat domain having a percentage of basic amino acid residues of more than 7.0% among all amino acid residues contained in the repeat domain; and Step (b) comprises: (1) substituting at least one basic amino acid residue in the repeat domain of step (a) with a neutral or acidic amino acid residue; and (2) substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; The modified ankyrin repeat domain has a percentage of basic amino acid residues of 7.0% or less among all amino acid residues contained in the repeat domain.

[0193] In another more particular embodiment, the method according to the invention comprises steps (a) and (b), Step (a) is a step of providing an ankyrin repeat domain having a percentage of basic amino acid residues of more than 7.0% among all amino acid residues contained in the repeat domain; and step (b) is a step of substituting at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; The modified ankyrin repeat domain has a percentage of basic amino acid residues of 7.0% or less among all amino acid residues contained in the repeat domain.

[0194] In one embodiment, the method according to the present invention comprises steps (a) and (b), Step (a) is a step of providing 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 step (b) comprising substituting at least one basic amino acid residue with a neutral or acidic amino acid residue among the framework residues of the repeat domain of step (a); The modified ankyrin repeat domain has a percentage of basic amino acid residues of 6.1% or less among the framework residues contained in the repeat domain.

[0195] In a more particular embodiment, the method according to the invention comprises steps (a) and (b), Step (a) is a step of providing 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 Step (b) is (1) substituting at least one basic amino acid residue with a neutral or acidic amino acid residue among the framework residues of the repeat domain of step (a); and (2) substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; The modified ankyrin repeat domain has a percentage of basic amino acid residues of 6.1% or less among the framework residues contained in the repeat domain.

[0196] In another more particular embodiment, the method according to the invention comprises steps (a) and (b), Step (a) is a step of providing 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 step (b) is a step of substituting at least one basic amino acid residue with a neutral or acidic amino acid residue among the framework residues of the repeat domain of step (a); The modified ankyrin repeat domain has a percentage of basic amino acid residues of 6.1% or less among the framework residues contained in the repeat domain.

[0197] In another embodiment 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 less than or equal to 0.44.

[0198] In another embodiment of the method according to the invention, the repeat domain in step (a) has a KR / DE ratio among framework residues higher than 0.36 and the modified repeat domain has a KR / DE ratio of 0.36 or less.

[0199] In another embodiment of the method according to the invention, the repeat domain in step (a) has a KR / DE ratio among all residues of the N-terminal capping module higher than 0.66 and the modified repeat domain has a KR / DE ratio among all residues of the N-terminal capping module lower than or equal to 0.66.

[0200] In other embodiments of the method according to the invention, the repeat domain of step (a) has a KR / DE ratio higher than 0.44 and the modified repeat domain has a KR / DE ratio of 0.44 or less, and / or the repeat domain of step (a) has a KR / DE ratio among framework residues higher than 0.36 and the modified repeat domain has a KR / DE ratio of 0.36 or less, and / or the repeat domain of step (a) has a KR / DE ratio among all residues of the N-terminal capping module higher than 0.66 and the modified repeat domain has a KR / DE ratio among all residues of the N-terminal capping module of 0.66 or less.

[0201] 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): step (a) providing an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.6, and / or a percentage of basic amino acid residues among all amino acid residues contained in the repeat domain higher than 12.0%, and / or a percentage of Arg and Lys residues among all amino acid residues contained in the repeat domain higher than 8.0%, and / or a percentage of basic amino acid residues among framework residues contained in the repeat domain higher than 9.6%, and / or a percentage of Arg and Lys residues among framework residues contained in the repeat domain higher than 6.7%, and Step (b) comprises: (1) substituting 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) substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; The modified repeat domain has the following list of characteristics: (i) an isoelectric point (pI) of pH 4.6 or less; (ii) the percentage of basic amino acid residues among all amino acid residues contained in the repeat domain is 12.0% or less; (iii) the percentage of Arg residues and Lys residues among all amino acid residues contained in the repeat domain is 8.0% or less; (iv) the percentage of basic amino acid residues among the framework residues contained in the repeat domain is 9.6% or less; and (v) the percentage of Arg residues and Lys residues among the framework residues contained in the repeat domain is 6.7% or less.

[0202] For clarity, the pI and percentage values in step (a) of the method preferably correlate with the pI and percentage values in properties (i)-(v), so that in some embodiments, if a different value is selected in one of properties (i)-(v), the value can be applied to the corresponding feature of the repeat domain of step (a). Thus, if one of properties (i)-(v) includes a feature that is "less than or equal to" X, the corresponding feature in step (a) must be "higher than" X.

[0203] For further clarity, at least one of the properties listed for the ankyrin repeat domain provided in step (a) is modified by step (b) such that the corresponding features (i) to (v) are achieved.

[0204] In one embodiment, the method according to the present invention comprises steps (a) and (b), step (a) providing an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.6, and / or a percentage of basic amino acid residues among all amino acid residues contained in the repeat domain higher than 12.0%, and / or a percentage of Arg and Lys residues among all amino acid residues contained in the repeat domain higher than 8.0%, and / or a percentage of basic amino acid residues among framework residues contained in the repeat domain higher than 9.6%, and / or a percentage of Arg and Lys residues among framework residues contained in the repeat domain higher than 6.7%, and Step (b) comprises: (1) substituting at least one basic amino acid residue in the repeat domain of step (a) with a neutral or acidic amino acid residue; and (2) substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; The modified repeat domain has the following list of characteristics: (i) an isoelectric point (pI) of pH 4.6 or less; (ii) the percentage of basic amino acid residues among all amino acid residues contained in the repeat domain is 12.0% or less; (iii) the percentage of Arg residues and Lys residues among all amino acid residues contained in the repeat domain is 8.0% or less; (iv) the percentage of basic amino acid residues among the framework residues contained in the repeat domain is 9.6% or less; and (v) the percentage of Arg residues and Lys residues among the framework residues contained in the repeat domain is 6.7% or less.

[0205] In another embodiment, the method according to the present invention comprises steps (a) and (b), step (a) providing an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.6, and / or a percentage of basic amino acid residues among all amino acid residues contained in the repeat domain higher than 12.0%, and / or a percentage of Arg and Lys residues among all amino acid residues contained in the repeat domain higher than 8.0%, and / or a percentage of basic amino acid residues among framework residues contained in the repeat domain higher than 9.6%, and / or a percentage of Arg and Lys residues among framework residues contained in the repeat domain higher than 6.7%, and step (b) is a step of substituting at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; The modified repeat domain has the following list of characteristics: (i) an isoelectric point (pI) of pH 4.6 or less; (ii) the percentage of basic amino acid residues among all amino acid residues contained in the repeat domain is 12.0% or less; (iii) the percentage of Arg residues and Lys residues among all amino acid residues contained in the repeat domain is 8.0% or less; (iv) the percentage of basic amino acid residues among the framework residues contained in the repeat domain is 9.6% or less; and (v) the percentage of Arg residues and Lys residues among the framework residues contained in the repeat domain is 6.7% or less.

[0206] In a further embodiment, the method according to the invention comprises steps (a) and (b), step (a) providing an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.6, and / or a percentage of basic amino acid residues among all amino acid residues contained in the repeat domain higher than 12.0%, and / or a percentage of Arg and Lys residues among all amino acid residues contained in the repeat domain higher than 8.0%, and / or a percentage of basic amino acid residues among framework residues contained in the repeat domain higher than 9.6%, and / or a percentage of Arg and Lys residues among framework residues contained in the repeat domain higher than 6.7%, and (2) step (b) is a step of substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; The modified repeat domain has the following list of characteristics: (i) an isoelectric point (pI) of pH 4.6 or less; (ii) the percentage of basic amino acid residues among all amino acid residues contained in the repeat domain is 12.0% or less; (iii) the percentage of Arg residues and Lys residues among all amino acid residues contained in the repeat domain is 8.0% or less; (iv) the percentage of basic amino acid residues among the framework residues contained in the repeat domain is 9.6% or less; and (v) the percentage of Arg residues and Lys residues among the framework residues contained in the repeat domain is 6.7% or less.

[0207] The present invention encompasses embodiments in which the modified repeat domain in the methods of the invention has any combination of features (i)-(v) above. Thus, in some embodiments, the repeat domain has at least features (i), (ii), (iii), (iv), or (v). In other embodiments, the repeat domain has 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), (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).

[0208] In some preferred embodiments, the modified repeat domain has an isoelectric point (pI) within the range of pH 3.0 to pH 4.6, preferably pH 3.3 to pH 4.6, more preferably pH 3.50 to pH 4.53. In some preferred embodiments, the percentage in (ii) is 8.7% or less. In some most preferred embodiments, the percentage in (ii) is 8.06% or less. In some preferred embodiments, the percentage in (iii) is 4.7% or less. In some most preferred embodiments, the percentage in (iii) is 4.03% or less. In some preferred embodiments, the percentage in (iv) is 6.7% or less. In some most preferred embodiments, the percentage in (iv) is 6.1% or less. In some preferred embodiments, the percentage in (v) is 5.7% or less. In some most preferred embodiments, the percentage in (v) is 4.85% or less.

[0209] In one embodiment, the method according to the present invention comprises steps (a) and (b), step (a) providing an ankyrin repeat domain with an isoelectric point (pI) higher than pH 4.6; and Step (b) comprises: (1) substituting 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) substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; The modified repeat domain has an isoelectric point (pI) of pH 4.6 or less.

[0210] In a more particular embodiment, the method according to the invention comprises steps (a) and (b), step (a) providing an ankyrin repeat domain with an isoelectric point (pI) higher than pH 4.6; and Step (b) comprises: (1) substituting at least one basic amino acid residue in the repeat domain of step (a) with a neutral or acidic amino acid residue; and (2) substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; The modified repeat domain has an isoelectric point (pI) of pH 4.6 or less.

[0211] In another more particular embodiment, the method according to the invention comprises steps (a) and (b), step (a) providing an ankyrin repeat domain with an isoelectric point (pI) higher than pH 4.6; and step (b) is a step of substituting at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; The modified repeat domain has an isoelectric point (pI) of pH 4.6 or less.

[0212] In another more particular embodiment, the method according to the invention comprises steps (a) and (b), step (a) providing an ankyrin repeat domain with an isoelectric point (pI) higher than pH 4.6; and (2) step (b) is a step of substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; The modified repeat domain has an isoelectric point (pI) of pH 4.6 or less.

[0213] In one embodiment, the method according to the present invention comprises steps (a) and (b), Step (a) is a step of providing 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 step (b) comprising substituting at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; The modified repeat domain has a percentage of basic amino acid residues of 12.0% or less among all amino acid residues contained in the repeat domain.

[0214] In a more particular embodiment, the method according to the invention comprises steps (a) and (b), Step (a) is a step of providing 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 Step (b) comprises: (1) substituting at least one basic amino acid residue in the repeat domain of step (a) with a neutral or acidic amino acid residue; and (2) substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; The modified repeat domain has a percentage of basic amino acid residues of 12.0% or less among all amino acid residues contained in the repeat domain.

[0215] In another more particular embodiment, the method according to the invention comprises steps (a) and (b), Step (a) is a step of providing 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 step (b) is a step of substituting at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; The modified repeat domain has a percentage of basic amino acid residues of 12.0% or less among all amino acid residues contained in the repeat domain.

[0216] In one embodiment, the method according to the present invention comprises steps (a) and (b), Step (a) is a step of providing an ankyrin repeat domain having a percentage of Arg and Lys residues of more than 8.0% among all amino acid residues contained in the repeat domain; and step (b) comprising substituting at least one Arg and Lys residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; The modified repeat domain has a proportion of Arg residues and Lys residues of 8.0% or less among all amino acid residues contained in the repeat domain.

[0217] In a more particular embodiment, the method according to the invention comprises steps (a) and (b), Step (a) is a step of providing an ankyrin repeat domain having a percentage of Arg and Lys residues of more than 8.0% among all amino acid residues contained in the repeat domain; and wherein step (b) comprises: (1) substituting at least one Arg or Lys residue in the repeat domain of step (a) with a neutral or acidic amino acid residue; and (2) substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; The modified repeat domain has a proportion of Arg residues and Lys residues of 8.0% or less among all amino acid residues contained in the repeat domain.

[0218] In another more particular embodiment, the method according to the invention comprises steps (a) and (b), Step (a) is a step of providing an ankyrin repeat domain having a percentage of Arg and Lys residues of more than 8.0% among all amino acid residues contained in the repeat domain; and step (b) substituting at least one Arg and Lys residue in the repeat domain of step (a) with a neutral or acidic amino acid residue; The modified repeat domain has a proportion of Arg residues and Lys residues of 8.0% or less among all amino acid residues contained in the repeat domain.

[0219] In one embodiment, the method according to the present invention comprises steps (a) and (b), Step (a) is a step of providing 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 step (b) comprising substituting at least one basic amino acid residue of the repeat domain of step (a) with a neutral or acidic amino acid residue; The modified repeat domain has a percentage of basic amino acid residues of 9.6% or less among the framework residues contained in the repeat domain.

[0220] In a more particular embodiment, the method according to the invention comprises steps (a) and (b), Step (a) is a step of providing 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 Step (b) is (1) substituting at least one basic amino acid residue with a neutral or acidic amino acid residue among the framework residues of the repeat domain of step (a); and (2) substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; The modified repeat domain has a percentage of basic amino acid residues of 9.6% or less among the framework residues contained in the repeat domain.

[0221] In another more particular embodiment, the method according to the invention comprises steps (a) and (b), Step (a) is a step of providing 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 step (b) is a step of substituting at least one basic amino acid residue with a neutral or acidic amino acid residue among the framework residues of the repeat domain of step (a); The modified repeat domain has a percentage of basic amino acid residues of 9.6% or less among the framework residues contained in the repeat domain.

[0222] In one embodiment, the method according to the present invention comprises steps (a) and (b), Step (a) is to provide an ankyrin repeat domain having a percentage of Arg and Lys residues among the framework residues contained in the repeat domain that is higher than 6.7%; and step (b) comprising substituting at least one Arg or Lys residue among the framework residues of the repeat domain of step (a) with a neutral or acidic amino acid residue; The modified repeat domain has a percentage of Arg and Lys residues of 6.7% or less among the framework residues contained in the repeat domain.

[0223] In a more particular embodiment, the method according to the invention comprises steps (a) and (b), Step (a) is to provide an ankyrin repeat domain having a percentage of Arg and Lys residues among the framework residues contained in the repeat domain that is higher than 6.7%; and Step (b) is (1) substituting at least one Arg or Lys residue among the framework residues of the repeat domain of step (a) with a neutral or acidic amino acid residue; and (2) substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; The modified repeat domain has a percentage of Arg and Lys residues of 6.7% or less among the framework residues contained in the repeat domain.

[0224] In another more particular embodiment, the method according to the invention comprises steps (a) and (b), Step (a) is to provide an ankyrin repeat domain having a percentage of Arg and Lys residues among the framework residues contained in the repeat domain that is higher than 6.7%; and step (b) substituting at least one Arg or Lys residue among the framework residues of the repeat domain of step (a) with a neutral or acidic amino acid residue; The modified repeat domain has a percentage of Arg and Lys residues of 6.7% or less among the framework residues contained in the repeat domain.

[0225] In another embodiment 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 less than or equal to 0.44.

[0226] In another embodiment of the method according to the invention, the repeat domain in step (a) has a KR / DE ratio among framework residues higher than 0.36 and the modified repeat domain has a KR / DE ratio of 0.36 or less.

[0227] In another embodiment of the method according to the invention, the repeat domain in step (a) has a KR / DE ratio among all residues of the N-terminal capping module higher than 0.66 and the modified repeat domain has a KR / DE ratio among all residues of the N-terminal capping module lower than or equal to 0.66.

[0228] In other embodiments of the method according to the invention, the repeat domain of step (a) has a KR / DE ratio higher than 0.44 and the modified repeat domain has a KR / DE ratio of 0.44 or less, and / or the repeat domain of step (a) has a KR / DE ratio among framework residues higher than 0.36 and the modified repeat domain has a KR / DE ratio of 0.36 or less, and / or the repeat domain of step (a) has a KR / DE ratio among all residues of the N-terminal capping module higher than 0.66 and the modified repeat domain has a KR / DE ratio among all residues of the N-terminal capping module of 0.66 or less.

[0229] 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, or at least 15 residues of the repeat domain in step (a) are substituted.

[0230] 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 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%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, 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% are substituted. When such percentages are converted to the corresponding number of residues, conventional rounding of the calculated residues to the nearest integer may be used.

[0231] 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 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%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, 0%, 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% substituted. In the above embodiment, the wording per repeat module includes an N-terminal capping module, a C-terminal capping module, and / or an internal repeat module(s).

[0232] In one embodiment, the method according to the invention further comprises the step of linking a drug moiety to the modified repeat domain. In some embodiments, the drug moiety is covalently or non-covalently linked to the modified repeat domain. In a more particular embodiment, the drug moiety is linked to the modified repeat domain by a chelator. An appropriate chelator can be selected depending on the application. In a particular embodiment, the chelator is DTPA. In a more particular embodiment, the drug moiety is a radionuclide. The choice of radionuclide can depend on the intended application of the modified repeat domain (e.g., diagnostic or therapeutic).

[0233] 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 one more particular embodiment, the radionuclide is indium-111.

[0234] In other embodiments, the drug moiety is a diagnostic moiety. In a more particular embodiment, the diagnostic moiety is a fluorophore, chromophore, imaging agent, or radionuclide.

[0235] The residues selected for substitution can be present at randomized or non-randomized positions in the repeat domain. Thus, in some embodiments, the substituted residues are selected from among residues present at non-randomized positions in the repeat domain. In other embodiments, the substituted residues are selected from among residues present at randomized positions in the repeat domain. In some embodiments, the substituted residues are selected from among residues present at randomized and non-randomized positions in the repeat domain. In other embodiments, the residues selected for substitution can be framework residues or target interaction residues. Thus, in some embodiments, the substituted residues are selected from among framework residues contained in the repeat domain. In other aspects, the substituted residues are selected from among target interaction residues contained in the repeat domain. In other embodiments, the substituted residues are selected from among all residues contained in the repeat domain. All residues in this sense shall mean either target interaction residues or framework residues contained in the repeat domain. In some embodiments, the randomized positions in the repeat domain correspond to the positions of potential target interaction residues. In further embodiments, the non-randomized positions correspond to the positions of framework residues. Preferred positions of framework residues are shown in Table 4. Table 5 shows preferred positions of potential target interaction residues.

[0236] [Table 5]

[0237] In certain embodiments, the substitution occurs outside the structural core residues of the ankyrin repeat domain, for example, in the beta loop connecting the alpha helices. In certain embodiments, the substitution occurs 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, such as a consensus sequence selected from the following: XDXXGXTPLXXAXXXGXLXIXXVLLXAGADVNA (SEQ ID NO: 32), DLGXXLLXAAXXGQLDXVRXLLXAGADVNA (SEQ ID NO: 20), and QDXXGXTPAXXAAXXGXXXIAXVLQXAA (SEQ ID NO: 41), where "X" represents any amino acid. In one embodiment, the substitution occurs at the residue designated as "X". In another embodiment, the substitution occurs outside the residue designated as "X". In a preferred embodiment, the substituted amino acid is not cysteine, glycine, or proline. In some embodiments, the term "any amino acid" defined for a particular position "X" in the disclosed sequences of the present invention corresponds to "any naturally occurring amino acid."

[0238] The repeat domain in step (a) can be any repeat domain commonly known to those skilled in the art. Preferably, the repeat domain in step (a) is a designed ankyrin repeat domain. In some embodiments, the repeat domain in step (a) comprises an N-terminal capping module, one or more internal repeat modules, and a C-terminal capping module. Preferably, the repeat domain in 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 in step (a) may comprise a sequence selected from SEQ ID NOs: 19-25, 57-60, 65-81, 31-35, 61, 62, 92-94, 40-42, 63, 64, and 82-91. In some embodiments, each internal repeat module comprised in the repeat domain in step (a) may independently comprise a sequence selected from SEQ ID NOs: 31-35, 61, 62, and 92-94. 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, and 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, 64, and 82 to 91.

[0239] 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.

[0240] In one embodiment, the repeat domain of step (a) specifically binds to the target. In some embodiments, the repeat domain of step (a) specifically binds to the target. -5 M or less, about 10 -6 M or less, about 10 -7 M or less, about 10 -8 M or less, about 10 -9 M or less, about 10 -10 M or less, about 10 -11 M or less, about 10 -12M or less, about 10 -13 M or less, about 10 -14 The dissociation constant (K D ) and binds to the target.

[0241] In preferred embodiments, the modified repeat domains produced by the methods of the present invention maintain the binding properties of the repeat domain of step (a). Thus, in some embodiments, the modified repeat domain maintains the binding properties of the repeat domain of step (a). (1) The K D or (2) the K at which the repeat domain of step (a) binds to the target D K that is less than 100 times, less than 10 times, less than 5 times, or less than 2 times higher than D binds to the target.

[0242] In some embodiments, the modified repeat domain produced by the methods of the invention has an isoelectric point (pI) that is at least 0.1 pH points lower than the repeat domain of step (a), hi some embodiments, said 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).

[0243] In one embodiment, the modified repeat domain produced by the methods of the invention is for use in a drug, wherein the drug comprises the modified repeat domain linked to a drug moiety, which is optionally a radionuclide or a cytotoxic agent.

[0244] The properties described in connection with the designed ankyrin repeat domains of the invention are equally applicable to the modified repeat domains obtained by the methods of the invention.

[0245] In some embodiments, the modified repeat domains produced by the methods of the invention have a pI of pH 4.9 or less, pH 4.8 or less, pH 4.7 or less, pH 4.6 or less, pH 4.5 or less, pH 4.4 or less, pH 4.3 or less, pH 4.2 or less, pH 4.1 or less, pH 4.0 or less, pH 3.9 or less, pH 3.8 or less, pH 3.7 or less, pH 3.6 or less, or pH 3.5 or less.

[0246] In some embodiments, the modified repeat domains produced by the methods of the invention have a pI of 4.53 or less, pH 4.52 or less, pH 4.50 or less, pH 4.48 or less, pH 4.46 or less, pH 4.44 or less, pH 4.42 or less, pH 4.40 or less, pH 4.38 or less, pH 4.36 or less, pH 4.34 or less, pH 4.32 or less, pH 4.30 or less, pH 4.28 or less, pH 4.26 or less, pH 4.24 or less, pH 4.22 or less, pH 4.20 or less, pH 4.18 or less, pH 4.16 or less, pH 4.14 or less, pH 4.12 or less, pH 4.10 or less, pH 4.08 or less, pH 4.09 or less, pH 4.16 or less, pH 4.17 or less, pH 4.19 or less, pH 4.20 or less, pH 4.21 or less, pH 4.22 or less, pH 4.20 or less, pH 4.23 or less, pH 4.24 or less, pH 4.25 or less, pH 4.26 or less, pH 4.27 or less, pH 4.28 or less, pH 4.29 or less, pH 4.30 or less, pH 4.31 or less, pH 4.32 or less, pH 4.30 or less, pH 4.33 or less, pH 4.34 or less, pH 4.32 or less, pH 4.30 or less, pH 4.28 or less, pH 4.26 or less, pH 4.24 or less, pH 4.22 or less, pH 4.20 or less, pH 4.18 or less, pH 4.16 or less, pH 4.1 0.06 or less, pH 4.04 or less, pH 4.02 or less, pH 4.00 or less, pH 3.98 or less, pH 3.96 or less, pH 3.94 or less, pH 3.92 or less, pH 3.90 or less, pH 3.88 or less, pH 3.86 or less, pH 3.84 or less, pH 3.82 or less, pH 3.80 or less, pH 3.78 or less, pH 3.76 or less, pH 3.74 or less, pH 3.72 or less, pH 3.70 or less, pH 3.68 or less, pH 3.66 or less, pH 3.64 or less, pH 3.62 or less, pH 3.60 or less, pH 3.58 or less, pH 3.56 or less, pH 3.54 or less, pH 3.52 or less, or pH 3.50 or less.

[0247] In some embodiments, the modified repeat domains produced by the methods of the present invention have a pI of between pH 3.00 and pH 4.60, pH 3.10 and pH 4.60, pH 3.20 and pH 4.60, pH 3.30 and pH 4.60, pH 3.40 and pH 4.60, pH 3.50 and pH 4.60, pH 3.50 and pH 4.58, pH 3.50 and pH 4.56, pH 3.50 and pH 4.54, pH 3.50 and pH 4.53, pH 3.50 and pH 4.52, pH 3.50 and pH 4.50 , pH3.50~pH4.48, pH3.50~pH4.46, pH3.50~pH4.44, pH3.50~pH4.42, pH3.50~pH4.40, pH3.50~pH4.38, pH3.50~pH4.36, pH3.5 0~pH4.34, pH3.50~pH4.32, pH3.50~pH4.30, pH3.60~pH4.60, pH3.60~pH4.58, pH3.60~pH4.56, pH3.60~pH4.54, pH3.60~pH4.5 3, pH3.60~pH4.52, pH3.60~pH4.50, pH3.60~pH4.48, pH3.60~pH4.46, pH3.60~pH4.44, pH3.60~pH4.42, pH3.60~pH4.40, pH3. 60~pH4.38, pH3.60~pH4.36, pH3.60~pH4.34, pH3.60~pH4.32, pH3.60~pH4.30, pH3.70~pH4.60, pH3.70~pH4.58, pH3.70~pH4 .56, pH3.70 to pH4.54, pH3.70 to pH4.53, pH3.70 to pH4.52, pH3.70 to pH4.50, pH3.70 to pH4.48, pH3.70 to pH4.46, pH3.70 to pH4.44, pH3.70 to pH4.42, pH3.70 to pH4.40, pH3.70 to pH4.38, pH3.70 to pH4.36, pH3.70 to pH4.34, pH3.70 to pH4.32, or pH3.70 to pH4.30.

[0248] In some embodiments, the modified repeat domains produced by the methods of the invention have a percentage of basic amino acid residues of 12.0% or less, 11.5% or less, 11.0% or less, 10.5% or less, 10.0% or less, 9.5% or less, or 9.0% or less.

[0249] In some embodiments, the modified repeat domains produced by the methods of the invention have a percentage of basic amino acid residues 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 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, 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, or 4.80% or less.

[0250] In some embodiments, the modified repeat domains produced by the methods of the invention have a percentage of Arg and Lys residues of 8.0% or less, 7.5% or less, 7.0% or less, 6.5% or less, or 6.0% or less.

[0251] In some embodiments, the modified repeat domains produced by the methods of the invention have a percentage of Arg and Lys residues of 5.65%, 5.60%, 5.50%, 5.40%, 5.30%, 5.20%, 5.10%, 5.00%, 4.90%, 4.80%, 4.70%, 4.60%, 4.50%, 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% 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, 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.

[0252] In some embodiments, the modified repeat domains produced by the methods of the invention have a percentage of basic amino acid residues in their 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, or 8.0% or less, 7.8% or less.

[0253] In some embodiments, the modified repeat domain produced by the methods of the invention has a percentage of basic amino acid residues among its framework residues of 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, 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.

[0254] In some embodiments, the designed ankyrin repeat domain of the invention has a percentage of Arg and Lys residues among its framework residues of 6.7% or less, 6.6% or less, 6.5% or less, 6.4% or less, 6.3% or less, 6.2% or less, 6.1% or less, 6.0% or less, or 5.9% or less.

[0255] In some embodiments, the modified repeat domains produced by the methods of the invention have a percentage of Arg and Lys residues among its framework residues of 5.83%, 5.80%, 5.70%, 5.60%, 5.50%, 5.40%, 5.30%, 5.20%, 5.10%, 5.00%, 4.90%, 4.80%, 4.70%, 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% 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, 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 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.

[0257] In some embodiments, the total number of basic amino acid residues contained in the modified repeat domain generated by the methods of the 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 of n or less, 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+3R, 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, where R is the number of internal repeat modules comprised in the engineered repeat domain. In such embodiments, "xR" means x multiplied by R.

[0258] In another embodiment, the total number of Arg and Lys residues in the modified repeat domains generated by the methods of the invention is m or less, and 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, where R is the number of internal repeat modules contained in the engineered repeat domain.

[0259] In some embodiments, the engineered repeat domain generated by the methods of the invention has a total number of basic amino acid residues in its framework residues that is less than or equal to x, where 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, where R is the number of internal repeat modules comprised in the engineered repeat domain.

[0260] In another embodiment, the engineered repeat domain produced by the methods of the invention has a total number of Arg and Lys residues in its framework residues that is y or less: 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 comprised in the engineered repeat domain.

[0261] Preferably, the modified repeat domains produced by the methods of the present invention specifically bind to a target. In a preferred embodiment, the modified repeat domains bind to a target of about 10-5 M or less, about 10 -6 M or less, about 10 -7 M or less, about 10 -8 M or less, about 10 -9 M or less, about 10 -10 M or less, about 10 -11 M or less, about 10 -12 M or less, about 10 -13 M or less, about 10 -14 The dissociation constant (K D ) and binds to the target.

[0262] In some embodiments, the modified repeat domains produced by the methods of the invention have 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 0.3 or less.

[0263] In some embodiments, the modified repeat domain produced by the methods of the invention has a KR / DE ratio among its framework residues 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. Preferably, the KR / DE ratio among the framework residues is 0.25 or less.

[0264] In some embodiments, the modified repeat domain produced by the methods of the invention has a KR / DE ratio among all residues of its N-terminal capping module of 0.66 or less, 0.64 or less, 0.62 or less, 0.60 or less, 0.58 or less, 0.56 or less, 0.54 or less, 0.52 or less, 0.50 or less, 0.48 or less, 0.46 or less, 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, 0.08 or less, 0.06 or less, 0.04 or less, or 0.02 or less. Preferably, the KR / DE ratio among all residues of the N-terminal capping module is 0.5 or less.

[0265] In some embodiments, the modified repeat domains generated by the methods of the invention have no Arg or Lys residues in the framework residue positions.

[0266] In some embodiments, the modified repeat domain produced by the methods of the 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 of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 or less. Preferably, the total number of basic residues is 14 or less. More preferably, the total number of basic residues is 10 or less. In another particular embodiment, the modified repeat domain comprising two internal repeat modules may have a total number of Arg and Lys residues of 1, 2, 3, 4, 5, 6, 7, 8, or 9 or less. Preferably, the total number of Arg and Lys residues is 9 or less. More preferably, the total number of Arg and Lys residues is 5 or less. In another particular embodiment, the modified repeat domain comprising two internal repeat modules may have a total number of Arg and Lys residues in the framework residues of 1, 2, 3, 4, 5, or 6 or less. Preferably, the total number of Arg and Lys residues in the framework residues is 6 or less. More preferably, the total number of Arg and Lys residues in the framework residues is 5 or less.

[0267] In some embodiments, the modified repeat domain produced by the methods of the invention comprises an N-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID NOs: 25, and 115-122; and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue in any of SEQ ID NOs: 25, and 115-122 is replaced by another amino acid.

[0268] 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, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue in either of SEQ ID NOs: 115 and 121 is substituted with another amino acid.

[0269] In further embodiments, the N-terminal capping module has a sequence selected from the group consisting of: (1) SEQ ID NO: 115, and (2) SEQ ID NO: 115 in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue is substituted by another amino acid.

[0270] Instead of 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: 123-130, and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue in any of SEQ ID NOs: 123-130 is replaced by another amino acid.

[0271] 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, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue in either of SEQ ID NOs: 123 and 126 is substituted by another amino acid.

[0272] In further embodiments, the C-terminal capping module has a sequence selected from the group consisting of: (1) SEQ ID NO: 123, and (2) SEQ ID NO: 123 in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue is substituted by another amino acid.

[0273] In one particular embodiment, the modified repeat domain produced by the method of the invention comprises: 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, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue in either of SEQ ID NOs: 115 and 121 is substituted with another amino acid; and 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, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue in either SEQ ID NOs: 123 and 126 is replaced by another amino acid.

[0274] In another particular embodiment, the modified repeat domain produced by the method of the invention comprises: an N-terminal capping module having a sequence selected from the group consisting of: (1) SEQ ID NO: 115, and (2) SEQ ID NO: 115, in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue is substituted by another amino acid; and 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, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue in SEQ ID NO: 123 is replaced by another amino acid.

[0275] In a further embodiment, the modified repeat domain produced by the method of the invention comprises one or more internal repeat modules, each independently comprising a sequence selected from the group consisting of: (1) SEQ ID NO: 31; and (2) a sequence in which up to 5, 4, 3, 2, or 1 framework residue other than positions 1, 10, 13, 17, 19, 21, 22, and 26 in SEQ ID NO: 31 is substituted by another amino acid; and / or an N-terminal capping module, each independently comprising a sequence selected from the group consisting of: (1) SEQ ID NO: 19; and (2) a sequence in which up to 5, 4, 3, 2, or 1 framework residue other than positions 1, 10, 13, 17, 19, 21, 22, and 26 in SEQ ID NO: 31 is substituted by another amino acid. and / or a C-terminal capping module comprising a sequence selected from the group consisting of: (1) SEQ ID NO: 40; and (2) a C-terminal capping module comprising a sequence selected from the group consisting of sequences in which up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue other than positions 10, 11, 17, 18, 19, 22, and 26 of SEQ ID NO: 40 are substituted with another amino acid.

[0276] In certain embodiments, the modified repeat domain of the present invention comprises a sequence selected from SEQ ID NOs: 19-25, 57-60, 65-81, 31-35, 61, 62, 92-94, 40-42, 63, 64, and 82-91.

[0277] In certain embodiments, each internal repeat module comprised in a modified repeat domain generated by the method of the invention may independently comprise a sequence selected from SEQ ID NOs: 31-35, 61, 62, and 92-94. Alternatively or additionally, an N-terminal capping module comprised in a modified repeat domain generated by the method of the invention may comprise a sequence selected from SEQ ID NOs: 19-25, 57-60, and 65-81. Alternatively or additionally, a C-terminal capping module comprised in a modified repeat domain generated by the method of the invention may comprise a sequence selected from SEQ ID NOs: 40-42, 63, 64, and 82-91.

[0278] In certain embodiments, each internal repeat module comprised in a modified repeat domain generated by the method of the invention independently comprises a sequence selected from SEQ ID NOs: 31-35, 61, 62, 92-94, and 131-142. Alternatively or additionally, an N-terminal capping module comprised in a modified repeat domain generated by the method of the invention comprises a sequence selected from SEQ ID NOs: 19-25, 57-60, 65-81, and 115-122. Alternatively or additionally, a C-terminal capping module comprised in a modified repeat domain generated by the method of the invention comprises a sequence selected from SEQ ID NOs: 40-42, 63, 64, 82-91, and 123-130.

[0279] In a further embodiment, each internal repeat module comprised in the modified repeat domain generated by the method of the invention independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 31-35, 61, 62, and 92-94; and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue in any of SEQ ID NOs: 31-35, 61, 62, and 92-94 is substituted by another amino acid. Alternatively or additionally, the N-terminal capping module contained in the modified repeat domain generated by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 19-25, 57-60, and 65-81; and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue in any of SEQ ID NOs: 19-25, 57-60, and 65-81 is replaced by another amino acid. Alternatively or additionally, the C-terminal capping module contained in the modified repeat domain generated by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 40-42, 63, 64, and 82-91, and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue in any of SEQ ID NOs: 40-42, 63, 64, and 82-91 is replaced by another amino acid.

[0280] In certain embodiments, each internal repeat module comprised in the modified repeat domain generated by the methods of the invention independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 31 and 32; and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue in either of SEQ ID NOs: 31 and 32 is substituted by another amino acid. Alternatively or additionally, the N-terminal capping module comprised in the modified repeat domain generated by the methods of the invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 19 and 20; and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue in either of SEQ ID NOs: 19 and 20 is substituted by another amino acid. Alternatively or additionally, the C-terminal capping module comprised in the modified repeat domain generated by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 40 and 41, and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue in either SEQ ID NOs: 40 and 41 is replaced by another amino acid.

[0281] In further embodiments, each internal repeat module contained in the modified repeat domain generated by the method of the present invention may independently comprise a sequence selected from the group consisting of (1) SEQ ID NOs: 26-30, and (2) sequences in which 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids in any of SEQ ID NOs: 26-30 are substituted by other amino acids. Alternatively or additionally, the N-terminal capping module contained in the modified repeat domain generated by the method of the present invention may comprise a sequence selected from the group consisting of (1) SEQ ID NOs: 15-18, and (2) sequences in which 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids in any of SEQ ID NOs: 15-18 are substituted by other amino acids. Alternatively or additionally, the C-terminal capping module contained in the modified repeat domain generated by the method of the present invention may comprise a sequence selected from the group consisting of (1) SEQ ID NOs: 36-39, and (2) sequences in which 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids in any of SEQ ID NOs: 36-39 are substituted by other amino acids.

[0282] In further embodiments, each internal repeat module comprised in the modified repeat domain generated by the method of the present invention may further independently comprise a sequence selected from the group consisting of: (1) SEQ ID NOs: 46-52; and (2) sequences in which 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids in any of SEQ ID NOs: 46-52 are substituted by other amino acids. Alternatively or additionally, each N-terminal capping module comprised in the modified repeat domain generated by the method of the present invention may further comprise a sequence selected from the group consisting of: (1) SEQ ID NOs: 43-45; and (2) sequences in which 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids in any of SEQ ID NOs: 43-45 are substituted by other amino acids. Alternatively or additionally, the C-terminal capping module contained in the modified repeat domain generated by the method of the present invention may further comprise a sequence selected from the group consisting of: (1) SEQ ID NOs: 53 to 56, and (2) sequences in which 1, 2, 3, 4, 5, 6, 7, 8, or 9 amino acids in any of SEQ ID NOs: 53 to 56 are substituted with other amino acids.

[0283] In a further embodiment, each internal repeat module comprised in the modified repeat domain generated by the method of the present invention independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 26-30, and 46-52; and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 26-30, and 46-52 is substituted with 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 NOs: 15-18, 43-45, and 99-101, and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 15-18, 43-45, and 99-101 is substituted with 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 comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 36 to 39, 53 to 56, 102, and 103, and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 36 to 39, 53 to 56, 102, and 103 is substituted with another amino acid.

[0284] In a further embodiment, each internal repeat module comprised in the modified repeat domain generated by the method of the present invention independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 26-30, and 47-48; and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 26-30, 47, and 48 is substituted with another amino acid. Alternatively or additionally, the N-terminal capping module contained in the modified repeat domain generated by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 15-18, 43, and 99-101, and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 15-18, 43, and 99-101 is substituted with 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 comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 36 to 39, 54, 102, and 103, and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 36 to 39, 54, 102, and 103 is replaced by another amino acid.

[0285] In a further embodiment, each internal repeat module comprised in the modified repeat domain produced by the method of the invention independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 47 and 48; and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 47-48 is substituted with another amino acid. Alternatively or additionally, the N-terminal capping module comprised in the modified repeat domain produced by the method of the invention comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 43, 99, 100, and 101; and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 43, 99, 100, and 101 is substituted with 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, 102, and 103; and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 54, 102, and 103 is replaced by another amino acid.

[0286] In a further embodiment, each internal repeat module comprised in the modified repeat domain produced by the method of the invention independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 29, 30, 47-50; and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 29, 30, 47-50 is substituted with another amino acid. Alternatively or additionally, the N-terminal capping module comprised in the modified repeat domain produced by the method of the invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 18, 43, 44, and 99-101; and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 18, 43, 44, and 99-101 is substituted with 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: 39, 54, 55, 102, and 103; and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 39, 54, 55, 102, and 103 is substituted with another amino acid.

[0287] In a further embodiment, each internal repeat module comprised in the modified repeat domain produced by the method of the invention independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 26-28, and 46; and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 26-28 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 invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 15-17; and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 15-17 is substituted by another amino acid. Alternatively or additionally, the C-terminal capping module contained in the modified repeat domain generated 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, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 36 to 38, and 53 is replaced by another amino acid.

[0288] In a further embodiment, each internal repeat module comprised in the modified repeat domain generated by the method of the invention independently comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 51 and 52, and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in either of SEQ ID NOs: 51 and 52 is substituted by another amino acid. Alternatively or additionally, the N-terminal capping module comprised in the modified repeat domain generated by the method of the invention comprises a sequence selected from the group consisting of: (1) SEQ ID NO: 45, and (2) a sequence in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 framework residue 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 up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in SEQ ID NO: 56 is substituted with another amino acid.

[0289] In some embodiments, each internal repeat module comprised in an engineered repeat domain generated by the methods of the invention comprises one or more residues selected from the group consisting of: (i) Gln at position 1, and (ii) Glu at position 26, numbered relative to SEQ ID NO: 48.

[0290] In some embodiments, the N-terminal capping module comprised in the modified repeat domain generated by the methods of the invention comprises one or more residues 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, numbered relative to SEQ ID NO: 43.

[0291] In some embodiments, the C-terminal capping module comprised in the modified repeat domain generated by the methods of the invention comprises one or more residues 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, numbered relative to SEQ ID NO: 54.

[0292] Thus, in an exemplary embodiment, the modified repeat domain produced by the methods 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 comprising 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.

[0293] In another exemplary embodiment, the modified repeat domain produced by the method of the 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 comprising Gln at position 1 and Glu at position 26, respectively, 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.

[0294] In some further 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: 104-114, 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 to any of SEQ ID NOs: 104-114. In some 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 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 to either 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 to SEQ ID NO:104.

[0295] 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: 2-5, 8-10, 12, and 95-98; 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: 2-5, 8-10, 12, and 95-98. 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: 2-5, 8-10, 12, and 95-98, and (2) a sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 2-5, 8-10, 12, and 95-98.

[0296] 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: 8-10, 12, and 95-98; 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: 8-10, 12, and 95-98. 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: 8-10, 12, and 95-98, and (2) a sequence having at least 80% amino acid sequence identity to SEQ ID NOs: 8-10, 12, and 95-98.

[0297] In a specific embodiment, the engineered repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 8-10 and 95-98, 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: 8-10 and 95-98. Thus, in a more specific embodiment, the engineered repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 8-10 and 95-98, and (2) a sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 8-10 and 95-98.

[0298] In a specific embodiment, the engineered repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 8-9 and 95-98, 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: 8-10 and 95-98. Thus, in a more specific embodiment, the engineered repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 8-9 and 95-98, and (2) a sequence having at least 80% amino acid sequence identity to SEQ ID NOs: 8-9 and 95-98.

[0299] In a specific embodiment, the engineered repeat domain produced by the method of the present invention comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 95-98, 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: 95-98. Thus, in a more specific embodiment, the engineered repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 95-98, and (2) a sequence having at least 80% amino acid sequence identity to any of SEQ ID NOs: 95-98.

[0300] In a particular embodiment, the engineered repeat domain produced by the method of the 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 particular embodiment, the engineered 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 about 80% amino acid sequence identity to SEQ ID NO: 12.

[0301] In a specific embodiment, the engineered 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 engineered 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 identity to any one of SEQ ID NOs: 2 to 5.

[0302] In a particular embodiment, the designed ankyrin repeat domain produced by the method of the 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 particular 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 identity to any one of SEQ ID NOs: 2, 3, and 5.

[0303] In a further embodiment, the designed ankyrin repeat domain produced by the method of the invention comprises one or more residues selected from the group consisting of: (i) Thr at position 4, (ii) Gln at position 5, (iii) Leu, Val, He, 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, numbered relative to SEQ ID NO: 6.

[0304] Thus, in an exemplary embodiment, the designed ankyrin repeat domain generated by the method 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.

[0305] In another exemplary embodiment, the designed ankyrin repeat domain generated by the method of the 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.

[0306] In some embodiments, the modified repeat domain produced by the methods of the invention is linked to a drug moiety. In some embodiments, the drug moiety is covalently or non-covalently linked to the modified repeat domain produced by the methods of the invention. In a more particular embodiment, the drug moiety is linked to the modified repeat domain produced by the methods of the invention by a chelator. The appropriate chelator can be selected depending on the use of the modified repeat domain of the invention. In a particular embodiment, the chelator is DTPA. In a more particular embodiment, the drug moiety is a radionuclide. The choice of radionuclide can depend on the intended use (e.g., diagnostic or therapeutic) of the repeat domain produced by the methods of the invention.

[0307] In one particular 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 particular embodiment, the radionuclide is indium-111. In another particular embodiment, the drug moiety is a diagnostic moiety. In a more particular embodiment, the diagnostic moiety is a fluorophore, a chromophore, an imaging agent, or a radionuclide.

[0308] In a preferred embodiment, the modified repeat domain produced by the method of the invention is a modified engineered ankyrin repeat domain.

[0309] Recombinant proteins In one aspect, the present invention provides a recombinant protein comprising an engineered repeat domain of the invention.

[0310] The engineered repeat domains of the present invention can be genetically fused to further components, such as drug moieties, proteins, or drugs; such fusions are also referred to as "recombinant proteins." Linkers known in the art can be used between the repeat domains in such repeat proteins (see, for example, WO 2021 / 116469) or between the repeat domain and the further component. Such recombinant proteins are particularly envisioned for use in medicine.

[0311] In some embodiments, recombinant proteins of the present invention comprise a repeat domain of the present invention linked to a drug moiety. In some embodiments, additional drug moieties may be linked to such recombinant proteins. In other embodiments, recombinant proteins of the present invention comprise a repeat domain of the present invention that is not linked to a drug moiety. In more specific embodiments, one or more drug moieties may be linked to the recombinant protein.

[0312] In some embodiments, the recombinant protein of the invention comprises one or more additional engineered ankyrin repeat domains.

[0313] Nucleic acids, vectors and host cells In another aspect, the present invention relates to the amino acid sequence of a designed repeat domain of the invention or an isolated nucleic acid encoding the amino acid sequence of a designed repeat domain of the invention. In one aspect, the present invention relates to an isolated nucleic acid encoding the amino acid sequence of a recombinant protein of the invention. In one embodiment, the present invention relates to an isolated nucleic acid encoding the amino acid sequence of a designed repeat domain of the invention.

[0314] Furthermore, the present invention relates to a vector comprising any of the nucleic acids of the present invention. Accordingly, in another aspect, the present invention provides a recombinant expression vector comprising a nucleic acid according to the present invention, wherein the vector optionally comprises an expression control sequence that allows expression of a polypeptide encoded by the nucleic acid operably linked to the nucleic acid in a prokaryotic or eukaryotic host cell. The nucleic acid sequence can be inserted into the recombinant vector by methods well known to those skilled in the art, for example, the methods described in MOLECULAR CLONING: A LABORATORY MANUAL, Sambrook et al., 4th Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001.

[0315] Nucleic acids are well known to those skilled in the art and have been used to produce engineered ankyrin repeat domains or recombinant binding proteins of the invention in E. coli, as further described, for example, in U.S. Patent No. 7,417,130.

[0316] In another aspect, the present 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, or a yeast cell such as Saccharomyces, an insect cell, a mammalian cell such as a Chinese Hamster Ovary (CHO) cell, a Cl27 mouse cell line, a BHK cell line of Syrian hamster cells, or a Human Embryonic Kidney 293 (HEK293) cell. In some embodiments, the host cell is a CHO cell or a HEK293 cell. The host cell can be used, for example, to express a recombinant protein of the present invention.

[0317] composition The present invention further 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 therapeutic and diagnostic methods using the pharmaceutical compositions disclosed herein. The methods and uses encompassed by the present invention are described in more detail below.

[0318] The pharmaceutical compositions described herein can be prepared using methods known in the art.

[0319] The pharmaceutical composition may comprise a pharmaceutically acceptable carrier or excipient or diluent. Typical pharmaceutical carriers include phosphate buffered saline solutions, water, emulsions such as oil / water or water / oil emulsions, and various types of wetting agents.

[0320] Pharmaceutical compositions may contain, for example, acidifying agents, additives, adsorbents, aerosol propellants, air displacing agents, alkalizing agents, anti-hardening agents, anticoagulants, antimicrobial preservatives, antioxidants, disinfectants, bases, binders, buffers, chelating agents, coating agents, colorants, desiccants, detergents, diluents, disinfectants, disintegrants, dispersants, dissolution enhancers, dyes, emollients, emulsifiers, emulsion stabilizers, fillers, film formers, seasonings, flavorings, flow enhancers, gel Any other pharmaceutically acceptable ingredients may be included, including a thickening agent, a granulating agent, a moisturizing agent, a lubricant, a mucoadhesive agent, an ointment base, an ointment, an oily vehicle, an organic base, a pastel base, a pigment, a plasticizer, an abrasive, a preservative, a sequestering agent, a skin penetration agent, a solubilizer, a solvent, a stabilizer, a suppository base, a surfactant, a surface-active substance, a suspending agent, a sweetener, a therapeutic agent, a thickening agent, a tonicity agent, a toxicity agent, a thickening agent, a water-absorbing agent, a water-miscible cosolvent, a softening agent, or a humectant. See, for example, Handbook of Pharmaceutical Excipients, Third Edition, A.H. Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated by reference in its entirety. Remington's Pharmaceutical Sciences, Sixteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980), which is incorporated by reference in its entirety.

[0321] 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.

[0322] Therapeutic and diagnostic uses and methods according to the present invention In the context of the present invention, the terms "medical condition," "disease," and "disorder" are used interchangeably and include, but are not limited to, cancer. In a preferred embodiment, the medical condition is cancer.

[0323] In one aspect, the present invention provides a method of 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 a designed repeat domain of the invention, a recombinant protein of the invention, a nucleic acid of the invention, or a pharmaceutical composition of the invention.

[0324] Further provided is an engineered repeat domain, recombinant protein, nucleic acid, or pharmaceutical composition of the invention for use in a method of treating and / or diagnosing a medical condition.

[0325] 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 according to the present invention for the treatment and / or diagnosis of a disease, for which purpose the designed repeat domain, a recombinant protein, a nucleic acid or a pharmaceutical composition according to the present invention is administered to a patient in need thereof in a therapeutically and / or diagnostically effective amount.

[0326] In one embodiment, the present invention relates to a method for the treatment and / or diagnosis of a medical condition, comprising the step of administering to a patient in need of such treatment and / or diagnosis a therapeutically and / or diagnostically effective amount of a designed repeat domain, recombinant protein, nucleic acid or pharmaceutical composition of the invention.

[0327] In one embodiment, the present invention relates to the use of an engineered repeat domain, a recombinant protein, a nucleic acid, or a pharmaceutical composition of the invention for the treatment and / or diagnosis of a medical condition.

[0328] In one embodiment, the invention relates to a designed repeat domain, a recombinant protein, a nucleic acid, or a pharmaceutical composition of the invention for use in the treatment and / or diagnosis of a medical condition. In one embodiment, the invention relates to the use of a designed repeat domain, a recombinant protein, a nucleic acid, or a pharmaceutical composition of the invention for the treatment and / or diagnosis of a medical condition.

[0329] In one embodiment, the present invention relates to a process for treating and / or diagnosing a medical condition using a designed repeat domain, recombinant protein, nucleic acid, or pharmaceutical composition of the invention. In one embodiment, the present invention relates to a process for the manufacture of a medicament for the treatment and / or diagnosis of a medical condition, wherein the designed repeat domain, recombinant protein, nucleic acid, or pharmaceutical composition of the invention is the active ingredient of the medicament.

[0330] In one aspect, the invention relates to the use of an engineered repeat domain, a recombinant protein, a nucleic acid, or a pharmaceutical composition of the invention for the manufacture of a medicament.

[0331] In one embodiment, the present invention relates to the use of an engineered repeat domain, recombinant protein, nucleic acid, or pharmaceutical composition of the invention for the manufacture of a medicament for the treatment and / or diagnosis of a medical condition.

[0332] In a further embodiment, the present invention relates to the use of the designed repeat domain, recombinant protein, nucleic acid or pharmaceutical composition of the invention for the manufacture of a medicament used for the treatment of a medical condition, preferably a neoplastic disease, more preferably cancer.

[0333] In a preferred embodiment, the medical condition is cancer.

[0334] In some embodiments, the subject is a mammal. In preferred embodiments, the subject is a human.

[0335] In some embodiments, a single administration of an engineered ankyrin repeat domain, recombinant protein, nucleic acid or pharmaceutical composition of the invention may be sufficient. In other embodiments, multiple administrations may be necessary. Various factors, such as the age and general health of the subject, as well as the nature and typical administration regime of any drug moiety contained in such an engineered ankyrin repeat domain, recombinant protein, nucleic acid or pharmaceutical composition of the invention, will influence the number and frequency of administration.

[0336] The designed repeat domains, recombinant proteins, nucleic acids, or pharmaceutical compositions described herein can be used in combination with another therapeutic and / or diagnostic agent. Each therapeutic and / or diagnostic agent may be administered simultaneously (e.g., in the same formulation or simultaneously), in parallel (i.e., in separate formulations where one is administered immediately after the other, in any order), or sequentially in any order. Sequential administration is useful when the therapeutic and / or diagnostic agents in the combination therapy and / or combination diagnosis are in different dosage forms (e.g., one formulation is a tablet or capsule and another is a sterile liquid) and / or when administered on different dosing schedules (e.g., a pain reliever administered at least daily and a biotherapeutic administered less frequently, such as once a week or once every two weeks).

[0337] Administration can include topical, oral, and parenteral administration, with parenteral administration being the typical route of administration.

[0338] In certain embodiments, oral administration is excluded from any of the above methods or uses according to the present invention. Thus, in some specific embodiments, 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 for use in a method of treating and / or diagnosing a medical condition according to the present invention is not administered orally. In some embodiments, the present invention provides a method of treating and / or diagnosing a medical condition, comprising administering 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 to a patient in need of treating and / or diagnosing the medical condition.

[0339] In some further embodiments, the designed repeat domains, recombinant proteins, or pharmaceutical compositions described herein can be used in radiopharmaceutical therapy or diagnosis. Exemplary approaches and indications 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.

[0340] In some alternative embodiments, the designed repeat domains, recombinant proteins, or pharmaceutical compositions described herein may be used in therapeutic and / or diagnostic approaches in which antibody-drug conjugates may also be used. Such approaches and indications 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.

[0341] The present invention is not limited to the specific embodiments described in the examples. [Example]

[0342] material Chemicals were purchased from Sigma-Aldrich (USA). Oligonucleotides were purchased from Microsynth (Switzerland). Unless otherwise stated, DNA polymerases, restriction enzymes, and buffers were purchased 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 (eBlocks) were obtained from IDT (USA). Maleimide-DTPA was purchased from Chematech, metal-free PBS was purchased from VWR, and Chelex 100 chelating resin was purchased from BioRad.

[0343] molecular biology Unless otherwise stated, the methods are carried out according to known protocols (see, for example, Sambrook J., Fritsch EF and Maniatis T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory 1989, New York).

[0344] Designed ankyrin repeat protein library Methods for generating designed ankyrin repeat protein libraries are described, for example, in U.S. Pat. No. 7,417,130; Binz et al., 2003, supra; Binz et al., 2004, supra. Such methods allow the construction of designed ankyrin repeat protein libraries with randomized ankyrin repeat modules and / or randomized capping modules. Thus, for example, such libraries can be assembled based on a fixed or randomized N-terminal capping module, one or more randomized repeat modules, and a fixed or randomized C-terminal capping module (see, for example, the N-terminal and C-terminal capping modules provided in International Patent Applications WO 2021 / 116462 and WO 2021 / 116469). Preferably, such libraries are assembled so that the randomized positions of the repeat or capping modules are free of the amino acids C, G, M, N (before a G residue), and P.

[0345] Furthermore, such randomized modules in such libraries can contain additional polypeptide loop insertions with randomized amino acid positions. Examples of such polypeptide loop insertions are complement-determining region (CDR) loop libraries of antibodies or de novo generated peptide libraries. For example, such loop insertions could be designed using the structure of the N-terminal ankyrin repeat domain of human ribonuclease L as guidance (Tanaka, N., Nakanishi, M., Kusakabe, Y., Goto, Y., Kitade, Y., Nakamura, K.T., EMBO J. 23(30), 3929-3938, 2004). Similar to this ankyrin repeat domain in which 10 amino acids are inserted into a beta turn located near the boundary between two ankyrin repeats, ankyrin repeat protein libraries can contain randomized loops (with fixed and randomized positions) of variable length (e.g., 1 to 20 amino acids) inserted into one or more beta turns of the ankyrin repeat domain.

[0346] An N-terminal capping module of an ankyrin repeat protein library preferably has a RILLAA, RILLKA or RELLKA motif, and any such C-terminal capping module of an ankyrin repeat protein library preferably has a KLN, KLA or KAA motif.

[0347] The design of such ankyrin repeat protein libraries can be guided by known structures of ankyrin repeat domains that interact with targets. Examples of such structures, identified by unique Protein Data Bank (PDB) accession or identification codes (PDB-IDs), are 1WDY, 3V31, 3V30, 3V2X, 3V2O, 3UXG, 3TWQ-3TWX, 1N11, 1S70, and 2ZGD.

[0348] Examples of designed ankyrin repeat protein libraries have been described, such as N2C and N3C designed ankyrin repeat protein libraries (U.S. Patent No. 7,417,130; Binz et al., 2003, supra; Binz et al., 2004, supra). The N2C and N3C numbers describe the number of randomized repeat modules present between the N- and C-terminal capping modules.

[0349] The nomenclature used to define the repeat units and positions within modules is based on Binz et al., (2004) (cited above), with the modification that the boundaries between ankyrin repeat modules and ankyrin repeat units are shifted by one amino acid position. For example, position 1 of the ankyrin repeat module of Binz et al., (2004) (cited above) corresponds to position 2 of the ankyrin repeat module of the present disclosure; consequently, position 33 of the ankyrin repeat module of Binz et al., (2004) (cited above) corresponds to position 1 of the following ankyrin repeat module of the present disclosure.

[0350] Experimental conditions for some examples are further described in WO 2012 / 069654, WO 2016 / 156596, and WO 2021 / 116462.

[0351] Example 1: Construction, expression and purification of DARPin variants DARPins with defined amino acid sequences can be produced by gene synthesis of the corresponding back-translated nucleic acid sequence, subcloning into an appropriate expression vector for an expression system (e.g., an E. coli expression system), protein expression and purification. Such methods are already known to those skilled in the art.

[0352] Charge-engineered DARPin variants were constructed and investigated by assessing which amino acid positions allow for charge-reducing substitutions of parent amino acid residues while still retaining favorable biophysical properties and / or biological activity (target binding). Specific amino acid substitutions were investigated in Simeon, RA et al., Protein Engineering, Design and Selection, 34, (2021) with the aim of increasing the protease stability of bacterial exotoxin-specific DARPins.

[0353] Overall, a set of nine DARPin mutants with different numbers of positively and negatively charged amino acid residues was designed, and these mutants and their parent DARPins were subsequently generated and characterized as described in the following paragraphs.

[0354] Three different parent DARPins were selected as starting points for engineering the DARPin charge variants: DARPin01 (SEQ ID NO: 1), DARPin06 (SEQ ID NO: 6), and parent DARPin11 (SEQ ID NO: 1). Parent DARPin06 and DARPin11 have already been described in WO 2018 / 054971, and parent DARPin01 in WO 2020 / 245746 and WO 2020 / 245175. DARPin07 was subsequently designed to be used as a negative control. DARPin07 has a higher pI and a higher percentage of basic amino acids among the framework residues compared to its parent DARPin06. Details regarding the generated DARPin variants and parent DARPins are shown in Table 6 and Figure 1.

[0355] [Table 6]

[0356] DNA encoding each of the designed ankyrin repeat domains consisting of SEQ ID NOs: 1-12 was cloned into a pQE (QIAgen, Germany)-based expression vector providing an N-terminal 6xHis tag to facilitate simple protein purification as described below. Proteins containing SEQ ID NOs: 1-12, respectively, fused to a His-TEV tag (SEQ ID NO: 13) at their N-terminus and a GSGSC tag (SEQ ID NO: 14) at their C-terminus were expressed in E. coli and purified on an IMAC column followed by desalting on a HiLoad 26 / 600 Superdex 200 column. The main fractions were pooled, and 4 mL of DARPin (at different concentrations) was digested with 290 μL of TEV (Sigma-Aldrich, >3 kU / mg TEV) at room temperature (2 h) and then at 4°C (overnight). Samples were taken after 2 h and overnight digestion and analyzed by SDS-PAGE to assess the extent of cleavage. Uncleaved DARPin, still containing the His-tag, and His-tagged TEV protease were removed by incubation with 5 mL of IMAC resin on a roller shaker for 2 hours before centrifugation and removal of the IMAC resin by decanting and filtration. The supernatant / flow-through was purified in a size-exclusion chromatography step and then concentrated. The final purified sample was stored in PBS. Detailed methods for protein production and purification are well known to those skilled in the art.

[0357] Example 2: Size Exclusion Chromatography Analysis The sample from Example 1 was analyzed on a GE Superdex Increase 200 150 / 5 column on an Agilent 1200 HPLC system in PBS at a flow rate of 0.5 mL / min. Of each protein, 0.1 mL was analyzed at 100 micromolar concentration. One of the proteins from SEQ ID NOS: 1-12 and the C-terminal GSGSC tag (SEQ ID NOS: 14) eluted as a partial dimer peak due to oxidation of the C-terminal cysteine to form homodimers. At least 95% of the area under the curve corresponded to the combined monomer and dimer fractions. The results are shown in Figure 2. These results demonstrate that the proteins exhibit favorable biophysically behavior.

[0358] Example 3: DTPA coupling to the C-terminal cysteine of a DARPin and 111 In loading A His-tag-free DARPin containing a C-terminal Cys was first reduced by incubating approximately 5 mg / mL of protein solution with a 10-fold excess of 0.5 M TCEP (pH adjusted to 7.6). The reaction was shaken at room temperature for 4 h. The reduced DARPin solution was then mixed with 0.5 M EDTA in a 1:1 molar ratio and stirred for 15 min. A 5-fold molar excess of 50 mM maleimide-DT PA (2,2'-(1-carboxy-2-(carboxymethyl)-13-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-10-oxo-2,5,8,11-tetraazatridecan-5,8-diyl)diacetic acid) dissolved in DMSO was then added and stirred at room temperature for 1 h. Samples were probed and used for analysis by ESI-MS to assess the coupling efficiency of the reaction. If the coupling efficiency was greater than 90%, the sample was desalted into metal-free PBS on a PD-10 column according to the manufacturer's instructions. The protein solution was then concentrated and diluted three times with metal-free PBS through an Amicon Ultra-15 centrifugal filter (3K) to further desalt the protein. The final concentration was determined by UV absorption and probed for another ESI-MS analysis.

[0359] To load the DTPA-conjugated DARPin with the radioisotope Indium-111, use 10-20 MBq. 111 The In was mixed with 5 μL of 1 M ammonium acetate buffer, followed by the addition of a DARPin coupled to DTPA (50-200 μL of approximately 1 mg / mL DARPin-DTPA construct). The solution was stirred at 37.5 °C for 18 h, and then 1 μL of 0.5 M EDTA was added to remove the unbound complex. 111 In was added. 111 The labeling efficiency of In-labeled DARPins was checked by HPLC, and only if the labeling yield was less than 90%, the labeled protein was purified on a PD-10 desalting column to obtain the free protein. 111The In was removed. Finally, non-radioactive loaded DTPA-DARPin was added to adjust the final concentration to reach a specific activity of 7500 Bq / µg of DARPin. The overall process for generating radioactively loaded DTPA-coupled DARPin is shown in Figure 3.

[0360] Example 4: SPR single trace element analysis Using surface plasmon resonance (SPR) assays, it was shown that the charge-engineered DARPin variants were still able to bind to HER2, as were the parent DARPin06 (SEQ ID NO: 6) and the parent DARPin11 (SEQ ID NO: 7). All SPR data were generated using a Bruker Sierra SPR-32 instrument with PBS-T (0.005% Tween 20) as running buffer. A new Bruker BTC chip was conditioned according to the manufacturer's protocol. The chip was coated with biotinylated target (bio-HER2) to reach a signal intensity of approximately 600 RU. All analytes (500 nM) were injected sequentially for 120 s and dissociation was recorded for 180 s (25 μL / min). Each injection was followed by a 60 s regeneration step with glycine pH 2.0. Data were double-referenced (control spot and buffer injection) and fitted to a 1:1 Langmuir model. All charge-engineered variants derived from the parent DARPin06 and DARPin11 exhibited a 10 s dissociation when coupled to DTPA. -7 K less than M DThe DTPA-coupled parent binders DARPin06 and DARPin11 were also able to bind to their targets. Table 7 shows details of the constructs tested in this SPR assay. DTPA-coupled DARPins are also referred to as "constructs." SPR curves are shown in Figure 4, with plots 1 and 2 showing the profiles of the HER2-binding parent DARPin06 and DARPin11 (SEQ ID NOs: 6 and 11, respectively). Plots 3, 4, 5, 6, and 7 show the profiles 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), respectively. Plots 8 and 9 show construct 11 (DARPin11-GSGSC-DTPA) and construct 12 (DARPin12-GSGSC-DTPA), respectively. A GSGSC tag (SEQ ID NO: 14) was fused to the C-terminus of the DARPin.

[0361] [Table 7]

[0362] Example 5: 111 Renal accumulation studies of In-labeled DARPins This example describes experiments carried out to investigate the renal accumulation of a DARPin according to the invention when administered as a radiolabeled compound.

[0363] As described in Example 3, DTPA-coupled DARPins were catalyzed with indium-111 ( 111 In), and radiolabeled DARPin 12, referred to as radiolabeled DARPin 01 to radiolabeled DARPin 12, were loaded. 111 In-labeled DARPins were obtained by injecting these radiolabeled DARPins (approximately 150 KBq, 1 mg / kg) into the tail vein of wild-type Balb / c mice (female, 7 weeks old, control).

[0364] A total of 54 mice were divided into 12 groups and treated with radiolabeled DARPins 01-12 as detailed in Table 8. Each radiolabeled DARPin was formulated in PBS+0.05% Tween 20.

[0365] Renal accumulation was monitored 4 hours after injection. Mice were euthanized by CO2 inhalation and cervical dislocation. Kidneys were extracted, weighed, and radioactivity was measured using a gamma counter (Packard Cobra II Gamma D5010, GMI, USA). Data are expressed as injected activity per gram of tissue mass (%IA / g) and are shown in Figure 5.

[0366] For autoradiography imaging, kidneys were embedded in OCT and frozen at -80°C. After 24 hours, frozen kidney sections were prepared on a cryostat and mounted on glass slides. The sections were placed in an X-ray cassette and exposed to a phosphor screen for 45 minutes.

[0367] [Table 8]

[0368] As shown in Figure 5, the charge-engineered DARPin variants according to the present invention (radiolabeled DARPin02, DARPin03, DARPin04, DARPin05, DARPin08, DARPin09, DARPin10, and DARPin12) show lower renal accumulation of radioactivity compared to their respective parent DARPins (DARPin01, DARPin06, and DARPin11). The main factors influencing renal accumulation in our experiments are the isoelectric point (pI) of the DARPin and the number of basic amino acids (positively charged at physiological pH; particularly Arg and Lys) in the entire ankyrin repeat domain or among the framework residues contained in the ankyrin repeat domain. Radiolabeled DARPin07 was used as a negative control and accumulated more than its parent DARPin06. This observation is consistent with our hypothesis, as DARPin07 has a higher pI and a higher percentage of basic amino acids among the framework residues compared to its parent DARPin06. DARPin07 is not a DARPin according to the present invention.

[0369] Table 9 provides the percent reduction in renal uptake for each variant compared to the corresponding parent DARPin.

[0370] [Table 9]

[0371] Example 6: In tumor-bearing mice 111 Biodistribution studies of In-labeled DARPins This example describes experiments performed to investigate the tissue accumulation of a selection of DARPins described in Example 5 when administered as radiolabeled compounds to mice bearing HER2-expressing 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.

[0372] DARPin01, DARPin02, DARPin06, DARPin, DARPin, and DARPin08 were prepared and coupled to DTPA as described in Examples 1 to 4. 111 The resulting radiolabeled DARPins 01, 02, 06, and 08 were injected into the tail vein (approximately 150 KBq, 1 mg / kg body weight) of mice (female, 9-12 weeks old, control: CD1-Foxn1nu) bearing HER2-expressing SKOV3ip tumors, as described in further detail below. Compounds were formulated in PBS + 0.05% Tween 20.

[0373] SKOV3ip tumor cells (5 × 10 in PBS) 6 The mice were randomized into different treatment groups (6 animals per group) and grew to approximately 180 mm after 2 weeks of implantation. 3 or tumor volume 3 weeks after implantation (approximately 360 mm 3 tumor volume) 111 In-labeled DARPins were injected intravenously. Tumors and organs were harvested, and %IA / g (equivalent to %ID / g) was determined. Data analysis of mice injected 2 weeks after tumor cell implantation and mice injected 3 weeks after tumor cell implantation showed similar results and were pooled for presentation. Table 10 shows a summary of the treatments.

[0374] Accumulation in organs and tumors was measured 4 hours after injection. Mice were euthanized by CO2 inhalation and cervical dislocation. Organs and tumors were extracted, weighed, and radioactivity was measured using a gamma counter (Packard Cobra II Gamma D5010, GMI, USA). Data are expressed as mean injected activity per gram of tissue mass (%IA / g) and are shown in Figures 6A-6C. Error bars indicate standard deviation.

[0375] [Table 10]

[0376] As shown in Figure 6A, the charge-modified DARPin variants according to the invention used in this experiment (i.e., radiolabeled DARPins 02 and 08) show lower renal accumulation of radioactivity compared to the respective parent DARPins (i.e., radiolabeled DARPins 01 and 06). The percent reduction in renal uptake for each variant compared to the corresponding parent DARPin is shown in Table 11.

[0377] [Table 11]

[0378] The accumulation of radioactivity in tumors and various organs was quantified, as well as in the kidney. The difference in tumor accumulation between the parent DARPin and the engineered variants was negligible, as shown in Figure 6B. Only very small amounts of nonspecific accumulation of unconjugated DARPin01 and DARPin02 were observed in the tumor, whereas much higher tumor accumulation was observed for the HER2-specific DARPin06 and DARPin08, likely due to target-specific binding (Figure 6B).

[0379] Table 12 further shows the tumor-to-kidney ratios measured for the HER2-binding DARPins used in this experiment. Charge engineering performed on DARPin08 resulted in a 3.9-fold increase in the tumor-to-kidney ratio compared to the unengineered parent DARPin06.

[0380] [Table 12]

[0381] The accumulation of DARPins in further tissues or organs (blood, heart, lung, spleen, liver, small intestine, large intestine, muscle, bone, and tail) was measured and is shown in Figure 6C. Based on these results, a similarly low accumulation of the engineered variants (DARPin02 and DARPin08) was observed in these organs and tissues compared to the respective parent DARPins (DARPin01 and DARPin06).

[0382] Based on this study, charge engineering of DARPins according to the present invention does not impair target-specific tumor accumulation of radiolabeled DARPins, while providing a significant reduction in radiolabeled DARPin accumulation in the kidney. Furthermore, charge engineering of DARPins does not result in significant adverse accumulation in the organs analyzed.

[0383] Example 7: In tumor-bearing mice 111 Time course biodistribution of In-labeled DARPin The retention kinetics of radiolabeled DARPin06 and DARPin08 upon administration to mice bearing HER2-expressing tumors was assessed in a time-course biodistribution experiment. Accumulation in tissues of interest (kidney, blood, and tumor) was measured at 1, 4, 24, 48, or 96 hours after administration.

[0384] DARPin06 and DARPin08 used in this study were coupled to DTPA and loaded with indium-111 as described in Examples 1-3.

[0385] SKOV3ip tumor cells (5 × 10 in PBS) 6 The 4- to 5-week-old CD1-Foxn1 mice (control: CD1-Foxn1 mice) were subcutaneously implanted into the flanks. The mice were randomized into different treatment groups (four animals per group) and implanted at approximately 350 mm 3 At tumor volumes of 1000-12000 mg / kg, radiolabeled DARPin was injected intravenously (single dose) (same injection activity and formulation as in Example 6). Tumors and organs were harvested and measured 1, 4, 24, 48, or 96 hours after administration according to the same protocol as in Example 6. Table 13 shows a summary of the treatments and conditions.

[0386] [Table 13]

[0387] The data obtained are expressed as the mean injected activity per gram of tissue mass (% IA / g) and are shown in Figure 7. Error bars indicate the standard deviation.

[0388] The areas under the curve (AUC) over the measurement time points (1 hour to 96 hours) and the corresponding kidney-to-tumor ratios are shown in Table 14. Kidney accumulation is reduced by 76% (AUC) in the group treated with engineered DARPin06 compared to parent DARPin08, while tumor accumulation and blood retention remain similar between these groups.

[0389] [Table 14]

[0390] Example 8: Design and biodistribution of additional charge-engineered DARPin variants Based on the observed reduction in kidney accumulation caused by radiolabeled DARPin08, further charge-engineered DARPin variants with binding specificity for HER2 were designed and tested.

[0391] Four further DARPin variants (DARPins 13, 14, 15 and 16) were designed and produced according to the same methods as in Examples 1 and 2. Sequence details are shown in Figure 8 and Table 15.

[0392] [Table 15]

[0393] The parent DARPin 06, mutant DARPin 08, and DARPin mutants 13–16 were coupled to DTPA. Specifically, the His-tagged DARPins containing a C-terminal Cys were first reduced by incubating approximately 5 mg / mL of protein solution with a 10-fold excess of 0.5 M TCEP (pH adjusted to 7.6). The reaction was shaken at room temperature for 30 min. TCEP was subsequently removed from the reduced protein by gel filtration (Zeba spin column, 5 mL). After adding 0.5 M EDTA to the purified protein to a final concentration of 10 mM and stirring for 15 min, a 5-fold molar excess of 50 mM maleimide-DTPA (2,2'-(1-carboxy-2-(carboxymethyl)-13-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-10-oxo-2,5,8,11-tetraazatridecan-5,8-diyl)diacetic acid) dissolved in DMSO was added and stirred at room temperature for 1 h. Coupling efficiency was determined by reverse-phase HPLC (RP-HPLC), and samples were only taken if the coupling efficiency was greater than 95%. Excess chelator was removed by another gel filtration step (Zeba spin column, 5 mL), and the protein was rebuffered in metal-free PBS. The final concentration was determined by UV absorption and probed for ESI-MS analysis.

[0394] The results are shown in Table 16. The binding kinetics of the DTPA-coupled DARPin variants were determined as detailed below (Section 8.1). The constructs were subsequently coupled to the DTPA-coupled DARPin variants according to the methods detailed in Example 3. 111 Radiolabeled DARPins were generated by loading In. The resulting radiolabeled DARPins 13-16, as well as radiolabeled DARPin 06 and radiolabeled DARPin 08 shown in the previous examples, were tested in mouse biodistribution experiments, as described in further detail below (Section 8.2).

[0395] [Table 16]

[0396] 8.1 Binding kinetics The binding kinetics of the constructs to the target protein HER2 were measured in a multi-trace SPR assay. A new Xantec NAHLC200M chip was prepared according to the manufacturer's protocol. The chip was coated with biotinylated target (bio-HER2, 10 μg / mL) for 900 seconds to reach a signal intensity of approximately 1200-1300 RU. Three-fold dilutions of the analyte (50, 16.667, 5.556, 1.852, and 0.617 nM) were injected for 300 seconds, and dissociation was recorded for 1500 seconds (25 μL / min). Each injection was followed by a 60-second regeneration step with glycine pH 2.0. The signal (i.e., resonance unit (RU) value) of the blank surface and the reference injection (i.e., injection of running buffer only) were subtracted from the RU trace obtained after the injection of the construct (double referencing). Dissociation constants (K) were calculated from the globally fitted on- and off-rates using a standard 1:1-Langmuir model. D ) was calculated.

[0397] All charge-engineered mutants in this experiment exhibited 10 sigma when coupled to DTPA. -9 K less than M D The binding properties measured are shown in Table 17 and the SPR curves are shown in Figure 9.

[0398] [Table 17]

[0399] 8.2 In tumor-bearing mice 111 Biodistribution studies of In-labeled DARPins As described in further detail below, radiolabeled DARPins 06, 08, 13, 14, 15, and 16 were injected (approximately 150 KBq, 1 mg / kg body weight) into the tail vein of female mice (6-7 weeks old, control: CD1-Foxn1nu) bearing HER2-expressing SKOV3ip tumors. Compounds were formulated in PBS + 0.05% Tween 20.

[0400] SKOV3ip tumor cells (5 × 10 in PBS)6 The mice were randomized into different treatment groups (four animals per group) and implanted subcutaneously in the flanks of approximately 350 mm 3 With a tumor volume of 111 In-labeled DARPins were injected intravenously (single dose). Table 18 shows the treatment summary.

[0401] Accumulation in organs and tumors was measured 4 hours after injection. Mice were euthanized by CO2 inhalation and cervical dislocation. Kidneys and tumors were extracted and weighed, and radioactivity was measured using a gamma counter (Packard Cobra II Gamma D5010, GMI, USA). Data are expressed as the average injected activity per gram of tissue mass (%IA / g, equivalent to %ID / g) and are shown in Figure 10 (error bars indicate standard deviation).

[0402] [Table 18]

[0403] As shown in Figure 10A, the charge-modified DARPin variants according to the invention used in this experiment (i.e., radiolabeled DARPin 08 and 13-16) show lower renal accumulation of radioactivity compared to the parent DARPin (i.e., radiolabeled DARPin 06). The percent reduction in renal uptake for each variant compared to the parent DARPin is shown in Table 19.

[0404] [Table 19]

[0405] The accumulation of radioactivity in the tumors was quantified as well as in the kidneys. The difference in tumor accumulation between the parent DARPin and the engineered variants is negligible, as shown in Figure 10B. Table 20 further shows the tumor-to-kidney ratios measured for the HER2-binding DARPins used in this experiment. Charge engineering performed on DARPins 08, 13, 14, 15, and 16 resulted in an increase in the tumor-to-kidney ratio of 4.62-fold, 7.44-fold, 9.88-fold, 7.74-fold, and 6.03-fold, respectively, compared to the unengineered parent DARPin 06.

[0406] [Table 20]

[0407] In this study, further charge-modified DARPins according to the invention were successfully produced. As also observed in Example 6, this study shows that charge engineering of DARPins according to the invention does not impair target-specific tumor accumulation of radiolabeled DARPins, while providing a significant reduction in radiolabeled DARPin accumulation in the kidney. Furthermore, charge engineering of DARPins does not result in significant adverse accumulation in the further analyzed organs.

[0408] All autoradiography measurements shown in Examples 5 to 8 were decay-corrected. Data analysis was performed using Prism 9 (GraphPad Software LLC, San Diego, CA, USA).

[0409] The specification is most thoroughly understood in light of the teachings of the references cited within the specification. The embodiments within the specification provide illustrations of embodiments of the invention, but should not be construed as limiting the scope of the invention. One of ordinary skill in the art will readily recognize that many other embodiments are encompassed by the present invention. All publications, patents, and GenBank sequences cited in this disclosure are incorporated by reference in their entirety. To the extent that material incorporated by reference contradicts or is inconsistent with the specification, the specification will supersede any such material. The citation of any reference herein is not an admission that such reference is prior art to the present invention.

[0410] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, that there are many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

[0411] [Table 21-1]

[0412] [Table 21-2]

[0413] [Table 21-3]

[0414] [Table 21-4]

[0415] [Table 21-5]

[0416]

Table 21-6

[0417]

Table 21-7

[0418]

Table 21-8

[0419]

Table 21-9

[0420]

Table 21-10

Claims

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

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

3. 3. The repeat domain according to claim 1 or 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 is －７ Dissociation constant (K Ｄ 6. The repeat domain of claim 5, which binds to the target at

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

9. 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: 131-142, and (2) sequences in which up to nine framework residues in any of SEQ ID NOs: 131-142 are replaced 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 115-122, and (2) sequences in which up to nine framework residues in any of SEQ ID NOs: 25, and 115-122 are replaced by another amino acid; and / or the C-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 123-130, and (2) sequences in which up to nine framework residues in any of SEQ ID NOs: 123-130 are replaced by another amino acid.

10. 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 five 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 SEQ ID NO: 19, in which up to five framework residues other than positions 5, 17, 20, and 23 are substituted by another amino acid; and / or the C-terminal capping module comprises: (1) SEQ ID NO: 40; and (2) a sequence in which up to five 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. 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: 31-35, 61, 62, 92-94, and 131-142, and / or the N-terminal capping module comprises a sequence selected from SEQ ID NOs: 19-25, 57-60, 65-81, and 115-122, and / or the C-terminal capping module comprises a sequence selected from SEQ ID NOs: 40-42, 63, 64, 82-91, and 123-130.

12. 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: 26-30, 47, and 48; and (2) the sequences in which up to 9 amino acids in any of SEQ ID NOs: 26-30, 47, and 48 are replaced by another amino acid; and / or wherein the N-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 15-18, 43, and 99-101; and (2) the sequences in which up to 9 amino acids in any of SEQ ID NOs: 15-18, 43, and 99-101 are replaced by another amino acid; and / or wherein the C-terminal capping module comprises a sequence selected from the group consisting of: (1) SEQ ID NOs: 36-39, 54, 102, and 103; and (2) the sequences in which up to 9 amino acids in any of SEQ ID NOs: 36-39, 54, 102, and 103 are replaced by another amino acid.

13. 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 112-114; and (2) a sequence having at least 80% amino acid sequence identity among framework residues of any one of SEQ ID NOs: 104, 108, 109, and 112-114.

14. The repeat domain according to 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, 9, and 95 to 98; and (2) a sequence having at least 80% amino acid sequence identity to any one of the amino acid sequences of SEQ ID NOs: 8, 9, and 95 to 98.

15. 1. A method for producing an engineered ankyrin repeat domain, the method comprising steps (a) and (b), step (a) providing an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.07, and / or a percentage of basic amino acid residues among all amino acid residues contained in said repeat domain higher than 7.0%, and / or a percentage of basic amino acid residues among the framework residues contained in said repeat domain higher than 6.1%, and Step (b) comprises: (1) substituting at least one basic amino acid residue of said repeat domain of step (a) with a neutral or acidic amino acid residue; and / or (2) substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; The modified ankyrin repeat domain may have the following list of features: (i) an isoelectric point (pI) of pH 4.07 or less; (ii) the percentage of basic amino acid residues among all amino acid residues contained in the repeat domain is 7.0% or less; and (iii) the percentage of basic amino acid residues among the framework residues contained in the repeat domain is 6.1% or less.

16. comprising steps (a) and (b), step (a) providing an ankyrin repeat domain with an isoelectric point (pI) higher than pH 4.07; and Step (b) comprises: (1) substituting at least one basic amino acid residue of said repeat domain of step (a) with a neutral or acidic amino acid residue; and / or (2) substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; 16. The method of claim 15, wherein the modified ankyrin repeat domain has an isoelectric point (pI) of pH 4.07 or less.

17. comprising steps (a) and (b), Step (a) is a step of providing an 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 step (b) comprising substituting at least one basic amino acid residue of said repeat domain of step (a) with a neutral or acidic amino acid residue; 16. The method of claim 15, wherein the modified ankyrin repeat domain has a percentage of basic amino acid residues of 7.0% or less among all amino acid residues contained in the repeat domain.

18. comprising steps (a) and (b), step (a) providing an ankyrin repeat domain having a percentage of basic amino acid residues higher than 6.1% among the framework residues contained in said repeat domain; and step (b) comprising substituting at least one basic amino acid residue with a neutral or acidic amino acid residue among the framework residues of the repeat domain of step (a); 16. The method of claim 15, wherein the modified ankyrin repeat domain has a percentage of basic amino acid residues of 6.1% or less among the framework residues contained in the repeat domain.

19. 19. The method of 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 of 0.44 or less; and / or the repeat domain in step (a) has a KR / DE ratio among the framework residues higher than 0.36 and the modified repeat domain has a KR / DE ratio of 0.36 or less; and / or the repeat domain in step (a) has a KR / DE ratio among all residues of the N-terminal capping module higher than 0.66 and the modified repeat domain has a KR / DE ratio among all residues of the N-terminal capping module of 0.66 or less.

20. The repeat domain of step (a) specifically binds to a target and optionally －７ Dissociation constant (K Ｄ The method according to any one of claims 15 to 19, wherein the binding is carried out by

21. The modified repeat domain may be 10 －７ K less than M Ｄ and preferably the modified repeat domain specifically binds to the target at K Ｄ K is approximately equal to Ｄ or (2) the repeat domain of step (a) binds to the target Ｄ K less than 100 times, less than 10 times, less than 5 times, or less than 2 times higher than Ｄ 21. The method of claim 20, wherein the target is bound by

22. 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. 1. A designed ankyrin repeat domain comprising an N-terminal capping module, at least one internal repeat module, and a C-terminal capping module, said repeat domain being linked to a drug moiety, said repeat domain having the following list of features: (a) an isoelectric point (pI) of pH 4.6 or less; (b) the percentage of basic amino acid residues among all amino acid residues contained in the repeat domain is 12.0% or less; (c) the percentage of Arg residues and Lys residues among all amino acid residues contained in the repeat domain is 8.0% or less; (d) the percentage of basic amino acid residues among the framework residues contained in the repeat domain is 9.6% or less; and (e) the percentage of Arg and Lys residues among the framework residues contained in the repeat domain is 6.7% or less.

24. 24. The repeat domain according to claim 23, wherein said repeat domain has at least characteristic (a), and wherein said isoelectric point (pI) is optionally in the range of pH 3.0 to pH 4.6, preferably in the range of pH 3.3 to pH 4.6, more preferably in the range of pH 3.50 to pH 4.

53.

25. 25. The repeat domain of claim 23 or 24, wherein the repeat domain has at least characteristic (b), and the percentage in characteristic (b) is optionally 8.7% or less.

26. 26. The repeat domain according to any one of claims 23 to 25, wherein said repeat domain has at least characteristic (c), optionally with a percentage of characteristic (c) not greater than 4.7%.

27. 27. The repeat domain according to any one of claims 23 to 26, wherein said repeat domain has at least characteristic (d), optionally with a percentage of characteristic (d) not greater than 6.7%.

28. 28. The repeat domain according to any one of claims 23 to 27, wherein said repeat domain has at least characteristic (e), optionally with a percentage of characteristic (e) not greater than 5.7%.

29. 29. The repeat domain according to any one of claims 23 to 28, wherein the repeat domain has a KR / DE ratio of 0.44 or less, and / or a KR / DE ratio among the framework residues of 0.36 or less, and / or a KR / DE ratio among all residues of the N-terminal capping module of 0.66 or less.

30. 30. The repeat domain of any one of claims 23 to 29, wherein the repeat domain has a total number of basic amino acid residues equal to or less than n, where n=4+5R, where R is the number of internal repeat modules comprised in the repeat domain.

31. 31. The repeat domain of any one of claims 23 to 30, wherein the repeat domain has a total number of Arg and Lys residues not greater than m, where m=5+2R, and R is the number of internal repeat modules comprised in the repeat domain.

32. 32. The repeat domain according to any one of claims 23 to 31, wherein the repeat domain has no Arg or Lys residues in the framework residue positions.

33. 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 according to any one of claims 23 to 33, wherein the repeat domain specifically binds to a target.

35. The repeat domain is 10 －７ Dissociation constant (K Ｄ 35. The repeat domain of claim 34, which binds to the target at

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. 38. The repeat domain of claim 37, wherein the toxin is a radionuclide.

39. 38. The repeat domain of claim 37, wherein the toxin is a cytotoxin.

40. 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: 104-114; (2) a sequence having at least 80% amino acid sequence identity among the framework residues of any one of SEQ ID NOs: 104-114.

41. 41. The repeat domain of 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: 104-112, (2) a sequence having at least 80% amino acid sequence identity among the framework residues of any one of SEQ ID NOs: 104-112.

42. The repeat domain of 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: 8-10, 12, and 95-98; (2) a sequence having at least 80% amino acid sequence identity to SEQ ID NOs: 8-10, 12, and 95-98.

43. 43. A repeat domain linked to a drug moiety for use in a method for treating and / or diagnosing a medical condition, said method comprising administering to a patient in need of said treatment and / or diagnosis a therapeutically and / or diagnostically effective amount of said repeat domain, optionally wherein said administration is not oral.

44. 44. A designed ankyrin repeat domain according to claim 43, linked to a drug moiety for use in a method for treating and / or diagnosing a medical condition, wherein the medical condition is cancer.

45. 1. A method for producing a modified ankyrin repeat domain linked to a drug moiety, said method comprising steps (a) and (b): step (a) providing an ankyrin repeat domain having an isoelectric point (pI) higher than pH 4.6, and / or a percentage of basic amino acid residues among all amino acid residues contained in said repeat domain higher than 12.0%, and / or a percentage of Arg and Lys residues among all amino acid residues contained in said repeat domain higher than 8.0%, and / or a percentage of basic amino acid residues among the framework residues contained in said repeat domain higher than 9.6%, and / or a percentage of Arg and Lys residues among the framework residues contained in said repeat domain higher than 6.7%, and Step (b) comprises: (1) substituting at least one basic amino acid residue of said repeat domain of step (a) with a neutral or acidic amino acid residue; and / or (2) substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; The modified repeat domain has the following list of features: (i) an isoelectric point (pI) of pH 4.6 or less; (ii) the percentage of basic amino acid residues among all amino acid residues contained in the repeat domain is 12.0% or less; (iii) the percentage of Arg residues and Lys residues among all amino acid residues contained in the repeat domain is 8.0% or less; (iv) the percentage of basic amino acid residues among the framework residues contained in the repeat domain is 9.6% or less; and (v) the percentage of Arg and Lys residues among the framework residues contained in the repeat domain is 6.7% or less.

46. comprising steps (a) and (b), step (a) providing an ankyrin repeat domain with an isoelectric point (pI) higher than pH 4.6; and Step (b) comprises: (1) substituting at least one basic amino acid residue of said repeat domain of step (a) with a neutral or acidic amino acid residue; and / or (2) substituting at least one neutral amino acid residue in the repeat domain of step (a) with an acidic amino acid; 46. The method of claim 45, wherein the modified repeat domain has an isoelectric point (pI) of pH 4.6 or less.

47. comprising steps (a) and (b), Step (a) is a step of providing an ankyrin repeat domain having a percentage of basic amino acid residues higher than 12.0% among all amino acid residues contained in said repeat domain; and step (b) comprising substituting at least one basic amino acid residue of said repeat domain of step (a) with a neutral or acidic amino acid residue; 46. The method of claim 45, wherein the modified repeat domain has a percentage of basic amino acid residues of 12.0% or less among all amino acid residues contained in the repeat domain.

48. comprising steps (a) and (b), Step (a) is a step of providing an ankyrin repeat domain having a percentage of Arg and Lys residues of more than 8.0% among all amino acid residues contained in said repeat domain; and step (b) comprising substituting at least one Arg and Lys residue of said repeat domain of step (a) with a neutral or acidic amino acid residue; 46. The method of claim 45, wherein the modified repeat domain has a proportion of Arg and Lys residues of 8.0% or less among all amino acid residues contained in the repeat domain.

49. comprising steps (a) and (b), step (a) providing an ankyrin repeat domain having a percentage of basic amino acid residues higher than 9.6% among the framework residues contained in said repeat domain; and step (b) comprising substituting at least one basic amino acid residue with a neutral or acidic amino acid residue among the framework residues of the repeat domain of step (a); 46. The method of claim 45, wherein the modified repeat domain has a percentage of basic amino acid residues of 9.6% or less among the framework residues contained in the repeat domain.

50. comprising steps (a) and (b), step (a) providing an ankyrin repeat domain having a percentage of Arg and Lys residues among the framework residues contained in said repeat domain that is higher than 6.7%; and step (b) comprising substituting at least one Arg or Lys residue among the framework residues of the repeat domain of step (a) with a neutral or acidic amino acid residue; 46. The method of claim 45, wherein the modified repeat domain has a percentage of Arg and Lys residues among the framework residues contained in the repeat domain of 6.7% or less.

51. 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 of 0.44 or less; and / or the repeat domain in step (a) has a KR / DE ratio among the framework residues higher than 0.36 and the modified repeat domain has a KR / DE ratio of 0.36 or less; and / or the repeat domain in step (a) has a KR / DE ratio among all residues of the N-terminal capping module higher than 0.66 and the modified repeat domain has a KR / DE ratio among all residues of the N-terminal capping module of 0.66 or less.

52. 52. The method of any one of claims 45 to 51, further comprising linking a drug moiety to the modified repeat domain.

53. The repeat domain of step (a) specifically binds to a target and optionally －７ Dissociation constant (K Ｄ The method according to any one of claims 45 to 52, wherein the binding is carried out at

54. The modified repeat domain may be 10 －７ K less than M Ｄ and preferably the modified repeat domain specifically binds to the target at K Ｄ K is approximately equal to Ｄ or (2) the repeat domain of step (a) binds to the target Ｄ K less than 100 times, less than 10 times, less than 5 times, or less than 2 times higher than Ｄ 54. The method of claim 53, wherein the target is bound by

55. 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 or obtained by a method according to any one of claims 15 to 22 or 45 to 55.

57. A recombinant protein comprising the designed repeat domain of any one of claims 1 to 14, 23 to 42 and 56.

58. 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 protein of claim 57.

59. 59. A recombinant expression vector comprising the nucleic acid of claim 58.

60. 60. A host cell comprising the recombinant expression vector of claim 59.

61. 19. A pharmaceutical composition comprising one or more of: (i) the repeat domain of any one of claims 1 to 14, 23 to 42, and 56; (ii) the recombinant protein of claim 57; (iii) the nucleic acid of claim 58; and / or (iv) the recombinant expression vector of claim 59, and optionally at least one pharmaceutically acceptable carrier or diluent.

62. 62. A method of treating and / or diagnosing a medical condition, comprising administering to a patient in need thereof a therapeutically and / or diagnostically effective amount of a designed repeat domain of any one of claims 1 to 14, 23 to 42 and 56, a recombinant protein of claim 57, a nucleic acid of claim 58, or a pharmaceutical composition of claim 61.

63. 63. The method of claim 62, wherein the medical condition is cancer.

64. 62. 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 for use in a method for treating and / or diagnosing a medical condition.

65. 65. The repeat domain, recombinant protein, nucleic acid or pharmaceutical composition for use according to claim 64, wherein the medical condition is cancer.

Images