Designed repeat domains with dual binding specificity and uses thereof

Abstract

The present invention relates to a method for producing a designed repeat domain with binding specificities for two targets, where the binding of such repeat domain to the two targets is mutually exclusive. Preferably, the designed repeat domain is a designed ankyrin repeat domain. The present invention also provides such designed repeat domains and recombinant proteins comprising such repeat domains, as well as the use of such repeat domains as molecular switches, e.g. switches for controlling the activation or deactivation of a therapeutic agent. The present invention further relates to nucleic acids encoding such repeat domains or recombinant proteins, pharmaceutical compositions, recombinant expression vectors and host cells comprising such recombinant proteins, repeat domains or nucleic acids, as well as 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 No. 21214519, filed December 14, 2021. The disclosures of these patent applications are incorporated herein by reference in their entireties for all purposes.

[0002] FIELD OF THEINVENTION The present invention relates to a method for producing a designed repeat domain with binding specificities for two targets, where the binding of such repeat domain to the two targets is mutually exclusive. Preferably, the designed repeat domain is a designed ankyrin repeat domain. The present invention also provides such designed repeat domains and recombinant proteins comprising such repeat domains, as well as the use of such repeat domains as molecular switches, e.g. switches for controlling the activation or deactivation of a therapeutic agent. The present invention further relates to nucleic acids encoding such repeat domains or recombinant proteins, pharmaceutical compositions, recombinant expression vectors and host cells comprising such recombinant proteins, repeat domains or nucleic acids, as well as the use of such proteins, nucleic acids or pharmaceutical compositions in methods for treating diseases, such as cancer. [Background technology]

[0003] Finding the optimal balance between drug efficacy and safety is an ongoing challenge in targeted drug development. Numerous strategies have been explored to improve the precision and specificity of drugs, thereby limiting their adverse side effects. Although some strategies may have reached preclinical proof-of-concept, many of them face barriers when moving to clinical application. Therefore, finding mechanisms to reliably control the speed, time and / or location of therapeutic activity of drug molecules in the body remains highly desirable.

[0004] Antibodies and their inherent affinity and selectivity properties are widely used in therapy. Making antibodies activatable under specific conditions can provide a layer of control to focus their therapeutic effect at the desired site of action. To do so, antibodies can be engineered to respond to endogenous or exogenous stimuli such as light, temperature, pH, enzymatic activity, ions, combinations of effector molecules and antigens (Lucchi, R. et al., 2021, ACS Central Science, 7(5), 724-738).

[0005] The tumor microenvironment (TME) provides various biochemical cues that can potentially be exploited as triggers for drug activation. For example, antibody on / off switch mechanisms have been developed that sense the levels of extracellular adenosine triphosphate (ATP), which is significantly elevated in the TME compared to healthy tissue. Such Switch Antibody™ (or Switch-Ig™) molecules have been reported to initiate binding to their targets in the presence of 10 mM ATP, and have consistently shown activity comparable to non-switched control antibodies at 100 mM ATP and negligible activity at 1 mM ATP. Nevertheless, their production requires time- and labor-consuming procedures, including identifying anti-ATP antibodies to be used as library templates, designing and constructing synthetic libraries of antibodies with ATP-binding motifs in their complementarity determining regions (CDRs), and identifying ATP-dependent antigen-binding antibodies by phage panning (Mimoto, F. et al., 2020, Cell Reports, 33(12), 108542).

[0006] Further approaches to drug activation switches have been developed in the context of chimeric antigen receptor (CAR) T cell therapy (Yu, S. et al., 2019, Molecular Cancer, 18(1), 1-13). CAR on-switches have been designed in which the activation of engineered T cells depends on the presence of a small molecule that induces heterodimerization of the target antigen and the CAR module. Neither small molecules nor antigens alone activate CAR T cells. These small molecule-dependent on-switch approaches can reduce toxicity by controlling the timing, location, and dose of T cell activity (Wu, CY et al., 2015, Science, 350(6258); Zajc, CU et al., 2020, PNAS, 117(26), 14926-14935). Off-switch mechanisms can also be implemented by relying on protein degradation regulation. An example of such an off-switch relies on lenalidomide (Revlimid®), which can induce CAR degradation. To turn off CAR signaling, lenalidomide induces ubiquitination and subsequent proteasomal degradation of degron-tagged CAR. As the level of the small molecule decreases, CAR signaling can be restored by translation of the CAR protein (Jan, M. et al., 2021, Science Translational Medicine, 13(575); WO 2019 / 089592 A1). However, such coordinated mechanisms to mitigate adverse effects in adoptive cell therapy are not always applicable in non-cell therapy modalities.

[0007] Another control mechanism is provided by reversible on / off molecular switches based on allosteric regulation. Allosteric proteins can act as molecular switches in which the binding of a molecule to a site distinct from the active site of the protein changes the conformation of the protein. Although more prevalent as biosensors, modular allosteric switches have been designed to control the activity of proteins via effector molecules. In one such design, a synthetic biligand molecule with mutually exclusive binding sites is fused to the protein of interest via a linker. In the absence of the effector molecule, the biligand binds to the active site of the protein, thereby inhibiting the activity of the protein. In the presence of the effector, binding of the effector to the ligand molecule releases the inhibitor of the protein, thereby activating the protein (Schena, A. et al., 2015, Nature Communications, 6(1), 1-10; WO 2015 / 067302 A1). However, the application of this approach to therapeutically relevant targets is limited by the need to select or identify allosteric inhibitor molecules specific for the target to be switched on / off. Further limitations may arise from the design requirements of the fused biligand, which in this particular case includes a fluorescent dye.

[0008] Modular protein switches comprising alternative binding scaffolds (monobodies and designed ankyrin repeat proteins (or DARPins)) have also been proposed (Nicholes, N. et al., 2016, Protein Engineering, Design and Selection, 29(2), 77-85; WO 2003 / 078575 A2). In this approach, the binding scaffold comprises an input domain, such as a DARPin with binding affinity for maltose binding protein (MBP), and further comprises an output domain, such as a β-lactamase enzyme, fused to a selected site in the input domain, such as between the first and second internal repeats of the DARPin. Although some expected switch activity (i.e., scaffold-bound ligand-dependent enzymatic activity of β-lactamase) was observed, fused DARPins that showed switch properties suffered a significant loss of affinity for their ligands compared to the corresponding non-fused DARPins. Also, when attempting to generalize the applicability of these switch constructs to additional ligands by mutagenesis, only limited success was observed in redirecting the specificity of the binding scaffold.

[0009] In summary, currently available molecular drug switch approaches are time-, cost- and / or labor-intensive and limited in their capabilities and applications. Therefore, new molecular switch designs are still needed. Such switch designs may be useful in controlling protein activity (e.g., protein-based therapeutics). Summary of the Invention

[0010] The present invention provides a method for producing a designed repeat domain with binding specificity for two targets, where the binding of the domain to both targets is mutually exclusive. Preferably, the designed repeat domain is a designed ankyrin repeat domain. The present invention also provides such designed ankyrin repeat domains and recombinant proteins comprising such repeat domains, as well as the use of such repeat domains as molecular switches, e.g. switches for controlling the activation or deactivation of therapeutic agents. In addition, the present invention provides nucleic acids encoding such repeat domains or recombinant proteins, pharmaceutical compositions comprising such recombinant proteins, repeat domains or nucleic acids, recombinant expression vectors and host cells, the use of such proteins, nucleic acids or pharmaceutical compositions in methods for treating diseases such as cancer in mammals, including humans.

[0011] In one aspect, the invention provides a method for producing a designed repeat domain, the method comprising: (i) providing a first repeat domain and a second repeat domain, wherein the first repeat domain has binding specificity for a first target and the second repeat domain has binding specificity for a second target; (ii) covalently combining at least one repeat module of the first repeat domain with at least one repeat module of the second repeat domain, thereby linking a part of the first repeat domain with a part of the second repeat domain to form a designed repeat domain with dual binding specificity, The designed repeat domain has binding specificity for a first target and binding specificity for a second target, and the binding of the designed repeat domain to the first and second targets is mutually exclusive.

[0012] In another aspect, the present invention provides designed repeat domains obtainable by the above methods.

[0013] In another aspect, the invention provides an engineered repeat domain having a first binding specificity for a first target and a second binding specificity for a second target, wherein binding of the repeat domain to the first and second targets is mutually exclusive.

[0014] The designed repeat domains of the present invention bind to their two targets only in a mutually exclusive manner, i.e., once a repeat domain binds to one target molecule, binding of that domain to the other target molecule is prevented primarily by steric hindrance effects.

[0015] The repeat domain of the present invention can be advantageously used as a molecular on / off switch mechanism that allows conditional activation or inactivation of the biological activity of a therapeutic agent. Such biological activity of a therapeutic agent may be, for example, the binding of the therapeutic agent to a biological target. In such a scenario, the repeat domain of the present invention is chemically linked to a therapeutic agent, the first target molecule of the repeat domain is the therapeutic agent, and the second target molecule is a sensor target molecule. Preferably, such a sensor target molecule is a molecule that is associated with a pathological condition, for example, selectively expressed or overexpressed in a pathological condition. One such pathological condition is cancer, and the sensor target molecule is selectively expressed or overexpressed in tumor tissue.

[0016] Thus, in another embodiment, the present invention provides a recombinant protein comprising such a designed repeat domain of the invention. In one particular embodiment, such a recombinant protein comprising such a designed repeat domain of the invention further comprises a therapeutic agent. In one embodiment, the therapeutic agent is a binding molecule. In a further embodiment, the binding molecule comprises an ankyrin binding domain.

[0017] The state (active / on or inactive / off) of the therapeutic agent in the recombinant protein of the invention depends on the binding of the designed repeat domain of the invention to its first and second targets. In one aspect, in the on state, the designed repeat domain of the invention cannot bind to its first target (e.g., a therapeutic agent) due to the presence of a second target (e.g., a sensor target) that is substantially bound to the designed repeat domain (see, e.g., the on switch in FIG. 3A). Thus, the first target (e.g., a therapeutic agent) is not bound and is active. In the off state, the second target is substantially absent, and as a result, the designed repeat domain binds to the first target (e.g., a therapeutic agent), thereby preventing its normal activity.

[0018] In another embodiment, the present invention provides a recombinant protein comprising such a designed repeat domain of the present invention, which does not further comprise a therapeutic agent. The state (active / on or inactive / off) of such a recombinant protein of the present invention may also depend on the binding of the designed repeat domain of the present invention to its first and second targets. In the on state, the designed repeat domain of the present invention can bind to its first target (e.g. a cell surface receptor) because the second target (e.g. a sensor target) is not present in substantial amounts and therefore does not bind to the designed repeat domain (see, for example, the off switch in FIG. 3B). Thus, the first target (e.g. a cell surface receptor) is bound by the designed repeat domain of the present invention, thereby activating or inhibiting its biological activity. In the off state, the second target (e.g. a sensor target) is present in substantial amounts and therefore binds to the designed repeat domain, thereby preventing the binding of the designed repeat domain to the first target.

[0019] In another aspect, the present invention provides an isolated nucleic acid encoding a designed repeat domain of the invention or encoding a recombinant protein of the invention, a recombinant expression vector comprising such a nucleic acid, a host cell comprising such an expression vector, and a pharmaceutical composition comprising the designed repeat protein, recombinant protein, nucleic acid, recombinant expression vector and / or host cell of the invention and at least one pharma- ceutically acceptable carrier or diluent.

[0020] In another aspect, the present invention provides a method for treating a medical condition, comprising administering to a patient in need of treating the medical condition a therapeutically 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.

[0021] In another aspect, the invention provides an engineered repeat domain, recombinant protein, nucleic acid, or pharmaceutical composition of the invention for use in a method of treating a medical condition. In one particular aspect, the medical condition is cancer.

[0022] Based on the disclosure provided herein, those of ordinary skill 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.

[0023] E1. A method for producing an engineered repeat domain, comprising: (i) providing a first repeat domain and a second repeat domain, wherein the first repeat domain has binding specificity for a first target and the second repeat domain has binding specificity for a second target; (ii) linking a part of the first repeat domain with a part of the second repeat domain by covalently combining at least one repeat module of the first repeat domain with at least one repeat module of the second repeat domain to form a designed repeat domain with dual binding specificity, A method, wherein the designed repeat domain has binding specificity for a first target and binding specificity for a second target, and the binding of the designed repeat domain to the first and second targets is mutually exclusive.

[0024] E2. The method of E1, wherein covalently associating at least one repeat module of the first repeat domain with at least one repeat module of the second repeat domain comprises covalently connecting at least one repeat module of the first repeat domain with at least one repeat module of the second repeat domain.

[0025] E2.1. The method of E2, wherein at least one repeat module of the first repeat domain and at least one repeat module of the second repeat domain are covalently connected by a linker.

[0026] E2.2. The method of E2.1, wherein the linker is a peptide linker or a non-linked repeat module.

[0027] E3. The method of E1, wherein covalently combining at least one repeat module of the first repeat domain with at least one repeat module of the second repeat domain comprises covalently combining at least one repeat module of the first repeat domain with at least one repeat module of the second repeat domain.

[0028] E4. Any one of the methods of E1-E3, wherein the designed repeat domain comprises an N-terminal capping module and / or a C-terminal capping module and at least one internal repeat module.

[0029] E5. The method of E4, wherein the N-terminal capping module is at least 90% identical in amino acid sequence to the N-terminal capping module comprised in the first repeat domain and the C-terminal capping module is at least 90% identical in amino acid sequence to the C-terminal capping module comprised in the second repeat domain.

[0030] E6. Any one of the methods E1-E5, wherein the first repeat domain portion comprises an N-terminal capping module and at least one repeat module and the second repeat domain portion comprises a C-terminal capping module and at least one repeat module.

[0031] E7. The method of any one of E1-E6, wherein the N-terminal fragment of the first repeat domain is covalently associated with a C-terminal fragment of the second repeat domain.

[0032] E8. Any one of the methods of E1-E7, wherein step (ii) comprises removing the C-terminal capping module of the first repeat domain and removing the N-terminal capping module of the second repeat domain, such that the C-terminal capping module of the first repeat domain and the N-terminal capping module of the second repeat domain are not included in the designed repeat domain.

[0033] E9. The method of any one of E1-E8, wherein the designed repeat domain, when bound to the first target or the second target, inhibits a biological activity of the bound target.

[0034] E10. Any one of the methods E1-E9, wherein binding of the designed repeat domain to one of the first and second targets sterically hinders binding of the designed repeat domain to the other of the first and second targets.

[0035] E11. The designed repeat domain is -5 Dissociation constant (K D ) to bind to the first target.

[0036] E12. The designed repeat domain is -5 Dissociation constant (K D ) to bind to a second target.

[0037] E13. The designed repeat domain binds to one of the first and second targets with a first binding affinity and to the other of the first and second targets with a second binding affinity, and the ratio of the first binding affinity to the second binding affinity is between 1:1 and 1:10. 5 Any one of methods E1 to E12.

[0038] E14. The method of any one of E1-E13, wherein the first and second targets are located on different molecules.

[0039] E15. The method of any one of E1-E14, wherein the first repeat domain is an ankyrin repeat domain and the second repeat domain is an ankyrin repeat domain.

[0040] E16. The binding protein of any one of E1-E15, wherein the engineered repeat domain is an engineered ankyrin repeat domain.

[0041] E17. A designed repeat domain obtainable by any one of methods E1-E16.

[0042] E18. A designed repeat domain having a first binding specificity for a first target and a second binding specificity for a second target, wherein binding of the designed repeat domain to the first and second targets is mutually exclusive.

[0043] E19. An engineered repeat domain of E18, wherein the engineered repeat domain, when bound to a first target or a second target, inhibits a biological activity of the bound target.

[0044] E20. The designed repeat domain of any one of E18-E19, wherein binding of the designed repeat domain to one of the first and second targets sterically hinders binding of the designed repeat domain to the other of the first and second targets.

[0045] E21. The designed repeat domain is -5 Dissociation constant (K D ) any one of the designed repeat domains E18 to E20 that bind to the first target.

[0046] E22. The designed repeat domain is -5 Dissociation constant (K D ) any one of the designed repeat domains E18 to E21 that bind to a second target.

[0047] E23. The designed repeat domain binds to one of a first and a second target with a first binding affinity and binds to the other of the first and the second target with a second binding affinity, and the ratio of the first binding affinity to the second binding affinity is between 1:1 and 1:10. 5 Any one of the designed repeat domains E18 to E22.

[0048] E24. The designed repeat domain of any one of E18-E23, wherein the first and second targets are located on different molecules.

[0049] E25. The designed repeat domain of any one of E18 to E24, wherein the designed repeat domain comprises at least three repeat modules.

[0050] E26. The designed repeat domain of any one of E18 to E25, wherein the designed repeat domain comprises an N-terminal capping module and / or a C-terminal capping module and at least one internal repeat module, at least two internal repeat modules, at least three internal repeat modules or at least four internal repeat modules.

[0051] E27. The designed repeat domain of any one of E18 to E26, wherein all internal repeat modules comprised in the designed repeat domain contribute to the first and / or second binding specificity.

[0052] E28. The designed repeat domain of any one of E18 to E27, wherein the designed repeat domain comprises at least one internal repeat module that contributes to binding to a first target and wherein the designed repeat domain comprises at least one internal repeat module that contributes to binding to a second target.

[0053] E29. The designed repeat domain of any one of E18 to E28, wherein the designed repeat domain is an ankyrin repeat domain.

[0054] E30. The designed repeat domain of any one of E18 to E29, wherein the designed repeat domain comprises at least one, at least two, at least three, at least four, or at least five target interaction residues that bind to a first target, and the designed repeat domain comprises at least one, at least two, at least three, at least four, or at least five target interaction residues that bind to a second target.

[0055] E31. The designed repeat domain of any one of E18 to E30, wherein the designed repeat domain comprises at least one internal repeat module comprising a sequence selected from SEQ ID NOs: 75 to 81.

[0056] E32. Any one of the designed repeat domains of E18 to E31, comprising: (i) an N-terminal capping module comprising a sequence selected from the group consisting of (1) SEQ ID NOs: 35, 36, 47 to 63, and (2) SEQ ID NOs: 35, 36, 47, 53, 59, 60, 83, 84, in which up to nine amino acids have been replaced by other amino acids; and (ii) a C-terminal capping module comprising a sequence selected from the group consisting of (1) SEQ ID NOs: 37, 38, 64 to 74, and (2) SEQ ID NOs: 37, 38, 69, in which up to nine amino acids have been replaced by other amino acids.

[0057] E32.1. The designed repeat domain is Any one of the designed repeat domains E18 to E31, comprising at least one internal repeat module comprising a sequence selected from the group consisting of (i) (1) SEQ ID NOs: 35, 36, 47 to 63, 83 to 85, and 97 to 100, and (2) any one of SEQ ID NOs: 35, 36, 47, 53, 59, 60, 83, 84, in which up to 9 amino acids have been replaced by other amino acids; and / or (ii) (1) SEQ ID NOs: 37, 38, and 64 to 74, and (2) any one of SEQ ID NOs: 37, 38, 69, in which up to 9 amino acids have been replaced by other amino acids; and / or (iii) (1) SEQ ID NOs: 40 to 46, and (2) any one of SEQ ID NOs: 40 to 46, in which up to 9 amino acids have been replaced by other amino acids.

[0058] E33. The designed repeat domain is any one of E18 to E32.1, comprising at least one internal repeat module comprising a sequence selected from (1) SEQ ID NOs: 40 to 46 and (2) any one of SEQ ID NOs: 40 to 46 in which up to 9 amino acids are substituted with other amino acids.

[0059] E34. The designed repeat domain of any one of E18 to E33, comprising: (i) an N-terminal capping module comprising a sequence selected from the group consisting of (1) SEQ ID NOs: 35 to 36 and (2) any of SEQ ID NOs: 35 to 36 in which up to 9 amino acids have been replaced by other amino acids; and / or (ii) a C-terminal capping module comprising a sequence selected from the group consisting of (1) SEQ ID NO: 38 and (2) any of SEQ ID NO: 38 in which up to 9 amino acids have been replaced by other amino acids.

[0060] E35. The designed repeat domain of any one of E18 to E34, wherein the designed repeat domain comprises at least one internal repeat module and wherein residues at positions corresponding to positions 3, 4, 6, 11, 14, and / or 15 of SEQ ID NO: 39 are target interaction residues.

[0061] E36. The designed repeat domain of any one of E18 to E35, wherein the designed repeat domain comprises at least one N-terminal capping module and the residues at positions corresponding to positions 4, 5, 8, 11 and / or 12 of SEQ ID NO: 36 are target interaction residues.

[0062] E37. The designed repeat domain of any one of E18 to E36, wherein the designed repeat domain comprises at least one C-terminal capping module and wherein the residues at positions corresponding to positions 3, 4, 6, 14 and / or 15 of SEQ ID NO: 38 are target interaction residues.

[0063] E38. The designed repeat domain of any one of E18 to E37, wherein the designed repeat domain comprises two internal repeat modules, the N-terminal one of the two internal repeat modules comprising target interaction residues that bind to a first target and the C-terminal one of the two internal repeat modules comprising target interaction residues that bind to a second target.

[0064] E39. The designed repeat domain of any one of E18 to E37, wherein the designed repeat domain comprises three internal repeat modules, wherein the most N-terminal of the three internal repeat modules comprises target interaction residues that bind a first target, the most C-terminal of the three internal repeat modules comprises target interaction residues that bind a second target, and a central one of the three internal repeat modules comprises target interaction residues that bind the first and / or second target.

[0065] E40. The designed repeat domain of E39, comprising an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 6-10, and (2) a sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 6-10.

[0066] E41. The designed repeat domain of any one of E18 to E37, wherein the designed repeat domain comprises four internal repeat modules, wherein the most N-terminal of the four internal repeat modules comprises target interaction residues that bind a first target, the most C-terminal of the four internal repeat modules comprises target interaction residues that bind a second target, and wherein the second most N-terminal and second most C-terminal of the four internal repeat modules each comprise target interaction residues that bind the first target and / or the second target.

[0067] E42. The designed repeat domain of E41, comprising an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 11-13, and (2) a sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 11-13.

[0068] E43. A recombinant protein comprising the designed repeat domain of any one of claims 17-42.

[0069] E44. The protein of E43, wherein the protein further comprises a therapeutic agent.

[0070] E45. The protein of E44, wherein the therapeutic agent is a binding molecule.

[0071] E46. A protein of E45 containing an ankyrin repeat domain for which a binding molecule has been designed.

[0072] E47. The protein of E45 or E46, wherein the binding molecule is a T cell engager.

[0073] E48. An isolated nucleic acid encoding any one of the designed repeat domains E17 to E42 or any one of the proteins E43 to E47.

[0074] A recombinant expression vector comprising a nucleic acid of E49.E48.

[0075] A host cell comprising a recombinant expression vector of E50.E49.

[0076] E51. A pharmaceutical composition comprising one or more of: (i) a designed repeat domain of any one of E17 to E42, (ii) a recombinant protein of any one of E43 to E47, (iii) a nucleic acid described in E48, and / or (iv) a recombinant expression vector described in E49, and optionally a pharma- ceutically acceptable carrier or diluent.

[0077] E52. A method for treating a medical condition is provided, the method comprising the step of administering to a patient in need of treating the medical condition a therapeutically effective amount of a designed repeat domain of any one of E17 to E42, a recombinant protein of any one of E43 to E47, a nucleic acid of E48, or a pharmaceutical composition of E51.

[0078] E53. The method of E52, wherein the medical condition is cancer.

[0079] E54. A designed repeat domain of any one of E17 to E42, a recombinant protein of any one of E43 to E47, a nucleic acid of E48, or a pharmaceutical composition of E51 for use in a method for treating a medical condition.

[0080] E55. A designed repeat domain, recombinant protein, nucleic acid or pharmaceutical composition for use in E54, wherein the medical condition is cancer. [Brief description of the drawings]

[0081] [Figure 1]Description of the designed repeat domains of the invention, as exemplified by the designed ankyrin repeat domains of the invention, also called "2-in-1 DARPins" or "SwitchDARPins" (6). DARPins typically comprise an N-terminal cap (N-cap), one or more internal repeat modules (most commonly one, two or three), and a C-terminal cap (C-cap). The target (or antigen) binding paratope (or binding surface) (4, 5) is a continuous surface formed by one or several of these repeat modules. 2-in-1 DARPins can be formed by genetically combining the N-cap + one or several internal repeat modules of a first DARPin that binds to target A with the C-cap + one or several internal repeat modules from a second DARPin that binds to target B (two parent DARPins are shown as (1) and (2)). The resulting 2-in-1 DARPin (6) then typically contains an N-cap, a C-cap and at least one internal repeat module, most commonly two internal repeat modules (called N2C), three internal repeat modules (N3C) or four internal repeat modules (N4C). It may contain even more internal repeat modules (NxC). A 2-in-1 DARPin can bind to target A and target B, but not simultaneously (see FIG. 2). Furthermore, one or some of the internal repeat modules in a 2-in-1 DARPin may be a mixed (or merged) repeat module (7). A repeat module is called a mixed repeat module if its binding surface comprises at least one target interaction residue from a corresponding module of a first parent repeat protein and at least one target interaction residue from a corresponding module of a second parent repeat protein. [Diagram 2]The concept of mutually exclusive binding is illustrated: the proximity or overlap of the two paratopes of a 2-in-1 DARPin (overlap is represented by a diagonal line) allows the molecule to bind target A (Figure 2A) or target B (Figure 2B) individually, but not both targets simultaneously, since simultaneous binding of both targets A and B is sterically hindered (Figure 2C). The steric clash is represented by an asterisk between target A and target B. [Diagram 3] General examples of constructs comprising the 2-in-1 DARPin of the present invention. These exemplary constructs are also referred to as "Switchdrug DARPins". In FIG. 3A, an exemplary on-switch mechanism is shown, in which the 2-in-1 DARPin binds to target A (e.g., a sensor target) or target B (e.g., a drug molecule) in a mutually exclusive manner and is chemically linked to target B (e.g., a drug molecule). The state of target B (e.g., an active or inactive drug molecule) as part of the Switchdrug DARPin is conditionally regulated by the interaction of the 2-in-1 DARPin with target A, and the binding of target A by the 2-in-1 DARPin results in the release of the bound target B (and thus the active drug molecule). In the exemplary off-switch setting shown in FIG. 3B, the 2-in-1 DARPin is designed to bind to target A (e.g., a sensor target) or target B (e.g., a cell receptor). A downstream activity resulting from the interaction of a 2-in-1 DARPin with target B can be conditionally switched off upon binding of target A to the 2-in-1 DARPin. In such Switchdrug DARPins, the 2-in-1 DARPin can be further linked to other binding domains or molecules. [Figure 4]Exemplary design of Switchdrug DARPins according to the invention as on and off switches for conditionally regulating T cell engagers (TCEs). In the on-switch concept (Figure 4A), the T cell engager comprises a CD3-binding DARPin (target B) and a tumor-associated antigen (TAA)-binding DARPin. A 2-in-1 DARPin is used that can bind and block the paratope of the CD3-binding DARPin, which can alternatively bind to a molecule that is predominantly present in the tumor microenvironment, represented as target A. This 2-in-1 DARPin is chemically bound to the TCE to form a Switchdrug DARPin. A high concentration of target A induces the release of the CD3-binding DARPin (target B), allowing the recruitment of cytotoxic T cells and the formation of an immunological synapse between TAA-expressing tumor cells and T cells. In the exemplary off-switch concept (Figure 4B), a 2-in-1 DARPin is used that binds to target A (sensor target) and alternatively binds to target B (CD3). The TCE in this case comprises a tumor-associated antigen (TAA)-binding DARPin chemically linked to a 2-in-1 DARPin. At high concentrations of target A, the TCE can be silenced by binding to target A, thereby preventing the binding of TCE to CD3 on T cells. [Figure 5A]Detailed representation of a 2-in-1 DARPin that can be used as a VEGF-triggered T-cell engager Switchdrug DARPin. The 2-in-1 DARPin shown in FIG. 1 was created by combining a first VEGF-binding DARPin, also called "VEGF-binder" (SEQ ID NO: 1), which has high affinity for VEGF, with a second DARPin, also called "α-CD3 DARPin-blocker" (SEQ ID NO: 2), which targets the paratope of the CD3-binding DARPin. Thus, by combining the N-cap + one or two internal repeat modules from the VEGF-binder and one or two internal repeat modules + C-cap of the α-CD3 DARPin-blocker, a VEGF-modulating 2-in-1 DARPin can be generated. Amino acid sequence alignment of the VEGF-binder, α-CD3 DARPin-blocker and eight 2-in-1 DARPin designs according to the invention. Figure 5A shows the N3C 2-in-1 DARPins (i.e. 2-in-1 DARPins 11, 12, 13, 15 and 17 corresponding to SEQ ID NOs: 6, 7, 8, 9 and 10, respectively) and Figure 5B shows the N4C 2-in-1 DARPins (i.e. 2-in-1 DARPins 18, 19 and 20 corresponding to SEQ ID NOs: 11, 12 and 13, respectively). The black boxed numbers in the repeat numbering column indicate the paratope position. [Figure 5B]Detailed representation of a 2-in-1 DARPin that can be used as a VEGF-triggered T-cell engager Switchdrug DARPin. The 2-in-1 DARPin shown in FIG. 1 was created by combining a first VEGF-binding DARPin, also called "VEGF-binder" (SEQ ID NO: 1), which has high affinity for VEGF, with a second DARPin, also called "α-CD3 DARPin-blocker" (SEQ ID NO: 2), which targets the paratope of the CD3-binding DARPin. Thus, by combining the N-cap + one or two internal repeat modules from the VEGF-binder and one or two internal repeat modules + C-cap of the α-CD3 DARPin-blocker, a VEGF-modulating 2-in-1 DARPin can be generated. Amino acid sequence alignment of the VEGF-binder, α-CD3 DARPin-blocker and eight 2-in-1 DARPin designs according to the invention. Figure 5A shows the N3C 2-in-1 DARPins (i.e. 2-in-1 DARPins 11, 12, 13, 15 and 17 corresponding to SEQ ID NOs: 6, 7, 8, 9 and 10, respectively) and Figure 5B shows the N4C 2-in-1 DARPins (i.e. 2-in-1 DARPins 18, 19 and 20 corresponding to SEQ ID NOs: 11, 12 and 13, respectively). The black boxed numbers in the repeat numbering column indicate the paratope position. [Figure 5C]Detailed representation of a 2-in-1 DARPin that can be used as a VEGF-triggered T-cell engager Switchdrug DARPin. The 2-in-1 DARPin shown in Figure 1 was created by combining a first VEGF-binding DARPin, also called "VEGF-binder" (SEQ ID NO: 1), which has a high affinity for VEGF, with a second DARPin, also called "α-CD3 DARPin-blocker" (SEQ ID NO: 2), which targets the paratope of the CD3-binding DARPin. Thus, by combining the N-cap + one or two internal repeat modules from the VEGF-binder and one or two internal repeat modules + C-cap of the α-CD3 DARPin-blocker, a VEGF-modulating 2-in-1 DARPin can be generated. A method for visualizing the paratope of the DARPin is shown, representing the surface of the DARPin paratope in 3D (Figure 5C) and 2D (Figure 5D) formats. Figure 5C shows a cartoon representation of an N3C DARPin (a DARPin containing an N-cap, three internal repeats, and a C-cap) highlighting the paratope positions within each repeat module. Figure 5D provides a positional map showing which positions of the paratope contain framework residues or potential target interaction residues for each of the repeat modules. The dotted lines in Figure 5C or 5D indicate the relative orientation of the same residue in the 3D and 2D representations. [Figure 5D]Detailed representation of a 2-in-1 DARPin that can be used as a VEGF-triggered T-cell engager Switchdrug DARPin. The 2-in-1 DARPin shown in Figure 1 was created by combining a first VEGF-binding DARPin, also called "VEGF-binder" (SEQ ID NO: 1), which has a high affinity for VEGF, with a second DARPin, also called "α-CD3 DARPin-blocker" (SEQ ID NO: 2), which targets the paratope of the CD3-binding DARPin. Thus, by combining the N-cap + one or two internal repeat modules from the VEGF-binder and one or two internal repeat modules + C-cap of the α-CD3 DARPin-blocker, a VEGF-modulating 2-in-1 DARPin can be generated. A method for visualizing the paratope of the DARPin is shown, representing the surface of the DARPin paratope in 3D (Figure 5C) and 2D (Figure 5D) formats. Figure 5C shows a cartoon representation of an N3C DARPin (a DARPin containing an N-cap, three internal repeats, and a C-cap) highlighting the paratope positions within each repeat module. Figure 5D provides a positional map showing which positions of the paratope contain framework residues or potential target interaction residues for each of the repeat modules. The dotted lines in Figure 5C or 5D indicate the relative orientation of the same residue in the 3D and 2D representations. [Figure 5E-1]Detailed representation of a 2-in-1 DARPin that can be used as a VEGF-triggered T-cell engager Switchdrug DARPin. The 2-in-1 DARPin shown in Figure 1 was created by combining a first VEGF-binding DARPin, also called "VEGF-binder" (SEQ ID NO: 1), which has high affinity for VEGF, with a second DARPin, also called "α-CD3 DARPin-blocker" (SEQ ID NO: 2), which targets the paratope of the CD3-binding DARPin. Thus, by combining the N-cap + one or two internal repeat modules from the VEGF-binder and one or two internal repeat modules + C-cap of the α-CD3 DARPin-blocker, a VEGF-modulating 2-in-1 DARPin can be generated. The VEGF-binder, α-CD3 DARPin-blocker and the paratopes of the eight 2-in-1 DARPins from Figures 5A-5B are shown in 2D representation. The bars below the 2D representation indicate whether the repeat module is taken from a VEGF-binding agent, an α-CD3 DARPin blocking agent, or a mixed repeat module between an α-CD3 DARPin blocking agent and a VEGF-binding agent. [Figure 5E-2]Detailed representation of a 2-in-1 DARPin that can be used as a VEGF-triggered T-cell engager Switchdrug DARPin. The 2-in-1 DARPin shown in Figure 1 was created by combining a first VEGF-binding DARPin, also called "VEGF-binder" (SEQ ID NO: 1), which has high affinity for VEGF, with a second DARPin, also called "α-CD3 DARPin-blocker" (SEQ ID NO: 2), which targets the paratope of the CD3-binding DARPin. Thus, by combining the N-cap + one or two internal repeat modules from the VEGF-binder and one or two internal repeat modules + C-cap of the α-CD3 DARPin-blocker, a VEGF-modulating 2-in-1 DARPin can be generated. The VEGF-binder, α-CD3 DARPin-blocker and the paratopes of the eight 2-in-1 DARPins from Figures 5A-5B are shown in 2D representation. The bars below the 2D representation indicate whether the repeat module is taken from a VEGF-binding agent, an α-CD3 DARPin blocking agent, or a mixed repeat module between an α-CD3 DARPin blocking agent and a VEGF-binding agent. [Figure 6A] Surface plasmon resonance (SPR) data for individual VEGF-binding agents, an α-CD3 DARPin blocker, and eight 2-in-1-DARPins binding to VEGF and α-CD3 DARPin. Schematic of the three different types of SPR experiments in FIG. 6D. [Figure 6B] Surface Plasmon Resonance (SPR) data for individual VEGF-binding agents, an α-CD3 DARPin blocker, and eight 2-in-1-DARPins binding to VEGF and α-CD3 DARPin. Binding of the VEGF-binding agents to their target (VEGF165) was assessed in single trace SPR measurements (see US9458211 B1 for detailed data). [Figure 6C]Surface Plasmon Resonance (SPR) data for individual VEGF binders, α-CD3 DARPin blockers, and eight 2-in-1-DARPins binding to VEGF and α-CD3 DARPin. Multi-trace SPR data were recorded for the α-CD3 DARPin blockers against the target CD3-binding DARPin, but no binding to VEGF could be measured. [Figure 6D-1] Surface plasmon resonance (SPR) data for individual VEGF binders, α-CD3 DARPin blockers, and eight 2-in-1-DARPins binding to VEGF and α-CD3 DARPin. Multi-trace SPR data for eight 2-in-1 DARPin-bound immobilized VEGF or α-CD3 DARPin, as well as single-trace SPR data for α-CD3 DARPin binding in the absence and presence of 130 nM soluble VEGF. [Figure 6D-2] Surface plasmon resonance (SPR) data for individual VEGF binders, α-CD3 DARPin blockers, and eight 2-in-1-DARPins binding to VEGF and α-CD3 DARPin. Multi-trace SPR data for eight 2-in-1 DARPin-bound immobilized VEGF or α-CD3 DARPin, as well as single-trace SPR data for α-CD3 DARPin binding in the absence and presence of 130 nM soluble VEGF. [Figure 6D-3] Surface plasmon resonance (SPR) data for individual VEGF binders, α-CD3 DARPin blockers, and eight 2-in-1-DARPins binding to VEGF and α-CD3 DARPin. Multi-trace SPR data for eight 2-in-1 DARPin-bound immobilized VEGF or α-CD3 DARPin, as well as single-trace SPR data for α-CD3 DARPin binding in the absence and presence of 130 nM soluble VEGF. [Figure 7]Residual binding of Switchdrug DARPins to VEGF by ELISA. Data for eight His-tagged Switchdrug DARPins in two different formats binding to biotinylated VEGF165 bound to streptavidin-coated microtiter plates. Binding is detected via an HRP-labeled anti-His antibody. Four Switchdrug DARPins in format A ([α-CD123 binder]-[α-CD3 binder]-[2-in-1 DARPin]) and four in format B ([2-in-1 DARPin]-[α-CD123 binder]-[α-CD3 binder]) were analyzed, VEGF binders were used as controls. [Figure 8A] Switching properties analysis. ELISA data of the His-tagged Switchdrug DARPins of the invention, including the different formats of 2-in-1 DARPins 13, 15, 17 or 18, which bind to biotinylated scCD3εy (bio-scCD3εy) bound to streptavidin-coated microtiter plates. Binding is detected via an HRP-labeled anti-His antibody. Binding of Switchdrug DARPins 15-1, 17-1 or 18-1 (format 1) to bio-scCD3ε in the presence and absence of 100 nM VEGF. Format 1 shows the constructs formatted in the order [α-CD3 binder]-[2-in-1 DARPin]. Shift in the EC50 of the Switchdrug DARPins in the presence and absence of VEGF is shown. α-CD3 mono-DARPin in the presence and absence of 100 nM VEGF was used as a control. [Figure 8B]Switching properties analysis. ELISA data of the His-tagged Switchdrug DARPins of the invention, including different formats of 2-in-1 DARPins 13, 15, 17 or 18, which bind to biotinylated scCD3εy (bio-scCD3εy) bound to streptavidin-coated microtiter plates. Binding is detected via an HRP-labeled anti-His antibody. Binding of Switchdrug DARPins 13-3, 15-3, 17-3 or 18-3 (format 3) to bio-scCD3ε in the presence and absence of 100 nM VEGF. Format 3 shows the constructs formatted in the order [2-in-1 DARPin]-[Ni2C]-[α-CD3 binder], where Ni2C represents the non-binding DARPin. Shift in EC50 of Switchdrug DARPins in the presence and absence of VEGF is shown. α-CD3 mono-DARPin in the presence and absence of 100 nM VEGF was used as a control. [Figure 8C] Switching properties analysis. ELISA data of the His-tagged Switchdrug DARPins of the invention, including different formats of 2-in-1 DARPins 13, 15, 17 or 18, which bind to biotinylated scCD3εy (bio-scCD3εy) bound to streptavidin-coated microtiter plates. Binding is detected via an HRP-labeled anti-His antibody. Binding of Switchdrug DARPins 17-A and 18-A (format A) to bio-scCD3ε in the presence and absence of 100 nM VEGF. Format A shows the constructs formatted in the order [α-CD123 binder]-[α-CD3 binder]-[2-in-1 DARPin]. Shift in the EC50 of the Switchdrug DARPins in the presence and absence of VEGF is shown. α-CD3 mono-DARPin was used as a control. [Figure 8D]Switching properties analysis. ELISA data of the His-tagged Switchdrug DARPins of the invention, including different formats of 2-in-1 DARPins 13, 15, 17 or 18, which bind to biotinylated scCD3εy (bio-scCD3εy) bound to streptavidin-coated microtiter plates. Binding is detected via an HRP-labeled anti-His antibody. Binding of Switchdrug DARPins 17-B and 18-B (format B) to bio-scCD3ε in the presence and absence of 100 nM VEGF. Format B shows the constructs formatted in the order [2-in-1 DARPin]-[α-CD123 binder]-[α-CD3 binder]. Shift in the EC50 of the Switchdrug DARPins in the presence and absence of VEGF is shown. α-CD3 mono-DARPin was used as a control. [Figure 9A] VEGF titration analysis. ELISA data for His-tagged Switchdrug DARPin conjugated Bio-scCD3εy in format 1 ([α-CD3 binder]-[2-in-1 DARPin]) or format 3 ([2-in-1 DARPin]-[Ni2C]-[α-CD3 binder]). Switchdrug DARPin 17-1 was titrated (left graph) or kept at a constant concentration of 10 nM and soluble VEGF was titrated from 100 nM to approximately 0.1 nM (right graph). VEGF-dependent binding curves of Switchdrug DARPin 17-1 can be observed. [Figure 9B] VEGF titration analysis. ELISA data of His-tagged Switchdrug DARPin conjugated Bio-scCD3εy in format 1 ([α-CD3 binder]-[2-in-1 DARPin]) or format 3 ([2-in-1 DARPin]-[Ni2C]-[α-CD3 binder]). Same as 9A but with Switchdrug DARPin 17-3, where Ni2C is the unconjugated DARPin. Immobilization of Switchdrug DARPin 17-3 and titration of VEGF resulted in a strong VEGF-dependent binding curve. [Figure 9C]VEGF titration analysis. ELISA data for His-tagged Switchdrug DARPin conjugated Bio-scCD3εy in format 1 ([α-CD3 binder]-[2-in-1 DARPin]) or format 3 ([2-in-1 DARPin]-[Ni2C]-[α-CD3 binder]). Switchdrug DARPin 18-3 was titrated (left graph) or kept at a constant concentration of 100 nM with soluble VEGF titrated from 500 nM to approximately 0.5 nM (right graph). A strong VEGF-dependent binding signal was detected. [Figure 10A] Homogeneous time-resolved fluorescence (HTRF) data of the four Switchdrug DARPin constructs of the invention. Schematic of the HTRF setup in the absence of soluble VEGF. bio-scCD3εv binds to a terbium-labeled streptavidin molecule, whereas the Switchdrug DARPin constructs bind to a d2-labeled anti-His antibody. In the absence of VEGF, the Switchdrug DARPin is preferentially in its closed conformation and therefore a low signal intensity is expected. [Figure 10B] Homogeneous time-resolved fluorescence (HTRF) data for four Switchdrug DARPin constructs of the invention. Addition of soluble VEGF is expected to open the Switchdrug DARPin and increase the binding signal. [Figure 10C] Homogeneous time-resolved fluorescence (HTRF) data of the four Switchdrug DARPin constructs of the invention. The HTRF signal was x-fold higher than that of PBS for Switchdrug DARPin 13-3, 15-3, 17-3 or 18-3 in the absence or presence of 100 nM VEGF. Clear differences in signal intensity can be observed for all four constructs. [Figure 10D] Homogeneous time-resolved fluorescence (HTRF) data for four Switchdrug DARPin constructs of the invention.HTRF signal (fold over PBS) for titration of VEGF into a fixed concentration of the same Switchdrug DARPin (20 nM) as in FIG. 10C. [Figure 11]Jurkat cell binding experiment of Switchdrug DARPin 17-B in the absence or presence of VEGF (100 nM or 400 nM). A control construct in which the 2-in-1 DARPin was replaced by a VEGF binder was used as a control. [Figure 12A] T activation assay (CD69+ cells as % of CD8+ cells) of Switchdrug DARPins 13-A, 13-B, 15-A, 15-B, 17-A, 17-B, 18-A and 18-B. As indicated, format A (Figure 12A) corresponds to [α-CD123 binder]-[α-CD3 binder]-[2-in-1 DARPin] and format B (Figure 12B) corresponds to [2-in-1 DARPin]-[α-CD123 binder]-[α-CD3 binder]. In addition, a TCE control construct ([α-CD123 binder]-[α-CD3 binder]) and a Switchdrug format control ([α-CD123 binder]-[α-CD3 binder]-[α-VEGF binder] for format A and [α-VEGF binder]-[α-CD123 binder]-[α-CD3 binder] for format B) were used. T cell activation was determined in the absence and presence of 100 nM VEGF and the shift in EC50 upon addition of VEGF is shown in each graph. [Figure 12B]T activation assay (CD69+ cells as % of CD8+ cells) of Switchdrug DARPins 13-A, 13-B, 15-A, 15-B, 17-A, 17-B, 18-A and 18-B. As indicated, format A (Figure 12A) corresponds to [α-CD123 binder]-[α-CD3 binder]-[2-in-1 DARPin] and format B (Figure 12B) corresponds to [2-in-1 DARPin]-[α-CD123 binder]-[α-CD3 binder]. In addition, a TCE control construct ([α-CD123 binder]-[α-CD3 binder]) and a Switchdrug format control ([α-CD123 binder]-[α-CD3 binder]-[α-VEGF binder] for format A and [α-VEGF binder]-[α-CD123 binder]-[α-CD3 binder] for format B) were used. T cell activation was determined in the absence and presence of 100 nM VEGF and the shift in EC50 upon addition of VEGF is shown in each graph. [Figure 13] An exemplary profiling method is applied to evaluate the suitability of parent DARPins to be covalently combined to form a 2-in-1 DARPin. This method is used to determine the optimal starting point for the subsequent combinatorial design based on two parent repeat domains. The graph shows the evaluation for the N-terminal side of the parent DARPin. The evaluation for the C-terminal side follows the same logic and is similar. In each of steps 1, 2, and 4, a mutant of the parent DARPin is generated that contains one or more repeat modules of a non-templated repeat domain (such as SEQ ID NO: 5), and then the binding of the mutant to the target of the parent DARPin is tested. In step 3, a mutant of the parent DARPin is generated that contains a modified capping module and one less internal repeat module, and then the binding of the mutant to the target of the parent DARPin is tested. Further details are provided in Example 9. IR stands for internal repeat module, N stands for N-terminal capping module, and C stands for C-terminal capping module. [Figure 14A]Characterization of 2-in-1 DARPins with mutually exclusive binding specificities for maltose binding protein (MBP) and aminoglycoside phosphotransferase (APH), i.e. 2-in-1 DARPin 21 (SEQ ID NO: 90) and 2-in-1 DARPin 22 (SEQ ID NO: 91). Size-exclusion chromatography (SEC) profile. [Figure 14B] Characterization of 2-in-1 DARPins with mutually exclusive binding specificities for maltose binding protein (MBP) and aminoglycoside phosphotransferase (APH), namely 2-in-1 DARPin 21 (SEQ ID NO: 90) and 2-in-1 DARPin 22 (SEQ ID NO: 91). Response curves from multi-trace SPR analysis of 2-in-1 DARPin 21, 2-in-1 DARPin 22 and parent DARPin no. 1 (SEQ ID NO: 86) and 2 (SEQ ID NO: 87) binding either to target APH or to target MBP as shown. Parent DARPin no. 1 binds to MBP (plot 5) and parent DARPin no. 2 binds to APH (plot 6). [Figure 14C] Characterization of 2-in-1 DARPins with mutually exclusive binding specificities for maltose binding protein (MBP) and aminoglycoside phosphotransferase (APH), namely 2-in-1 DARPin 21 (SEQ ID NO: 90) and 2-in-1 DARPin 22 (SEQ ID NO: 91). Results of a first SPR competitive binding assay. 2-in-1 DARPin 21 and 2-in-1 DARPin 22 were pre-incubated with different concentrations of soluble MBP for 40 min before the samples were run over an APH-coated SPR chip (plots 1 and 3). MBP acts as a competitor for the binding of APH, as observed by the MBP-dependent response curves (plots 2 and 4). [Figure 14D]Characterization of 2-in-1 DARPins with mutually exclusive binding specificities for maltose binding protein (MBP) and aminoglycoside phosphotransferase (APH), namely 2-in-1 DARPin 21 (SEQ ID NO: 90) and 2-in-1 DARPin 22 (SEQ ID NO: 91). Results of a second SPR competitive binding assay. 2-in-1 DARPin 21 and 2-in-1 DARPin 22 were pre-incubated with different concentrations of soluble APH for 40 min before the samples were run over an MBP-coated SPR chip (plots 1 and 3). APH acts as a competitor for the binding of MBP, as observed by the APH-dependent response curves (plots 2 and 4). [Figure 14E] Characterization of 2-in-1 DARPins with mutually exclusive binding specificities for maltose binding protein (MBP) and aminoglycoside phosphotransferase (APH), namely 2-in-1 DARPin 21 (SEQ ID NO: 90) and 2-in-1 DARPin 22 (SEQ ID NO: 91). Results of a competitive ELISA-based binding assay. 2-in-1 DARPin 21 and 2-in-1 DARPin 22 at a final concentration of 100 nM were incubated with serial dilutions of MBP for 1 h before exposure to the APH target coated on an ELISA plate. MBP acts as a competitor for the binding of APH, as observed by the MBP-dependent response curve. [Figure 14F] Characterization of 2-in-1 DARPins with mutually exclusive binding specificities for maltose binding protein (MBP) and aminoglycoside phosphotransferase (APH), namely 2-in-1 DARPin 21 (SEQ ID NO: 90) and 2-in-1 DARPin 22 (SEQ ID NO: 91). Independent binding of the 2-in-1 DARPins to the targets MBP (plot 1) or APH (plot 2) by ELISA. Parent DARPin no. 1 and 2 were used as positive controls. The 2-in-1 DARPins and the parent DARPins were tested at different concentrations as indicated. [Figure 15A]Characterization of 2-in-1 DARPins (i.e. 2-in-1 DARPin 23 (SEQ ID NO: 92), 2-in-1 DARPin 24 (SEQ ID NO: 93) and 2-in-1 DARPin 25 (SEQ ID NO: 94) with mutually exclusive binding specificities for scCD3εy and TargetA). Size exclusion chromatography (SEC) profiles. [Figure 15B-1] Characterization of 2-in-1 DARPins (i.e. 2-in-1 DARPin 23 (SEQ ID NO: 92), 2-in-1 DARPin 24 (SEQ ID NO: 93) and 2-in-1 DARPin 25 (SEQ ID NO: 94) with mutually exclusive binding specificities for scCD3εy and Target A). Response curves from multitrace SPR analysis of 2-in-1 DARPin 23, 2-in-1 DARPin 24, 2-in-1 DARPin 25 and parental DARPins no. 3 (SEQ ID NO: 3) and 4 (SEQ ID NO: 88) binding to either target scCD3εy or Target A as indicated. Parental DARPin no. 4 binds to Target A (plot 7) and parental DARPin no. 3 binds to scCD3εy (plot 8). [Figure 15B-2] Characterization of 2-in-1 DARPins (i.e. 2-in-1 DARPin 23 (SEQ ID NO: 92), 2-in-1 DARPin 24 (SEQ ID NO: 93) and 2-in-1 DARPin 25 (SEQ ID NO: 94) with mutually exclusive binding specificities for scCD3εy and Target A). Response curves from multitrace SPR analysis of 2-in-1 DARPin 23, 2-in-1 DARPin 24, 2-in-1 DARPin 25 and parental DARPins no. 3 (SEQ ID NO: 3) and 4 (SEQ ID NO: 88) binding to either target scCD3εy or Target A as indicated. Parental DARPin no. 4 binds to Target A (plot 7) and parental DARPin no. 3 binds to scCD3εy (plot 8). [Figure 15C]Characterization of 2-in-1 DARPins (i.e. 2-in-1 DARPin 23 (SEQ ID NO: 92), 2-in-1 DARPin 24 (SEQ ID NO: 93) and 2-in-1 DARPin 25 (SEQ ID NO: 94) with mutually exclusive binding specificities for scCD3εy and Target A). Results of SPR competitive binding assay. 2-in-1 DARPin 23 was pre-incubated with various concentrations of soluble Target A for 4.5 hours before the samples were run over a bio-scCD3εy coated SPR chip (plot 1). Target A acts as a competitor for the binding of scCD3εy as observed by the Target A dependent response curve (plot 2). [Figure 15D] Characterization of 2-in-1 DARPins, i.e. 2-in-1 DARPin 23 (SEQ ID NO: 92), 2-in-1 DARPin 24 (SEQ ID NO: 93) and 2-in-1 DARPin 25 (SEQ ID NO: 94) with mutually exclusive binding specificities for scCD3εy and Target A. Alternatively, the mutual exclusivity of the binding of 2-in-1 DARPins to their two targets can be measured by a competitive binding assay based on HTRF. The binding of 2-in-1 DARPin 23, 2-in-1 DARPin 24 and 2-in-1 DARPin 25 to bio-scCD3εy in the presence of different concentrations of non-biotinylated Target A competitor was evaluated. A Target A concentration-dependent decrease in binding to scCD3εy is observed for all 2-in-1 DARPins tested. [Figure 15E] Characterization of 2-in-1 DARPins (i.e. 2-in-1 DARPin 23 (SEQ ID NO: 92), 2-in-1 DARPin 24 (SEQ ID NO: 93) and 2-in-1 DARPin 25 (SEQ ID NO: 94) with mutually exclusive binding specificities for scCD3εy and Target A). Independent binding of the 2-in-1 DARPins to targets Target A (plot 1) or scCD3εy (plot 2) by ELISA. Parental DARPins no. 3 and 4 were used as positive controls. The 2-in-1 DARPins and the parental DARPins were tested at different concentrations as indicated. [Figure 16-1] 2D paratope representation of 2-in-1 DARPin 21 (SEQ ID NO: 90), 2-in-1 DARPin 22 (SEQ ID NO: 91), 2-in-1 DARPin 23 (SEQ ID NO: 92), 2-in-1 DARPin 24 (SEQ ID NO: 93), and 2-in-1 DARPin 25 (SEQ ID NO: 94) and the parental DARP pins no. 1, 2, 3, and 4. Black or white cells indicate the origin of each residue. Residues in bold are consensus residues. Residues in italics are residues that have been mutated compared to the parental sequences and therefore do not come directly from either the first or second parent. [Figure 16-2] 2D paratope representation of 2-in-1 DARPin 21 (SEQ ID NO: 90), 2-in-1 DARPin 22 (SEQ ID NO: 91), 2-in-1 DARPin 23 (SEQ ID NO: 92), 2-in-1 DARPin 24 (SEQ ID NO: 93), and 2-in-1 DARPin 25 (SEQ ID NO: 94) and the parental DARP pins no. 1, 2, 3, and 4. Black or white cells indicate the origin of each residue. Residues in bold are consensus residues. Residues in italics are residues that have been mutated compared to the parental sequences and therefore do not come directly from either the first or second parent. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0082] As disclosed and exemplified herein, the present disclosure provides in a first aspect a method for producing a designed repeat domain, preferably a designed ankyrin repeat domain, with binding specificities for two targets, wherein the binding of the designed repeat domain to the two targets is mutually exclusive.

[0083] Designed ankyrin repeat domains are 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 beneficial components of recombinant binding proteins for the treatment of diseases.

[0084] Designed ankyrin repeat proteins are a class of binding molecules with the potential to overcome the limitations of monoclonal antibodies, thus enabling novel therapeutic approaches. Such ankyrin repeat proteins may contain a single designed ankyrin repeat domain or may contain 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, Vol. 13, 695-701, 2008; US Pat. 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 and the possibility to combine two, three, four, five or more designed ankyrin repeat domains in one protein make designed ankyrin repeat proteins ideal agonist, antagonist and / or inhibitory drug candidates. Moreover, 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. In summary, designed ankyrin repeat proteins are examples of the next generation of protein therapeutics with the potential to go beyond existing antibody drugs.

[0085] The binding specificity of designed ankyrin repeat domains is determined by the nature of the repeat modules contained in each repeat domain. Both internal repeat modules and terminal capping modules (C-cap or N-cap modules) can contribute to the specificity. Unexpectedly, the method of the present invention allows to produce a single designed repeat domain (or monodomain) with mutually exclusive binding specificities for two targets by combining the repeat modules of two parent repeat domains. Thus, the binding specificity for each of the first and second targets is inherited from the binding specificity for each target of the parent repeat domain. Such designed repeat domains of the present invention maintain the advantageous properties of conventional designed repeat domains, including binding properties. The designed repeat domains of the present invention can be used to further expand the applications of repeat proteins, for example as control mechanisms such as protein switches.

[0086] 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 are those well known and commonly used in the art.

[0087] The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms unless otherwise indicated. When an aspect of the invention describes a feature as "comprising," in an embodiment, the feature is also contemplated 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 illustrate the disclosure and do not impose limitations on the scope of the disclosure unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure. Other than in the examples provided, or unless otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about" as that term would be interpreted by one of ordinary skill in the art. As used herein, the term "about" corresponds to ±10% of a given numerical value, unless otherwise indicated.

[0088] 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 recited herein.

[0089] The term "nucleic acid" refers to a polynucleotide molecule, which can be a ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) molecule, either single-stranded or double-stranded, including 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.

[0090] In the context of the present invention, the term "protein" refers to a molecule that comprises 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 more than one polypeptide chain, 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.

[0091] The term "recombinant" as used in recombinant proteins and polypeptides means that the protein or polypeptide is produced by the use of recombinant DNA technology 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 DNA that allows for in vitro expression. For example, when such a recombinant bacterial expression plasmid is inserted into a suitable bacterium (e.g., E. coli), these bacteria can produce the polypeptide encoded by the recombinant DNA. The polypeptide or protein produced accordingly is called a recombinant polypeptide or protein.

[0092] In the context of the present invention, the term "polypeptide" refers to a molecule that consists 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.

[0093] The term "target" refers to an individual molecule, such as a nucleic acid, a polypeptide or protein, a carbohydrate, or any other natural or non-natural molecule, including any portion of such an individual molecule, or a complex of two or more of such molecules. A target may be a whole cell or tissue sample, or may be any non-natural molecule or moiety. Preferably, a target is a naturally occurring or non-natural polypeptide, or a polypeptide that includes a chemical modification, e.g., modified by natural or non-natural phosphorylation, acetylation, or methylation.

[0094] 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 uses of repeat domains. The term "repeat protein" refers to a protein that comprises 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. A repeat domain may be a binding domain.

[0095] The term "repeat domain" refers to a protein domain that comprises as a structural unit two or more consecutive repeat modules, the repeat modules having structural and sequence homology. Preferably, the repeat domain also comprises an N-terminal and / or a C-terminal capping module. For clarity, a capping module may be a repeat module and thus contribute to two or more consecutive repeat modules of a repeat domain. Such repeat domains, repeat modules and capping modules, sequence motifs and structural and sequence homologies are well known to the skilled person from the example of ankyrin repeat domains (Binz et al., J. Mol. Biol. 332, 489-503, 2003; Binz et al., 2004, loc.cit,; WO 2002 / 020565; WO 2012 / 069655), leucine-rich repeat domains (WO 2002 / 020565), tetratricopeptide repeat domains (Main, ER, Xiong, Y., Cocco, MJ, 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 repeated domains are distinct from proteins that contain repeated amino acid sequences, all of which can form individual domains (e.g., the FN3 domain of fibronectin).

[0096] The term "ankyrin repeat domain" refers to a repeat domain that comprises two or more consecutive ankyrin repeat modules as structural units, which ankyrin repeat modules have structural and sequence homology.

[0097] The term "part" as used in "part of a repeat domain" (and so on) includes both a part of a repeat domain or the entire repeat domain. Such a part may comprise one or more repeat modules comprised in such a repeat domain, or any fragment or stretch of amino acids comprised in the sequence of such a repeat domain. Preferably, a part of a repeat domain as described herein comprises 10 or more (more preferably 20 or more, even more preferably 30 or more) consecutive amino acids of the amino acid sequence of the repeat domain. More preferably, such a part comprises at least one entire repeat module of the repeat domain.

[0098] 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.

[0099] The term "repeat module" refers to the repeated amino acid sequence and structural unit of a designed repeat domain, originally derived from a repeat unit of a naturally occurring repeat protein. Each repeat module contained 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 contained in a repeat domain may contain a "repeat sequence motif" deduced from a homologous repeat module with the same target specificity, obtained from the selected repeat domain on a target. The repeat modules 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 by one repeat module at the N-terminus and another repeat module at the C-terminus.

[0100] Thus, the term "ankyrin repeat module" refers to a repeat module derived from the repeat unit of ankyrin repeat protein originally occurring in nature. Ankyrin repeat proteins are well known to those skilled in the art. Designed ankyrin repeat proteins have been described previously, see, for example, WO 2002 / 020565, WO 2010 / 060748, WO 2011 / 135067, WO 2012 / 069654, WO 2012 / 069655, WO 2014 / 001442, WO 2014 / 191574, WO 2014 / 083208, WO 2016 / 156596 and WO 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.

[0101] A repeat module may comprise positions with amino acid residues that are not randomized in the library for the purpose of selecting a target-specific repeat domain ("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 a target-specific repeat domain ("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, either any of the usual 20 naturally occurring amino acids are allowed, or most of the 20 naturally occurring amino acids are allowed, such as amino acids other than cysteine ​​or amino acids other than glycine, cysteine, and proline.

[0102] The term "paratope" or "binding surface" refers to the region of a repeat domain that provides binding specificity for one or more targets. As used herein, a paratope may include target interaction residues and framework residues. The positions of the paratope residues are defined as follows:

[0103] In the internal repeat module, positions corresponding to positions 3, 4, 6, 11, 14 and / or 15 of SEQ ID NO:39 contain target interaction residues and position corresponding to position 10 of SEQ ID NO:39 contains framework residues; in the N-terminal capping module, positions corresponding to positions 4, 5, 8, 11 and / or 12 of SEQ ID NO:36 contain target interaction residues and position corresponding to position 7 of SEQ ID NO:36 contains framework residues; in the C-terminal capping module, positions corresponding to positions 3, 4, 6, 14 and / or 15 of SEQ ID NO:38 contain target interaction residues and positions corresponding to positions 10 and 11 of SEQ ID NO:38 contain framework residues.

[0104] 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 positions of the framework residues of the repeat modules. Similarly, the target interaction residue positions correspond to the positions of the target interaction residues of the repeat modules. The repeat sequence motif comprises non-randomized positions and randomized positions.

[0105] 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 the folding of the protein. 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.

[0106] The term "target interaction residues" refers to amino acid residues of a repeat module that contribute to the direct interaction with the 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). In the context of the present invention, a residue is considered to contribute to the interaction with the target if at least 10% loss of binding interaction between the repeat domain and the target is due to a residue mutation in the domain compared to the binding interaction between the non-mutated repeat domain and the target. Target interaction residues can also be determined by structural analysis of the repeat domain bound to the target.

[0107] 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 influences on the polypeptide backbone structure found in α-helices or β-sheets, or participation in amino acid stretches forming linear polypeptides or loops.

[0108] 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.

[0109] 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," and the like, 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 5 Methods 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 of skill in the art. The K 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.

[0110] The binding of any molecule to another molecule is determined by two forces: the association rate (k on ) and dissociation rate (k off ) The affinity of any binder [B] for a target [T] is then governed by the equilibrium dissociation constant K D This can be expressed as k off / k on is the quotient.

[0111]

number

[0112] Various methods for measuring binding affinity are 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 expressed as K D The K value 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), K D The smaller the binding affinity, the stronger the binding affinity.

[0113] 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 that contain epitope binding domains) on their surface. D Another method for determining K is to use Bio-Layer Interferometry (see, e.g., Shah et al. J Vis Exp. 2014;(84):51383). DValues ​​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 the 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. The K of the designed repeat domains binding to the target by SPR analysis. D An exemplary and preferred determination of is described in Example 2.

[0114] The term "PBS" refers to an aqueous phosphate buffer solution containing 137 mM NaCl, 10 mM phosphate, and 2.7 mM KCl, with a pH of 7.4.

[0115] The term "dual binding specificity" or "dual specificity" refers to the property of the designed repeat domain of the present invention to have binding specificity for two targets, which may be two epitopes on a single target molecule or two epitopes each located on a different target molecule. Preferably, the epitopes are different from each other.

[0116] The term "covalently combining" or "covalent combination" used in covalently combining repeat modules includes "covalently connecting" and "covalently merging" of repeat modules. Covalently connecting two repeat modules refers to directly connecting the repeat modules via a chemical bond, such as a peptide bond. Covalently connecting encompasses a direct peptide bond between the repeat modules or a linker bond. Such a linker may be a non-peptide chemical linker or a peptide linker, which may be any suitable peptide linker comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids. Alternatively, the peptide linker is the entire non-linked repeat module. Examples of non-linked repeat modules are described herein. Covalent merging of two (parental) repeat modules refers to the process of generating a single hybrid repeat module from the two parental repeat modules. Such a hybrid repeat module contains sequence elements from both parental repeat modules.

[0117] The term "combined module" or "mixed module" refers to a repeat module, preferably ankyrin repeat module, which can be obtained by covalently combining two parent repeat modules and which comprises at least one target interaction residue of a first parent repeat module and at least one target interaction residue of a second parent repeat module.

[0118] The term "mutually exclusive" or "mutually exclusive" as used in mutually exclusive binding refers to the property of the designed repeat domain of the invention that it can bind to a first target and a second target only in a substantially non-simultaneous manner. In such mutually exclusive binding, the binding of the first target to the repeat domain affects the binding of the second target to the repeat domain (and / or vice versa) such that simultaneous binding of the first and second targets is substantially prevented. The underlying causes of this effect include steric hindrance and / or overlapping paratopes. In the context of the present invention, the binding interaction between the designed repeat domain of the invention and its first and second targets is considered to be mutually exclusive if, when bound to one of the first and second targets, the repeat domain is unable to bind more than 1%, more than 2%, more than 3%, more than 4%, more than 5%, more than 10%, more than 15%, more than 20%, more than 25%, more than 30%, more than 35%, more than 40%, more than 45% or more than 50% of the molar equivalent of the other target. The degree of such mutual exclusivity can be measured by a competitive binding assay in which the binding of a first target to the repeat domain is compared with the binding of a second target to the repeat domain. Such competitive binding assays can be adapted from standard competitive binding assays used in the field of antibodies, as known to those skilled in the art.

[0119] As used herein, the term "therapeutic agent" refers to a drug, molecule, nucleic acid, protein, composition or other substance that provides a therapeutic effect. The terms "therapeutic agent" and "drug" are used interchangeably.

[0120] Throughout this disclosure, the order or orientation of "first" and "second" entities, e.g., as used in first and second targets, first and second repeat domains, first and second binding specificities, etc., is not meant to specify one particular order or orientation, but encompasses alternatives, unless specifically defined otherwise. In other words, any reference to such first and second entities also encompasses alternative orders in which the first entity corresponds to the second entity, and the second entity corresponds to the first entity, unless specifically defined otherwise.

[0121] Manufacturing method according to the present invention In one aspect, the present invention provides a method for preparing a protein, the method comprising: (i) providing a first repeat domain and a second repeat domain, wherein the first repeat domain has binding specificity for a first target and the second repeat domain has binding specificity for a second target; (ii) linking a part of the first repeat domain with a part of the second repeat domain by covalently combining at least one repeat module of the first repeat domain with at least one repeat module of the second repeat domain to form a designed repeat domain with dual binding specificity, The method provides a method in which the designed repeat domain has binding specificity for a first target and binding specificity for a second target, and the binding of the designed repeat domain to the first and second targets is mutually exclusive.

[0122] DARPins (designed ankyrin repeat proteins) are repeat proteins that contain 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 (see, for example, Binz et al. 2003). As used herein, the term "repeat module" encompasses internal and terminal repeat modules (C-cap and N-cap modules). 27 of the 33 amino acid positions of a typical internal repeat module are highly conserved, while the other 6 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). The paratope of ankyrin repeat domains is formed by a continuous surface that is mainly formed by these variable positions of the internal repeat modules and sometimes the capping repeat modules.

[0123] The inventors of the present invention surprisingly discovered that a single designed repeat domain with dual and mutually exclusive binding specificities (i.e. a monodomain) can be generated by combining amino acid sequence elements, such as repeat modules, from two parent repeat domains, which parent repeat domains are preferably both ankyrin repeat domains, and thus the generated designed repeat domain with dual and mutually exclusive binding specificities is also ankyrin repeat domain.

[0124] The basic concept of the production method of the present invention is illustrated in Figure 1. As can be seen from Figure 1, a part of a first (parent) repeat domain (1) with binding specificity for a first target (target A) and a part of a second (parent) repeat domain (2) with binding specificity for a second target (target B) can be covalently combined to form a newly designed repeat domain. The respective paratopes or binding surfaces of the parent repeat domains are shown ((4) and (5)). Such newly formed designed repeat domains are also referred to herein as "2-in-1 DARPins" or "SwitchDARPins" (6). Such designed repeat domains can bind to two targets A and B, i.e. either target A or target B, in a mutually exclusive manner. In addition, one or some internal repeat modules within a designed repeat domain can be a mixed (or merged) repeat module (7). A repeat module is called a mixed repeat module if its paratope comprises at least one target interaction residue from the corresponding module in the first parent repeat protein and at least one target interaction residue from the corresponding module in the second parent repeat protein. The mutually exclusive binding properties of the designed repeat domains of the present invention are shown in Figure 2. Due to the proximity or overlap of the two paratopes of the designed repeat domains of the present invention, the designed repeat domains can bind to target A (Figure 2A) or target B (Figure 2B) individually, but cannot bind to both targets simultaneously (Figure 2C), as simultaneous binding of both targets A and B is sterically hindered. This mutually exclusive binding feature consequently allows the design of on or off switches for the conditional activation or inactivation of drug molecules or other target molecules (e.g., cell surface receptors, etc.), as illustrated in Figures 3 and 4. This mutually exclusive binding feature also distinguishes the designed repeat domains of the present invention from other ankyrin repeat protein formats (Mohan, K. et al., 2019, Science, 364(6442); WO 2020 / 190852 A2).

[0125] Covalent combination as used in the present invention includes covalently connecting or covalently merging parent repeat modules. This distinction depends on the nature and location of the target interaction residues contained in the repeat module(s) resulting from the covalent combination compared to the target interaction residues contained in the parent modules. Covalently connecting a first and a second parent repeat module means connecting both parent repeat modules by a chemical bond, such as a peptide bond. Alternatively, covalently connecting refers to connecting the first and second repeat modules by a linker.

[0126] By covalently merging the first and second parent repeat modules, a single merged (or mixed) repeat module is obtained, which contains at least one target interaction residue from the first parent repeat module and at least one target interaction residue from the second parent repeat module. Thus, k merged repeat modules can be obtained from covalently merging 2k parent repeat modules. The effect of such a covalent combination is that the resulting designed repeat domain can only bind both targets in a mutually exclusive manner due to steric hindrance between the two targets. In the case of covalent merging, the presence of any merged repeat module can further enhance the mutually exclusive binding effect of the designed repeat domain due to the fact that such a merged module can bind to one or the other target at a given time.

[0127] Thus, in one embodiment, covalently combining at least one repeat module of the first repeat domain with at least one repeat module of the second repeat domain comprises covalently connecting at least one repeat module of the first repeat domain with at least one repeat module of the second repeat domain. Covalently connecting such repeat modules can be achieved by methods well known in the art and can comprise any type of covalent bond. In a preferred embodiment, the covalently connecting comprises a peptide bond. In a preferred embodiment, the covalently combining comprises covalently connecting one repeat module of the first repeat domain with one repeat module of the second repeat domain. In another embodiment, the covalently combining comprises covalently connecting one repeat module of the first repeat domain with one repeat module of the second repeat domain by a linker. In one embodiment, the linker is a chemical non-peptide linker. In another embodiment the linker is a peptide linker, preferably the peptide linker comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 amino acids. In another embodiment the linker is a non-linked repeat module. An example of such a non-linked repeat module is provided by SEQ ID NO:89.

[0128] In one embodiment, covalently combining at least one repeat module of the first repeat domain with at least one repeat module of the second repeat domain comprises covalently combining at least one repeat module of the first repeat domain with at least one repeat module of the second repeat domain. In another embodiment, one repeat module of the first repeat domain may be covalently combined with one repeat module of the second repeat domain. In another embodiment, two repeat modules of the first repeat domain may be covalently combined with two repeat modules of the second repeat domain. In another embodiment, three repeat modules of the first repeat domain may be covalently combined with three repeat modules of the second repeat domain.

[0129] In some embodiments, the first and / or second repeat domain(s) may independently comprise at least one repeat module, at least two repeat modules, at least three repeat modules, at least four repeat modules, at least five repeat modules, or at least six repeat modules. In some embodiments, the first and / or second repeat domain(s) may independently comprise 1-6 repeat modules, 2-6 repeat modules, 3-6 repeat modules, 4-6 repeat modules, or 5-6 repeat modules. In some embodiments, the first and / or second repeat domain(s) may independently comprise an N-terminal capping module and / or a C-terminal capping module. In some embodiments, the first and / or second repeat domain(s) may independently comprise an N-terminal capping module and / or a C-terminal capping module and at least one internal repeat module, at least two internal repeat modules, at least three internal repeat modules, or at least four internal repeat modules. In some embodiments, the first and / or second repeat domain(s) may independently comprise an N-terminal capping module and / or a C-terminal capping module and 1-4 internal repeat modules, 1-3 internal repeat modules or 1-2 internal repeat modules.

[0130] In some preferred embodiments, the first repeat domain comprises 3-5 repeat modules and the second repeat domain comprises 3-5 repeat modules. In some more preferred embodiments, the first repeat domain comprising 3-5 repeat modules comprises, from N-terminus to C-terminus: an N-terminal capping module, 1-3 internal repeat modules and a C-terminal capping module, and the second repeat domain comprising 3-5 repeat modules comprises, from N-terminus to C-terminus: an N-terminal capping module, 1-3 internal repeat modules and a C-terminal capping module.

[0131] In some preferred embodiments, each of the first and second repeat domains comprises four repeat modules, in some more preferred embodiments, each of the first and second repeat domains comprising four repeat modules comprises, from N-terminus to C-terminus, an N-terminal capping module, two internal repeat modules and a C-terminal capping module.

[0132] In some preferred embodiments, each of the first and second repeat domains comprises five repeat modules, in some more preferred embodiments, each of the first and second repeat domains comprising five repeat modules comprises, from N-terminus to C-terminus, an N-terminal capping module, three internal repeat modules and a C-terminal capping module.

[0133] In some preferred embodiments, the first repeat domain comprises 4 repeat modules and the second repeat domain comprises 5 repeat modules. In some more preferred embodiments, the first repeat domain comprising 4 repeat modules comprises, from N-terminus to C-terminus: an N-terminal capping module, two internal repeat modules and a C-terminal capping module, and the second repeat domain comprising 5 repeat modules comprises, from N-terminus to C-terminus: an N-terminal capping module, three internal repeat modules and a C-terminal capping module.

[0134] In one embodiment, the designed repeat domain comprises an N-terminal and / or a C-terminal capping module and at least one internal repeat module. In one embodiment, the designed repeat domain comprises an N-terminal and / or a C-terminal capping module and one internal repeat module. In one embodiment, the designed repeat domain comprises an N-terminal and / or a C-terminal capping module and two internal repeat modules. In one embodiment, the designed repeat domain comprises an N-terminal and / or a C-terminal capping module and three internal repeat modules. In one embodiment, the designed repeat domain comprises an N-terminal and / or a C-terminal capping module and four internal repeat modules. In one embodiment, the designed repeat domain comprises an N-terminal and / or a C-terminal capping module and five internal repeat modules. In any of the above embodiments, the bond "and" is preferred. Thus, in some forms, the designed repeat domain comprises an N-terminal and a C-terminal capping module and at least one internal repeat module. In one embodiment, the designed repeat domain comprises an N-terminal and a C-terminal capping module and one internal repeat module. In one embodiment, the designed repeat domain comprises an N-terminal and a C-terminal capping module and two internal repeat modules. In one embodiment, the designed repeat domain comprises an N-terminal and a C-terminal capping module and three internal repeat modules. In one embodiment, the designed repeat domain comprises an N-terminal and a C-terminal capping module and four internal repeat modules. In one embodiment, the designed repeat domain comprises an N-terminal and a C-terminal capping module and five internal repeat modules.

[0135] In one embodiment the N-terminal capping module is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical in amino acid sequence to the N-terminal capping module comprised in the first repeat domain.

[0136] In one embodiment, the C-terminal capping module is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical in amino acid sequence to the C-terminal capping module comprised in the second repeat domain.

[0137] In one embodiment, the N-terminal capping module is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical in amino acid sequence to the N-terminal capping module comprised in the first repeat domain. and the C-terminal capping module is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical in amino acid sequence to the C-terminal capping module comprised in the second repeat domain.

[0138] Thus, in certain embodiments, the N-terminal capping module is at least 90% identical in amino acid sequence to the N-terminal capping module comprised in the first repeat domain and the C-terminal capping module is at least 90% identical in amino acid sequence to the C-terminal capping module comprised in the second repeat domain. In some embodiments, the number of repeat modules comprised in a designed repeat domain corresponds to the sum of the repeat modules comprised in part of the first repeat domain and the repeat modules comprised in part of the second repeat domain. In some embodiments, the number of repeat modules comprised in a designed repeat domain corresponds to nm, where n is the sum of the repeat modules comprised in part of the first repeat domain and the repeat modules comprised in part of the second repeat domain and m is the number of combined modules in the designed repeat domain.

[0139] In some embodiments, the portion of the first repeat domain comprises a chain of repeat modules comprising x repeat modules and / or the portion of the second repeat domain comprises a chain of repeat modules comprising y repeat modules, where x is an integer selected in the range from 1 to the total number of repeat modules comprised in the first repeat domain and y is an integer selected in the range from 1 to the total number of repeat modules comprised in the second repeat domain.

[0140] In some aspects, the portion of the first repeat domain may be selected from the group consisting of a chain of 2 repeat modules, a chain of 3 repeat modules, a chain of 4 repeat modules, a chain of 5 repeat modules, and a chain of 6 repeat modules comprised in the first repeat domain, and / or the portion of the second repeat domain may be selected from the group consisting of a chain of 2 repeat modules, a chain of 3 repeat modules, a chain of 4 repeat modules, a chain of 5 repeat modules, and a chain of 6 repeat modules comprised in the second repeat domain. In some embodiments, the chain is a contiguous chain of repeat modules compared to the order of the repeat modules comprised in the corresponding first or second repeat domain. In some aspects, the portion of the first repeat domain comprises the entire first repeat domain, and / or the portion of the second repeat domain comprises the entire second repeat domain. In the above embodiments, the length or size of the portion of the first repeat domain may be independent of the length or size of the portion of the second repeat domain.

[0141] In one embodiment, the portion of the first repeat domain comprises the N-terminal capping module and at least one internal repeat module of the first repeat domain and the portion of the second repeat domain comprises the C-terminal capping module and at least one internal repeat module of the second repeat domain. In one embodiment, the portion of the first repeat domain comprises the C-terminal capping module and at least one internal repeat module of the first repeat domain and the portion of the second repeat domain comprises the N-terminal capping module and at least one internal repeat module of the second repeat domain. In an alternative embodiment, the portion of the first repeat domain comprises at least one internal repeat module of the first repeat domain and the portion of the second repeat domain comprises the N-terminal capping module, the C-terminal capping module and at least one internal repeat module of the second repeat domain.

[0142] In some embodiments, the portion of the first repeat domain represents between 1% and 5%, between 1% and 10%, between 1% and 15%, between 1% and 20%, between 1% and 25%, between 1% and 30%, between 1% and 35%, between 1% and 40%, between 1% and 45%, between 1% and 50%, between 1% and 55%, between 1% and 60%, between 1% and 65%, between 1% and 70%, between 1% and 75%, between 1% and 80%, between 1% and 85%, between 1% and 90%, between 1% and 95%, or between 1% and 100% of the amino acid sequence of the first repeat domain. and / or the portion of the second repeat domain may represent 1%-5%, 1%-10%, 1%-15%, 1%-20%, 1%-25%, 1%-30%, 1%-35%, 1%-40%, 1%-45%, 1%-50%, 1%-55%, 1%-60%, 1%-65%, 1%-70%, 1%-75%, 1%-80%, 1%-85%, 1%-90%, 1%-95%, or 1%-100% of the amino acid sequence of the second repeat domain.

[0143] In some embodiments, the portion of the first repeat domain may represent between 1% and 100%, between 5% and 100%, between 10% and 100%, between 15% and 100%, between 20% and 100%, between 25% and 100%, between 30% and 100%, between 35% and 100%, between 40% and 100%, between 45% and 100%, between 50% and 100%, between 55% and 100%, between 60% and 100%, between 65% and 100%, between 70% and 100%, between 75% and 100%, between 80% and 100%, between 85% and 100%, between 90% and 100%, or between 95% and 100% of the amino acid sequence of the first repeat domain. , and / or the portion of the second repeat domain may represent 1%-100%, 5%-100%, 10%-100%, 15%-100%, 20%-100%, 25%-100%, 30%-100%, 35%-100%, 40%-100%, 45%-100%, 50%-100%, 55%-100%, 60%-100%, 65%-100%, 70%%-100%, 75%-100%, 80%-100%, 85%-100%, 90%-100%, or 95%-100%% of the amino acid sequence of the second repeat domain.

[0144] In some embodiments, the portion of the first repeat domain represents about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100% of the amino acid sequence of the first repeat domain. and / or the portion of the second repeat domain may represent about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100% of the amino acid sequence of the second repeat domain. In the above embodiments, the length or size of the portion of the first repeat domain may be independent of the length or size of the portion of the second repeat domain. In some embodiments, the portion is a contiguous amino acid sequence compared to the corresponding amino acid sequences of the first and second repeat domains.

[0145] In some embodiments, any such portion of the first repeat domain is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical in amino acid sequence to the corresponding amino acid sequence of the first repeat domain. and / or any such portion of the second repeat domain is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical in amino acid sequence to the corresponding amino acid sequence of the second repeat domain.

[0146] In one embodiment, the N-terminal fragment of the first repeat domain may be covalently associated with the C-terminal fragment of the second repeat domain, in another embodiment, the C-terminal fragment of the first repeat domain may be covalently associated with the N-terminal fragment of the second repeat domain.

[0147] In some embodiments, such a fragment of the first repeat domain may represent an amino acid sequence corresponding to 1%-5%, 1%-10%, 1%-15%, 1%-20%, 1%-25%, 1%-30%, 1%-35%, 1%-40%, 1%-45%, 1%-50%, 1%-55%, 1%-60%, 1%-65%, 1%-70%, 1%-75%, 1%-80%, 1%-85%, 1%-90%, 1%-95%, or 1%-100% of the consecutive amino acids comprised in the amino acid sequence of the first repeat domain. Frequently, and / or such a fragment of the second repeat domain may represent an amino acid sequence corresponding to 1%-5%, 1%-10%, 1%-15%, 1%-20%, 1%-25%, 1%-30%, 1%-35%, 1%-40%, 1%-45%, 1%-50%, 1%-55%, 1%-60%, 1%-65%, 1%-70%, 1%-75%, 1%-80%, 1%-85%, 1%-90%, 1%-95%, or 1%-100% of the consecutive amino acids comprised in the amino acid sequence of the second repeat domain.

[0148] In some embodiments, such a fragment of the first repeat domain represents a stretch of contiguous amino acids corresponding to between 1% and 100%, 5% and 100%, 10% and 100%, 15% and 100%, 20% and 100%, 25% and 100%, 30% and 100%, 35% and 100%, 40% and 100%, 45% and 100%, 50% and 100%, 55% and 100%, 60% and 100%, 65% and 100%, 70% and 100%, 75% and 100%, 80% and 100%, 85% and 100%, 90% and 100% or 95% and 100% of the contiguous amino acids comprised in the amino acid sequence of the first repeat domain. and / or such a fragment of the second repeat domain may represent an amino acid sequence corresponding to 1%-100%, 5%-100%, 10%-100%, 15%-100%, 20%-100%, 25%-100%, 30%-100%, 35%-100%, 40%-100%, 45%-100%, 50%-100%, 55%-100%, 60%-100%, 65%-100%, 70%-100%, 75%-100%, 80%-100%, 85%-100%, 90%-100% or 95%-100% of the consecutive amino acids comprised in the amino acid sequence of the second repeat domain.

[0149] In some embodiments, such a fragment of the first repeat domain may represent an amino acid sequence corresponding to about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100% of the consecutive amino acids in the amino acid sequence of the first repeat domain. Frequently, and / or such a fragment of the second repeat domain may represent an amino acid sequence corresponding to about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95% or about 100% of the consecutive amino acids comprised in the amino acid sequence of the second repeat domain. In the above embodiments, the length or size of the fragment of the first repeat domain may be independent of the length or size of the fragment of the second repeat domain.

[0150] In a preferred embodiment, the fragment of a repeat domain corresponds to an amino acid sequence starting at one end (either the N-terminus or the C-terminus) of a repeat domain (such as the first or second repeat domain in the above embodiments). As used herein, covalently combining an N-terminal fragment of a repeat domain with a C-terminal fragment of another repeat domain includes covalently connecting or covalently merging both fragments.

[0151] In some embodiments, any such fragment of the first repeat domain is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical in amino acid sequence to the corresponding amino acid sequence of the first repeat domain. and / or any such fragment of the second repeat domain is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical in amino acid sequence to the corresponding amino acid sequence of the second repeat domain.

[0152] In one embodiment, the method of the invention further comprises step (i)(2) after step (i), which comprises evaluating the first and / or second repeat domain for their suitability to be covalently combined at their N- and / or C-terminal sides with another repeat domain. The extent of the evaluation may vary depending on the structural properties of the first and second repeat domains and their respective targets. In some embodiments, the evaluation comprises at least determining whether the N- and / or C-terminal capping module of the first repeat domain contributes to the binding of the first repeat domain to a first target and / or determining whether the N- and / or C-terminal capping module of the second repeat domain contributes to the binding of the second repeat domain to a second target. In some embodiments, the evaluating comprises determining whether the N- and / or C-terminal capping module of a first repeat domain and internal repeat modules adjacent to the N- and / or C-terminal capping module contribute to binding of the first repeat domain to a first target and / or determining whether the N- and / or C-terminal capping module of a second repeat domain and internal repeat modules adjacent to the N- and / or C-terminal capping module contribute to binding of the second repeat domain to a second target.

[0153] An exemplary method for making this binding contribution determination is to make mutants of the first and / or second repeat domains and then perform a binding assay to determine whether the modifications made in the mutants affect their binding properties, such as resulting in loss of binding or maintained binding. Based on the results of the evaluation of the mutants, the covalent combination compatibility of the first and / or second repeat domains can be inferred. The assay used in the evaluation or binding contribution determination includes ELISA binding assays or any other suitable test assays known to those skilled in the art. When an ELISA binding assay is used, an EC50 value of about 50 nm, about 100 nm, about 150 nm, about 200 nm, about 250 nm, about 300 nm, about 350 nm or about 400 nm can be used as a threshold value to classify whether loss of binding has occurred or whether binding has been maintained. Preferably, an EC50 value of 300 nm is used as such a threshold value.

[0154] Such a determination of covalent combination compatibility is described in Example 9.1 (the method used is also called the profiling method).

[0155] Thus, in one embodiment, the invention provides a method of producing a designed repeat domain, the method comprising: (i) providing a first repeat domain and a second repeat domain, wherein the first repeat domain has binding specificity for a first target and the second repeat domain has binding specificity for a second target; (i)(2) determining whether the first repeat domain and / or the second repeat domain are compatible with being covalently associated at the N-terminus and / or C-terminus with another repeat domain; (ii) linking a part of the first repeat domain with a part of the second repeat domain by covalently combining at least one repeat module of the first repeat domain with at least one repeat module of the second repeat domain to form a designed repeat domain with dual binding specificity, The method comprises the steps of: (a) providing a first repeat domain having binding specificity for a first target and binding specificity for a second target, and wherein the binding of the designed repeat domain to the first and second targets is mutually exclusive.

[0156] In one embodiment, step (ii) of the method of the invention further comprises removing the C-terminal capping module of the first repeat domain and removing the N-terminal capping module of the second repeat domain, such that the C-terminal capping module of the first repeat domain and the N-terminal capping module of the second repeat domain are not comprised in the designed repeat domain.

[0157] In one embodiment, step (ii) of the method of the invention further comprises removing the C-terminal capping module of the first repeat domain and / or removing the N-terminal capping module of the second repeat domain, such that the C-terminal capping module of the first repeat domain and / or the N-terminal capping module of the second repeat domain are not included in the designed repeat domain.

[0158] In one embodiment, step (ii) of the method of the invention further comprises removing the N-terminal capping module of the first repeat domain and / or removing the C-terminal capping module of the second repeat domain, such that the N-terminal capping module of the first repeat domain and / or the C-terminal capping module of the second repeat domain are not included in the designed repeat domain.

[0159] In one embodiment, step (ii) of the method of the invention further comprises removing one or more repeat modules from the C-terminus of the first repeat domain and removing one or more repeat modules from the N-terminus of the second repeat domain, such that one or more repeat modules from the C-terminus of the first repeat domain and one or more repeat modules from the N-terminus of the second repeat domain are not included in the designed repeat domain. In one embodiment, step (ii) of the method further comprises removing one or more repeat modules from the N-terminus of the first repeat domain and removing one or more repeat modules from the C-terminus of the second repeat domain, such that one or more repeat modules from the N-terminus of the first repeat domain and one or more repeat modules from the C-terminus of the second repeat domain are not included in the designed repeat domain.

[0160] In one embodiment, step (ii) of the method of the invention further comprises removing one or more repeat modules from the C-terminus of the first repeat domain and / or removing one or more repeat modules from the N-terminus of the second repeat domain, such that one or more repeat modules from the C-terminus of the first repeat domain and / or one or more repeat modules from the N-terminus of the second repeat domain are not included in the designed repeat domain. In one embodiment, step (ii) of the method further comprises removing one or more repeat modules from the N-terminus of the first repeat domain and / or removing one or more repeat modules from the C-terminus of the second repeat domain, such that one or more repeat modules from the N-terminus of the first repeat domain and / or one or more repeat modules from the C-terminus of the second repeat domain are not included in the designed repeat domain.

[0161] In one embodiment, the method of the invention further comprises a step (ii)(2), which comprises determining whether the binding of the designed repeat domain formed in step (ii) to the first and second targets is mutually exclusive. An exemplary determination of such mutually exclusive target binding of the designed repeat domain is described in Example 9.

[0162] Thus, in one embodiment, the invention provides a method of producing a designed repeat domain, the method comprising: (i) providing a first repeat domain and a second repeat domain, wherein the first repeat domain has binding specificity for a first target and the second repeat domain has binding specificity for a second target; (ii) linking a part of the first repeat domain with a part of the second repeat domain by covalently combining at least one repeat module of the first repeat domain with at least one repeat module of the second repeat domain to form a designed repeat domain with dual binding specificity; (ii)(2) determining whether the binding of the designed repeat domains formed in step (ii) to the first and second targets is mutually exclusive; The method comprises the steps of: (a) providing a first repeat domain having binding specificity for a first target and binding specificity for a second target, and wherein the binding of the designed repeat domain to the first and second targets is mutually exclusive.

[0163] In another embodiment, the present invention provides a method for producing a designed repeat domain, the method comprising: (i) providing a first repeat domain and a second repeat domain, wherein the first repeat domain has binding specificity for a first target and the second repeat domain has binding specificity for a second target; (i)(2) determining whether the first repeat domain and / or the second repeat domain are compatible for covalent combination at the N-terminus and / or C-terminus with another repeat domain; (ii) linking a part of the first repeat domain with a part of the second repeat domain by covalently combining at least one repeat module of the first repeat domain with at least one repeat module of the second repeat domain to form a designed repeat domain with dual binding specificity; (ii)(2) determining whether the binding of the designed repeat domains formed in step (ii) to the first and second targets is mutually exclusive; The method comprises the steps of: (a) providing a first repeat domain having binding specificity for a first target and binding specificity for a second target, and wherein the binding of the designed repeat domain to the first and second targets is mutually exclusive.

[0164] In one embodiment the method of the invention further comprises the step (ib) of comparing the target interaction residues of the repeat modules comprised in the first repeat domain with the target interaction residues of the repeat modules comprised in the second repeat domain and selecting at least one repeat module of the first repeat domain and at least one repeat module of the second repeat domain for covalent combination.

[0165] Thus, in one embodiment, the invention provides a method of producing a designed repeat domain, the method comprising: (i) providing a first repeat domain and a second repeat domain, wherein the first repeat domain has binding specificity for a first target and the second repeat domain has binding specificity for a second target; (ib) comparing target interaction residues in the first repeat domain with target interaction residues in the second repeat domain and selecting a part of the first repeat domain and a part of the second repeat domain; (ii) linking a portion of the first repeat domain with a portion of the second repeat domain by covalently combining at least one repeat module of the first repeat domain with at least one repeat module of the second repeat domain to form a designed repeat domain with dual binding specificity, The method comprises the steps of: (a) providing a first repeat domain having binding specificity for a first target and binding specificity for a second target, and wherein the binding of the designed repeat domain to the first and second targets is mutually exclusive.

[0166] In some preferred embodiments, the first target and / or the second target are biologically active molecules. Many biologically active molecules are known to those skilled in the art. One example of a biologically active molecule is an agonist or antagonist or modulator of a cell surface receptor and the cell surface receptor itself. As known to those skilled in the art, a biologically active molecule may function, for example, by interacting with a receptor on the cell surface, thereby stimulating, inhibiting, or modulating the activity of the cell surface receptor. Such activity of the cell surface receptor is then often involved in an intracellular signaling pathway of the cell that has the receptor, which includes biologically active molecules such as enzymes, e.g., protein kinases, and transcription factors. More generally, a biologically active molecule(s) may exert their biological activity by binding to one or more binding partners. The binding partners of such molecules may be extracellular proteins, intracellular proteins, or transmembrane proteins (e.g., soluble receptors, membrane-bound receptors, ligands, cytokines, enzymes, binding proteins, or structural proteins). Thus, in one embodiment, the designed repeat domain, upon binding to the first target or the second target, inhibits the biological activity of the bound target. In another embodiment, the designed repeat domain, upon binding to the first target or the second target, activates the biological activity of the bound target. In another embodiment, the designed repeat domain, upon binding to the first target or the second target, modulates the biological activity of the bound target.

[0167] In the context of the present invention, preferred examples of biologically active target molecules are any synthetic or naturally occurring molecules (e.g., polypeptides or proteins) that are applicable for use in the diagnosis, cure, mitigation, treatment or prevention of disease, disorder, condition or infection, or that are present in living organisms. In one embodiment, the biologically active target molecule is a drug molecule. In one embodiment, the drug molecule is a T cell.

[0168] In one embodiment, binding of the designed repeat domain to one of the first and second targets sterically hinders binding of the designed repeat domain to the other of the first and second targets.

[0169] In some embodiments, the binding affinity of the designed repeat domain to the first target is determined by the dissociation constant (K D In one embodiment, the designed repeat domains can be described in terms of a 10 -5 Dissociation constant (K D In certain embodiments, the designed repeat domain binds to the first target at 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 M or less, about 10 -5 M~about 10 -15 M, about 10 -6 M~about 10 -15 M, about 10 -7 M~about 10 -15 M, about 10 -8 M~about 10 -15 M, about 10 -9 M~about 10 -15 M, about 10 -10 M~about 10 -15 M, about 10 -11 M~about 10 -15 M, about 10 -12 M~about 10 -15 M, about 10 -5 M~about 10 -14 M, about 10 -6 M~about 10 -14 M, about 10 -7 M~about 10 -14 M, about 10 -8 M~about 10 -14 M, about 10 -9 M~about 10 -14 M, about 10 -10 M~about 10 -14 M, about 10-11 M~about 10 -14 M, about 10 -12 M~about 10 -14 M, about 10 -5 M~about 10 -13 M, about 10 -6 M~about 10 -13 M, about 10 -7 M~about 10 -13 M, about 10 -8 M~about 10 -13 M, about 10 -9 M~about 10 -13 M, about 10 -10 M~about 10 -13 M, about 10 -11 M~about 10 -13 M, or about 10 -12 M~about 10 -13 K D In further embodiments, the designed repeat domains bind with a K of about 1000 nM, about 100 nM, about 50 nM, about 25 nM, about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 50 pM, about 25 pM, about 10 pM, about 5 pM, about 2 pM, about 1 pM, about 500 fM, about 250 fM, about 100 fM, about 50 fM, about 25 fM, about 10 fM, about 5 fM, about 2 fM, or about 1 fM or less. D In an exemplary embodiment, the designed repeat domain binds to the first target with a K value of about 1 nM or less. D In another exemplary embodiment, the designed repeat domain binds to the first target with a K value of about 100 pM or less. D In another exemplary embodiment, the designed repeat domain binds to the first target with a K value of about 10 pM or less. D In yet another exemplary embodiment, the designed repeat domain binds to the first target with a K value of about 1 pM or less. D The first target is bound at a value.

[0170] In some embodiments, the binding affinity of the designed repeat domain to the second target is determined by the dissociation constant (K DIn one embodiment, the designed repeat domains may be described in terms of a .times. ... -5 Dissociation constant (K D In certain embodiments, the designed repeat domain binds to the second target at 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 -1 M or less, about 10 -11 M or less, about 10 -12 M or less, about 10 -13 M or less, about 10 -14 M or less, about 10 -5 M~about 10 -15 M, about 10 -6 M~about 10 -15 M, about 10 -7 M~about 10 -15 M, about 10 -8 M~about 10 -15 M, about 10 -9 M~about 10 -15 M, about 10 -10 M~about 10 -15 M, about 10 -11 M~about 10 -15 M, about 10 -12 M~about 10 -15 M, about 10 -5 M~about 10 -14 M, about 10 -6 M~about 10 -14 M, about 10 -7 M~about 10 -14 M, about 10 -8 M~about 10 -14 M, about 10 -9 M~about 10 -14 M, about 10 -10 M~about 10 -14 M, about 10 -11 M~about 10 -14 M, about 10 -12 M~about 10 -14 M, about 10 -5 M~about 10 -13 M, about 10 -6 M~about 10 -13 M, about 10 -7 M~about 10 -13 M, about 10-8 M~about 10 -13 M, about 10 -9 M~about 10 -13 M, about 10 -10 M~about 10 -13 M, about 10 -11 M~about 10 -13 M, or about 10 -12 M~about 10 -13 K D In further embodiments, the designed repeat domain binds to the second target at a K of about 1000 nM, about 100 nM, about 50 nM, about 25 nM, about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 50 pM, about 25 pM, about 10 pM, about 5 pM, about 2 pM, about 1 pM, about 500 fM, about 250 fM, about 100 fM, about 50 fM, about 25 fM, about 10 fM, about 5 fM, about 2 fM, or about 1 fM or less. D In an exemplary embodiment, the designed repeat domain binds with a K of about 1 nM or less. D In another exemplary embodiment, the designed repeat domain binds to the second target with a K value of about 100 pM or less. D In another exemplary embodiment, the designed repeat domain binds to the second target with a K value of about 10 pM or less. D In yet another exemplary embodiment, the designed repeat domain binds to the second target with a K value of about 1 pM or less. D The second target is bound at a value.

[0171] In some embodiments, the binding affinity of the designed repeat domain to each of the first and second targets is determined by the dissociation constant (K D In such an embodiment, the K D 1 represents the dissociation constant between the designed repeat domain and the first target, K D 2 represents the dissociation constant between the designed repeat domain and the second target. In an exemplary embodiment, the designed repeat domain binds to each of the first and second targets and has a K D 1 and K D2はINDEPENDENT して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 -1 M or less, about 10 -11 M or less, about 10 -12 M or less, about 10 -13 M or less, about 10 -14 M or less, about 10 -5 M~about 10 -15 M, about 10 -6 M~about 10 -15 M, about 10 -7 M~about 10 -15 M, about 10 -8 M~about 10 -15 M, about 10 -9 M~about 10 -15 M, about 10 -10 M~about 10 -15 M, about 10 -11 M~about 10 -15 M, about 10 -12 M~about 10 -15 M, about 10 -5 M~about 10 -14 M, about 10 -6 M~about 10 -14 M, about 10 -7 M~about 10 -14 M, about 10 -8 M~about 10 -14 M, about 10 -9 M~about 10 -14 M, about 10 -10 M~about 10 -14 M, about 10 -11 M~about 10 -14 M, about 10 -12 M~about 10 -14 M, about 10 -5 M~about 10 _13 M, about 10 -6 M~about 10 -13 M, about 10 -7 M~about 10 -13 M, about 10 -8 M~about 10 -13 M, about 10 -9 M~about 10 -13 M, about 10 -10 M~about 10 -13 M, about 10-11 M~about 10 -13 M, or about 10 -12 M~about 10 -13 M. In an exemplary embodiment, K D 1 and K D 2 is independently about 100 nM, about 100 nM, about 50 nM, about 25 nM, about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 50 pM, about 25 pM, about 10 pM, about 5 pM, about 2 pM, about 1 pM, about 500 fM, about 250 fM, about 100 fM, about 50 fM, about 25 fM, about 10 fM, about 5 fM, about 2 fM, or about 1 fM or less. D 1 and K D 2 are independently about 1 nM or less. In another exemplary embodiment, K D 1 and K D 2 are independently about 100 pM or less. In another exemplary embodiment, K D 1 and K D In yet another exemplary embodiment of the method according to the invention, the designed repeat domains have K D 1 and K D 2 binds to the first and second targets, respectively.

[0172] In one embodiment, the designed repeat domain binds to one of the first and second targets with a first binding affinity and binds to the other of the first and second targets with a second binding affinity, the ratio of the first binding affinity to the second binding affinity being between 1:1 and 1:10. 5 In further embodiments, the ratio is about 1:1, 1:10, 1:10 2 , 1:10 3 , 1:10 4 Or 1:10 5 may be equal to

[0173] In one embodiment, the first and second targets are located on different molecules. In one embodiment, the first and second targets are epitopes located on different molecules. In one embodiment, the first and second targets are located on the same molecule. In one embodiment, the first and second targets are epitopes on the same molecule. In one embodiment, the first and second targets are different epitopes on the same molecule. In some preferred embodiments, the first target is a molecule present in the tumor microenvironment (such as VEGF), and the second target molecule is a paratope of a CD3 binding domain (e.g., a (designed) ankyrin repeat domain with binding specificity for CD3).

[0174] In one embodiment the first repeat domain is an ankyrin repeat domain and the second repeat domain is an ankyrin repeat domain.

[0175] In one embodiment the engineered repeat domain is an ankyrin repeat domain.

[0176] Repeat domains of the present invention In one aspect, the present invention provides a designed repeat domain obtainable or obtained by a method according to the present invention.

[0177] In one aspect, the invention provides a designed repeat domain having a first binding specificity for a first target and a second binding specificity for a second target, wherein binding of the designed repeat domain to the first and second targets is mutually exclusive.

[0178] The designed repeat domains of the invention are therefore bispecific molecules, i.e. molecules with dual binding specificities for two, preferably different, targets. The mutually exclusive binding properties of the designed repeat domains of the invention are illustrated in Figure 2 and explained in detail above in the context of the production methods of the invention as well as in the accompanying examples.

[0179] In some preferred embodiments, the first target and / or the second target are a molecule(s) with biological activity (as defined herein above). Thus, in one embodiment, the designed repeat domain, when bound to the first target or the second target, inhibits the biological activity of the bound target. In other words, the binding of the designed repeat domain to the target substantially or significantly interferes with or competes with the binding of the target to its natural binding partner, whereby the biological activity of the target is inhibited by binding to the designed repeat domain of the present invention. In an alternative embodiment, the designed repeat domain, when bound to the first target or the second target, induces a biological activity by binding to the target (as described herein above).

[0180] In one embodiment, binding of the designed repeat domain to one of the first and second targets sterically hinders binding of the designed repeat domain to the other of the first and second targets.

[0181] In some embodiments, the binding affinity of the designed repeat domain to the first target is determined by the dissociation constant (K D In one embodiment, the designed ankyrin repeat domain can be described in terms of a 10 -5 Dissociation constant (K D In some embodiments, the designed repeat domain binds to the first target at 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 -1 M or less, about 10 -11 M or less, about 10 -12 M or less, about 10 -13 M or less, about 10 -14 M or less, about 10 -5 M~about 10 -15 M, about 10 -6 M~about 10 -15 M, about 10 -7 M~about 10 -15 M, about 10 -8 M~about 10 -15M, about 10 _9 M~about 10 -15 M, about 10 -10 M~about 10 -15 M, about 10 -11 M~about 10 -15 M, about 10 minutes. 12 M~about 10 minutes 15 M, about 10 -5 M~about 10 -14 M, about 10 -6 M~about 10 -14 M, about 10 -7 M~about 10 -14 M, about 10 -8 M~about 10 -14 M, about 10 -9 M~about 10 -14 M, about 10 -10 M~about 10 -14 M, about 10 -11 M~about 10 -14 M, about 10 -12 M~about 10 -14 M, about 10 -5 M~about 10 -13 M, about 10 -6 M~about 10 -13 M, about 10 -7 M~about 10 -13 M, about 10 minutes. 8 M~about 10 -13 M, about 10 -9 M~about 10 -13 M, about 10 -10 M~about 10 -13 M, about 10 -11 M~about 10 -13 M, or about 10 -12 M~about 10 -13 K D In further embodiments, the designed repeat domain binds to the first target with a K of about 1000 nM, about 100 nM, about 50 nM, about 25 nM, about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 50 pM, about 25 pM, about 10 pM, about 5 pM, about 2 pM, about 1 pM, about 500 fM, about 250 fM, about 100 fM, about 50 fM, about 25 fM, about 10 fM, about 5 fM, about 2 fM, or about 1 fM or less.D In an exemplary embodiment, the designed repeat domain binds with a K of about 1 nM or less. D In another exemplary embodiment, the designed repeat domain binds to the first target with a K value of about 100 pM or less. D In another exemplary embodiment, the designed repeat domain binds to the first target with a K value of about 10 pM or less. D In yet another exemplary embodiment, the designed repeat domain binds to the first target with a K value of about 1 pM or less. D The first target is bound at a value.

[0182] In some embodiments, the binding affinity of the designed repeat domain to the second target is determined by the dissociation constant (K D In one embodiment, the designed repeat domains can be described in terms of a 10 -5 Dissociation constant (K D ) to bind to a second target. In certain embodiments, the designed repeat domain has a length 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 -1 M or less, about 10 -11 M or less, about 10 -12 M or less, about 10 -13 M or less, about 10 -14 M or less, about 10 -5 M~about 10 -15 M, about 10 -6 M~about 10 -15 M, about 10 -7 M~about 10 -15 M, about 10 -8 M~about 10 -15 M, about 10 -9 M~about 10 -15 M, about 10 -10 M~about 10 -15 M, about 10 -11 M~about 10 -15 M, about 10 -12 M~about 10 -15 M, about 10 -5 M~about 10 -14 M, about 10-6 M~about 10 -14 M, about 10 -7 M~about 10 -14 M, about 10 -8 M~about 10 -14 M, about 10 -9 M~about 10 -14 M, about 10 -10 M~about 10 -14 M, about 10 -11 M~about 10 -14 M, about 10 -12 M~about 10 -14 M, about 10 -5 M~about 10 -13 M, about 10 -6 M~about 10 -13 M, about 10 -7 M~about 10 minutes 13 M, about 10 -8 M~about 10 -13 M, about 10 -9 M~about 10 -13 M, about 10 -10 M~about 10 -13 M, about 10 -11 M~about 10 -13 M, or about 10 -12 M~about 10 -13 K D In further embodiments, the designed repeat domain binds to the first target with a K of about 1000 nM, about 100 nM, about 50 nM, about 25 nM, about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 50 pM, about 25 pM, about 10 pM, about 5 pM, about 2 pM, about 1 pM, about 500 fM, about 250 fM, about 100 fM, about 50 fM, about 25 fM, about 10 fM, about 5 fM, about 2 fM, or about 1 fM or less. D In an exemplary embodiment, the designed repeat domain binds to the first target with a K value of about 1 nM or less. D In another exemplary embodiment, the designed repeat domain binds to the first target with a K value of about 100 pM or less. D In another exemplary embodiment, the designed repeat domain binds to the first target with a K value of about 10 pM or less. DIn yet another exemplary embodiment, the designed repeat domain binds to the first target with a K value of about 1 pM or less. D The first target is bound at a value.

[0183] In some embodiments, the binding affinity of the designed repeat domain to each of the first and second targets is determined by the dissociation constant (K D In such an embodiment, the K D 1 represents the dissociation constant between the designed repeat domain and the first target, K D 2 represents the dissociation constant between the designed repeat domain and the second target. In an exemplary embodiment, the designed repeat domain binds to each of the first and second targets and has a K D 1 and K D 2 is about 10 independently -5 Below, 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 -1 M or less, about 10 -11 M or less, about 10 -12 M or less, about 10 -13 M or less, about 10 -14 M or less, about 10 -5 M~about 10 -15 M, about 10 -6 M~about 10 -15 M, about 10 -7 M~about 10 -15 M, about 10 -8 M~about 10 -15 M, about 10 -9 M~about 10 -15 M, about 10 -10 M~about 10 -15 M, about 10 -11 M~about 10 -15 M, about 10 -12 M~about 10 -15 M, about 10 -5 M~about 10 -14 M, about 10 -6 M~about 10 -14 M, about 10 -7 M~about 10 -14 M, about 10 -8 M~about 10-14 M, about 10 -9 M~about 10 -14 M, about 10 -10 M~about 10 -14 M, about 10 -11 M~about 10 -14 M, about 10 -12 M~about 10 -14 M, about 10 -5 M~about 10 -13 M, about 10 -6 M~about 10 -13 M, about 10 -7 M~about 10 -13 M, about 10 -8 M~about 10 -13 M, about 10 -9 M~about 10 -13 M, about 10 -10 M~about 10 -13 M, about 10 -11 M~about 10 minutes 13 M, or about 10 -12 M~about 10 -13 In an exemplary embodiment, K D 1 and K D 2 is about 1000 nM, about 100 nM, about 50 nM, about 25 nM, about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 50 pM, about 25 pM, about 10 pM, about 5 pM, about 2 pM, about 1 pM, about 500 fM, about 250 fM, about 100 fM, about 50 fM, about 25 fM, about 10 fM, about 5 fM, about 2 fM, or about 1 fM or less. D 1 and K D 2 are independently about 1 nM or less. In another exemplary embodiment, K D 1 and K D 2 are independently about 100 pM or less. In another exemplary embodiment, K D 1 and K D In yet another exemplary embodiment of the method according to the invention, the designed repeat domains have K D 1 and K D 2 binds to the first and second targets, respectively.

[0184] In one embodiment, the designed repeat domain binds to one of the first and second targets with a first binding affinity and binds to the other of the first and second targets with a second binding affinity, the ratio of the first binding affinity to the second binding affinity being between 1:1 and 1:10. 5 In further embodiments, the ratio is about 1:1, 1:10, 1:10 2 , 1:10 3 , 1:10 4 Or 1:10 5 may be equal to

[0185] In one embodiment, the first and second targets are located on different molecules. In one embodiment, the first and second targets are epitopes located on different molecules. In one embodiment, the first and second targets are located on the same molecule. In one embodiment, the first and second targets are epitopes on the same molecule. In a preferred embodiment, the first and second targets are different epitopes on the same molecule. In some preferred embodiments, the first target is a molecule present in the tumor microenvironment (such as VEGF) and the second target molecule is a paratope of the CD3 binding domain (such as a (designed) ankyrin repeat domain).

[0186] In some embodiments, the designed repeat domain comprises at least one repeat module, at least two repeat modules, at least three repeat modules, at least four repeat modules, at least five repeat modules or at least six repeat modules. In some embodiments, the designed repeat domain comprises 1-6 repeat modules, 2-6 repeat modules, 3-6 repeat modules, 4-6 repeat modules or 5-6 repeat modules. In some embodiments, the designed repeat domain comprises an N-terminal capping module and / or a C-terminal capping module. In some embodiments, the designed repeat domain comprises an N-terminal capping module and / or a C-terminal capping module and at least one internal repeat module, at least two internal repeat modules, at least three internal repeat modules or at least four internal repeat modules. In some aspects, the designed repeat domain comprises an N-terminal capping module and / or a C-terminal capping module and 1-4 internal repeat modules, 1-3 internal repeat modules or 1-2 internal repeat modules.

[0187] In one particular embodiment, the designed repeat domain comprises at least two repeat modules. In another particular embodiment, the designed repeat domain comprises at least three repeat modules. In another particular embodiment, the designed repeat domain comprises at least four repeat modules. In another particular embodiment, the designed repeat domain comprises at least five repeat modules. In another particular embodiment, the designed repeat domain comprises at least six repeat modules. In another particular embodiment, the designed repeat domain comprises at least seven repeat modules. In one particular embodiment, the designed repeat domain comprises an N-terminal and / or a C-terminal capping module and at least one internal repeat module. In one particular embodiment, the designed repeat domain comprises an N-terminal and / or a C-terminal capping module and at least two internal repeat modules. In one particular embodiment, the designed repeat domain comprises an N-terminal and / or a C-terminal capping module and at least three internal repeat modules. In one particular embodiment, the designed repeat domain comprises an N-terminal and / or a C-terminal capping module and at least four internal repeat modules.

[0188] In a particular embodiment, the designed repeat domain comprises an N-terminal and / or a C-terminal capping module and one internal repeat module. In a particular embodiment, the designed repeat domain comprises an N-terminal and / or a C-terminal capping module and two internal repeat modules. In a particular embodiment, the designed repeat domain comprises an N-terminal and / or a C-terminal capping module and three internal repeat modules. In a particular embodiment, the designed repeat domain comprises an N-terminal and / or a C-terminal capping module and four internal repeat modules. In any of the above embodiments, the "and" linkage is preferred, i.e. the designed repeat domain preferably comprises an N-terminal and a C-terminal capping module in addition to the internal repeat module(s).

[0189] Thus, in some aspects, the designed repeat domain comprises an N-terminal capping module, a C-terminal capping module, and at least one internal repeat module. In one embodiment, the designed repeat domain comprises an N-terminal capping module, a C-terminal capping module, and one internal repeat module. In one embodiment, the designed repeat domain comprises an N-terminal capping module, a C-terminal capping module, and two internal repeat modules. In one embodiment, the designed repeat domain comprises an N-terminal capping module, a C-terminal capping module, and three internal repeat modules. In one embodiment, the designed repeat domain comprises an N-terminal capping module, a C-terminal capping module, and four internal repeat modules. In one embodiment, the designed repeat domain comprises an N-terminal capping module, a C-terminal capping module, and five internal repeat modules. In one embodiment, the designed repeat domain comprises an N-terminal capping module, a C-terminal capping module, and six internal repeat modules. In one embodiment, the designed repeat domain comprises an N-terminal capping module, a C-terminal capping module, and seven internal repeat modules.

[0190] In one embodiment, all internal repeat modules comprised in the designed repeat domain contribute to the first and / or second binding specificity. In some embodiments, the first and / or second binding specificity comprises target interaction residues comprised in the internal repeat modules. In one embodiment, all repeat modules comprised in the designed repeat domain contribute to the first and / or second binding specificity. In some embodiments, the first and / or second binding specificity comprises target interaction residues comprised in the repeat modules. In one embodiment, the designed repeat domain comprises at least one internal repeat module that contributes to binding to a first target and the designed repeat domain comprises at least one internal repeat module that contributes to binding to a second target.

[0191] In one embodiment, the designed repeat domain comprises at least one, at least two, at least three, at least four, or at least five target interaction residues that bind to a first target and / or the designed repeat domain comprises at least one, at least two, at least three, at least four, or at least five target interaction residues that bind to a second target. In one embodiment, the designed repeat domain comprises at least one, at least two, at least three, at least four, or at least five target interaction residues that bind to a first target and the designed repeat domain comprises at least one, at least two, at least three, at least four, or at least five target interaction residues that bind to a second target. Any combination of the above embodiments is possible, for example, in one embodiment, the designed repeat domain comprises at least one target interaction residue that binds to a first target and the designed repeat domain comprises at least one target interaction residue that binds to a second target. Methods for determining such contributions or amino acid residues involved in binding interactions between proteins or between a protein and a peptide, such as, for example, alanine scanning mutagenesis, are well known to those skilled in the art.

[0192] In one embodiment the engineered repeat domain is an ankyrin repeat domain.

[0193] In one embodiment, the designed repeat domain comprises at least one internal repeat module comprising a sequence selected from SEQ ID NOs: 75-81.

[0194] In one embodiment, the designed repeat domain comprises: (i) an N-terminal capping module comprising a sequence selected from the group consisting of (1) SEQ ID NOs: 35, 36, 47-63 and 83-85; and (2) SEQ ID NOs: 35, 36, 47, 53, 59, 60, 83 and 84, 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 amino acid is replaced by another amino acid; and / or (ii) a C-terminal capping module comprising a sequence selected from the group consisting of (1) SEQ ID NOs: 37, 38 and 64-74; and (2) SEQ ID NOs: 37, 38 and 69, 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 amino acid is replaced by another amino acid. Thus, in one embodiment, the designed repeat domain comprises (i) an N-terminal capping module comprising a sequence selected from the group consisting of (1) SEQ ID NOs: 35, 36, 47 to 63 and 83 to 85 and (2) any of SEQ ID NOs: 35, 36, 47, 53, 59, 60, 83 and 84 in which up to 9 amino acids have been replaced by other amino acids, and / or (ii) a C-terminal capping module comprising a sequence selected from the group consisting of (1) SEQ ID NOs: 37, 38 and 64 to 74 and (2) any of SEQ ID NOs: 37, 38 and 69 in which up to 9 amino acids have been replaced by other amino acids.

[0195] In one embodiment, the designed repeat domain comprises: (i) an N-terminal capping module comprising a sequence selected from the group consisting of (1) SEQ ID NOs: 35, 36, 47-63, 83-85, and 97-100; and (2) SEQ ID NOs: 35, 36, 47, 53, 59, 60, 83, and 84, 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 amino acid is replaced by another amino acid; and / or (ii) a C-terminal capping module comprising a sequence selected from the group consisting of (1) SEQ ID NOs: 37, 38, and 64-74; and (2) SEQ ID NOs: 37, 38, and 69, 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 amino acid is replaced by another amino acid. Thus, in one embodiment, the designed repeat domain comprises (i) an N-terminal capping module comprising a sequence selected from the group consisting of (1) SEQ ID NOs: 35, 36, 47-63, 83-85 and 97-100, and (2) any of SEQ ID NOs: 35, 36, 47, 53, 59, 60, 83 and 84 in which up to 9 amino acids have been replaced by other amino acids, and / or (ii) a C-terminal capping module comprising a sequence selected from the group consisting of (1) SEQ ID NOs: 37, 38 and 64-74, and (2) any of SEQ ID NOs: 37, 38 and 69 in which up to 9 amino acids have been replaced by other amino acids.

[0196] In one embodiment, the designed repeat domain comprises: (i) an N-terminal amino acid 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%, at least 99%, or 100% amino acid sequence identity to any one of SEQ ID NOs: 35, 36, 47, 53, 59, 60, 83, and 84; and / or (ii) a C-terminal capping module comprising an amino acid sequence of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 80%, 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to any of SEQ ID NOs: 37, 38 and 69.

[0197] In one embodiment, the designed repeat domain comprises at least one internal repeat module comprising a sequence selected from the group consisting of: (1) SEQ ID NOs: 40 to 46 and (2) sequences 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 amino acid in any of SEQ ID NOs: 40 to 46 is replaced by another amino acid. Thus, in one embodiment, the designed repeat domain comprises at least one internal repeat module comprising a sequence selected from the group consisting of: (1) SEQ ID NOs: 40 to 46 and (2) sequences in which up to 9 amino acids in any of SEQ ID NOs: 40 to 46 are replaced by another amino acid. In one embodiment, the designed repeat domain comprises at least one internal repeat module comprising a sequence of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to any one of SEQ ID NOs: 40-46.

[0198] In one embodiment, the designed repeat domain comprises at least one internal repeat module comprising a sequence selected from the group consisting of: (1) SEQ ID NOs: 40 to 46, 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 amino acid in any of SEQ ID NOs: 40 to 46 is substituted with another amino acid. The designed repeat domain further comprises: (i) an N-terminal capping module comprising a sequence selected from the group consisting of (1) SEQ ID NOs: 35-36, (2) sequences 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 amino acid in any of SEQ ID NOs: 35-36 is replaced with another amino acid, and / or (ii) a C-terminal capping module comprising a sequence selected from the group consisting of (1) SEQ ID NO: 38, (2) sequences 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 amino acid in SEQ ID NO: 38 is replaced with another amino acid.

[0199] Thus, in one embodiment, the designed repeat domain comprises at least one internal repeat module comprising a sequence selected from the group consisting of: (1) SEQ ID NOs: 40 to 46, and (2) any of SEQ ID NOs: 40 to 46 in which up to 9 amino acids are substituted with other amino acids, wherein the designed repeat domain comprises: The nucleic acid further comprises (i) an N-terminal capping module comprising a sequence selected from the group consisting of (1) SEQ ID NOs: 35 to 36, (2) sequences in which up to 9 amino acids in any one of SEQ ID NOs: 35 to 36 are replaced with other amino acids, and / or (ii) a C-terminal capping module comprising a sequence selected from the group consisting of (1) SEQ ID NO: 38, (2) sequences in which up to 9 amino acids in SEQ ID NO: 38 are replaced with other amino acids.

[0200] In one embodiment, the designed repeat domain comprises at least one internal repeat module comprising a sequence of at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to any one of SEQ ID NOs: 40-46. (i) an N-terminal capping module comprising 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%, at least 99%, or 100% amino acid sequence identity to any of SEQ ID NOs: 35-36, and / or (ii) further comprising a C-terminal capping module comprising 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%, at least 99%, or 100% amino acid sequence identity to any one of SEQ ID NOs: 38.

[0201] In one embodiment, the designed repeat domain comprises: (i) an N-terminal capping module comprising a sequence selected from the group consisting of (1) SEQ ID NOs: 35-36 and (2) sequences 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 amino acid in any of SEQ ID NOs: 35-36 is replaced by another amino acid; and / or (ii) a C-terminal capping module comprising a sequence selected from the group consisting of (1) SEQ ID NO: 38 and (2) sequences 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 amino acid in SEQ ID NO: 38 is replaced by another amino acid.

[0202] Thus, in one embodiment, the designed repeat domain comprises: (i) an N-terminal capping module comprising a sequence selected from the group consisting of (1) SEQ ID NOs: 35-36 and (2) sequences in which up to 9 amino acids of any of SEQ ID NOs: 35-36 are replaced by other amino acids, and / or (ii) a C-terminal capping module comprising a sequence selected from the group consisting of (1) SEQ ID NO: 38 and (2) sequences in which up to 9 amino acids of SEQ ID NO: 38 are replaced by other amino acids.

[0203] In one embodiment, the engineered repeat domain comprises (i) an N-terminal capping module comprising 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%, at least 99%, or 100% amino acid sequence identity to any of SEQ ID NOs: 35-36, and / or (ii) a C-terminal capping module comprising 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%, at least 99%, or 100% amino acid sequence identity to any one of SEQ ID NOs: 38.

[0204] In any of the above embodiments regarding amino acid substitutions in a repeat module, the substitutions may occur at the target interaction residue positions and / or at framework residue positions. Thus, in some embodiments, the substitutions may occur at the target interaction residue positions. In other embodiments, the substitutions may occur at framework residue positions. In other embodiments, the substitutions may occur at both the target interaction residue positions and the framework residue positions.

[0205] More specifically, in the internal repeat module, the positions of the target interaction residues are those corresponding to positions 3, 4, 6, 11, 14 and 15 of SEQ ID NO: 39, and the positions of the framework residues are those corresponding to positions other than positions 3, 4, 6, 11, 14 and 15 of SEQ ID NO: 39. Thus, in any of the above embodiments concerning amino acid substitutions in an internal repeat module, the substitutions may occur at any such positions of the target interaction residues and / or at framework residue positions.

[0206] In the N-terminal capping module, the target interaction residue positions are those corresponding to positions 4, 5, 8, 11 and 12 of SEQ ID NO: 36, and the framework residue positions are those corresponding to positions other than positions 4, 5, 8, 11 and 12 of SEQ ID NO: 36. Thus, in any of the above embodiments relating to amino acid substitutions in the N-terminal capping module, substitutions may occur at any such positions of the target interaction residues and / or framework residue positions.

[0207] In the C-terminal capping module, the target interaction residue positions are those corresponding to positions 3, 4, 6, 14 and 15 of SEQ ID NO: 38, and the framework residue positions are those corresponding to positions other than positions 3, 4, 6, 14 and 15 of SEQ ID NO: 38. Thus, in any of the above embodiments relating to amino acid substitutions in the C-terminal capping module, substitutions may occur at any such positions of the target interaction residues and / or framework residue positions.

[0208] In one embodiment, the designed repeat domain comprises at least one internal repeat module, wherein the residues at positions corresponding to positions 3, 4, 6, 11, 14 and / or 15 of SEQ ID NO: 39 are target interaction residues and the residue at position corresponding to position 10 of SEQ ID NO: 39 is a framework residue.

[0209] In one embodiment, the designed repeat domain comprises at least one internal repeat module, wherein the residues at positions corresponding to positions 3, 4, 6, 11, 14 and / or 15 of SEQ ID NO: 39 are target interaction residues.

[0210] In one embodiment, the designed repeat domain comprises at least one N-terminal capping module, wherein the residues at positions corresponding to positions 4, 5, 8, 11 and / or 12 of SEQ ID NO: 36 are target interaction residues and the residue at position corresponding to position 7 of SEQ ID NO: 36 is a framework residue.

[0211] In one embodiment, the designed repeat domain comprises at least one N-terminal capping module, wherein the residues at positions corresponding to positions 4, 5, 8, 11 and / or 12 of SEQ ID NO: 36 are target interaction residues.

[0212] In one embodiment, the designed repeat domain comprises at least one C-terminal capping module, wherein the residues at positions corresponding to positions 3, 4, 6, 14 and / or 15 of SEQ ID NO: 38 are target interaction residues and the residues at positions corresponding to positions 10 and / or 11 of SEQ ID NO: 38 are framework residues.

[0213] In one embodiment, the designed repeat domain comprises at least one C-terminal capping module, wherein the residues at positions corresponding to positions 3, 4, 6, 14 and / or 15 of SEQ ID NO: 38 are target interaction residues.

[0214] In some embodiments, the designed repeat domain according to the invention comprises a paratope. Such a paratope is formed by the repeat modules contained in the repeat domain, more precisely by the target interaction residues contained in each of these repeat modules. Thus, in some embodiments, such a paratope is essentially separated into two regions, each of which contains a repeat module contributing to a first or second binding specificity. In such embodiments, the paratope regions do not overlap. In other embodiments, some repeat modules may contribute to both binding specificities due to one or more target interaction residues that interact with both targets, and therefore both regions of the paratope may overlap to some extent.

[0215] In one embodiment the designed repeat domain comprises two internal repeat modules, the N-terminus of one of the two internal repeat modules comprises target interaction residues which bind to a first target and the C-terminus of one of the two internal repeat modules comprises target interaction residues which bind to a second target.

[0216] In one embodiment, the designed repeat domain comprises three internal repeat modules, the most N-terminal of the three internal repeat modules comprises target interaction residues that bind a first target, the most C-terminal of the three internal repeat modules comprises target interaction residues that bind a second target, and the central of the three internal repeat modules comprises target interaction residues that bind the first and / or second target. In a particular embodiment, the designed repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 6-10 and (2) sequences 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%, at least 99%, or 100% amino acid sequence identity to any of SEQ ID NOs: 6-10. Thus, in one embodiment, the designed repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 6-10, and (2) a sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 6-10.

[0217] In one embodiment, the designed repeat domain comprises four internal repeat modules, the most N-terminal of the four internal repeat modules comprises target interaction residues that bind a first target, the most C-terminal of the four internal repeat modules comprises target interaction residues that bind a second target, and the second most N-terminal and second most C-terminal of the four internal repeat modules each comprise target interaction residues that bind the first target and / or the second target. In a particular embodiment, the designed repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 11-13 and (2) sequences with at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% amino acid sequence identity to any of SEQ ID NOs: 11-13. Thus, in one embodiment, the designed repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 11-13, and (2) a sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 11-13.

[0218] In one embodiment, the designed repeat domain comprises six internal repeat modules, the three most N-terminal of the six internal repeat modules contain target interaction residues that bind a first target, the two most C-terminal of the six internal repeat modules contain target interaction residues that bind a second target, and the fourth most N-terminal one (or the third most C-terminal one) of the six internal repeat modules contains target interaction residues that bind the first target and / or the second target. In particular embodiments, the designed repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NO: 92, 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%, at least 99%, or 100% amino acid sequence identity to SEQ ID NO: 92. Thus, in further particular embodiments, the designed repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NO: 92, and (2) a sequence having at least 80% amino acid sequence identity to SEQ ID NO: 92.

[0219] In one embodiment, the designed repeat domain comprises seven internal repeat modules, the three most N-terminal of the seven internal repeat modules comprise target interaction residues that bind a first target, the two most C-terminal of the seven internal repeat modules comprise target interaction residues that bind a second target, and the fourth and fifth most N-terminal, respectively (or the third and fourth most C-terminal, respectively) of the seven internal repeat modules comprise target interaction residues that bind the first target and / or the second target. In certain embodiments, the designed repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 11-13, 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%, at least 99%, or 100% amino acid sequence identity to any one of SEQ ID NOs: 11-13. Thus, in one embodiment, the designed repeat domain comprises an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 90, 91, 93, and 94, and (2) a sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs: 90, 91, 93, and 94.

[0220] Furthermore, any repeat domain sequence of the invention may optionally comprise at its N-terminus G, S or GS. Furthermore, any repeat domain sequence of the invention may optionally have an A in the penultimate position replaced with an L and / or an A in the last position replaced with an N.

[0221] Recombinant Proteins The engineered repeat domains of the present invention may be genetically fused to additional moieties (e.g., therapeutic agents), and such fusions are also referred to interchangeably as "recombinant proteins" or "constructs." In such constructs, the engineered repeat domains may act as an on or off switch for the fused therapeutic agent.

[0222] FIG. 3 shows general examples of constructs comprising the designed repeat domain of the invention. These exemplary constructs are also referred to as "Switchdrug DARPins". In the on-switch setting (FIG. 3A), the designed repeat domain of the invention binds to target A (e.g. a sensor target) or target B (e.g. a drug molecule) in a mutually exclusive manner and is chemically linked to target B (e.g. a drug molecule). The state of target B (e.g. an active or inactive drug molecule) as part of the Switchdrug DARPin is conditionally regulated by the interaction of the designed repeat domain of the invention with target A, and the binding of target A by the designed repeat domain of the invention results in the release of the bound target B (and thus the active drug molecule). In the exemplary off-switch setting shown in FIG. 3B, the designed repeat domain of the invention is designed to bind to target A (e.g. a sensor target) or target B (e.g. a cellular receptor). The downstream activity resulting from the interaction of the designed repeat domain of the invention with target B can be conditionally switched off when target A binds to the designed repeat domain of the invention. The designed repeat domain of the invention can be further linked to other binding domains or molecules.

[0223] Thus, in one aspect, the invention provides a recombinant protein comprising an engineered repeat domain of the invention. In one embodiment, the protein further comprises a therapeutic agent. In one embodiment, the therapeutic agent is a binding molecule. In a further embodiment, the binding molecule comprises an engineered ankyrin repeat domain.

[0224] Such recombinant proteins of the invention can be produced in different formats. In one embodiment, a therapeutic agent is fused N-terminally to a designed repeat domain of the invention. In one embodiment, a therapeutic agent is fused C-terminally to a designed repeat domain of the invention. In some embodiments, a therapeutic agent is directly linked to a designed repeat domain of the invention. In some embodiments, a peptide linker is used to link the designed repeat domain of the invention to the therapeutic agent. In some aspects, the therapeutic agent is separated from the designed repeat domain of the invention by an additional molecule, for example one or more ankyrin repeat domains.

[0225] In one embodiment, the recombinant protein of the invention comprises an additional repeat domain, for example, the recombinant protein of the invention comprises a designed repeat domain of the invention and two or more further ankyrin repeat domains, where the two or more ankyrin repeat domains, for example two or three ankyrin repeat domains, can be linked with a peptide linker. In one embodiment, the peptide linker is a proline-threonine rich peptide linker. In one embodiment, the peptide linker is a proline-threonine rich peptide linker of SEQ ID NO: 82. Other linkers known in the art can also be used to link the repeat domains (see, for example, WO 2021 / 116469).

[0226] In some embodiments, each of the first and second binding regions (or paratopes) included in the designed repeat domain of the invention may have a defined function. In one embodiment, the designed repeat domain is described as comprising a sensor portion and a blocker portion, the sensor portion corresponding to one of the first or second binding regions and the blocker portion corresponding to the other binding region of the repeat domain. As a result, in such a construct, the designed repeat domain of the invention has a first binding specificity for a sensor target and a second binding specificity for a blocker target. In a preferred embodiment, the blocker target is a therapeutic agent, and the sensor target can be selected as required depending on the therapeutic agent, application or situation. Based on the selected therapeutic agent and sensor target, the designed repeat domain can be generated as described in the present disclosure and then genetically fused to further components, in particular therapeutic agents, using methods known to those skilled in the art.

[0227] Immune cell engagers, such as T cell engagers (TCEs) or natural killer (NK) cell engagers, can be advantageously used as fusion partners in the recombinant proteins of the invention. TCEs typically comprise a CD3 binding domain and a tumor associated antigen (TAA) binding domain. Thus, in one preferred embodiment, the recombinant protein of the invention comprises an engineered repeat domain of the invention with a first binding specificity for the α-CD3 binding domain of the TCE (blocker target) and a second binding specificity for a sensor target. The engineered repeat domain binds to the TCE and inhibits its activity unless the sensor target binds to the engineered repeat domain. High concentrations of the sensor target induce the release of the CD3 binding domain (blocker target), allowing the recruitment of cytotoxic T cells and the formation of an immunological synapse between TAA-expressing tumor cells and T cells. Such an embodiment is shown in FIG. 4A.

[0228] The examples disclosed herein illustrate various recombinant proteins (or constructs) comprising a CD3 binding domain, preferably a CD3 binding DARPin, and optionally a TCE comprising a CD123 binding or non-binding (placeholder) domain, and an inventive designed repeat domain with a first binding specificity for a paratope of the CD3 binding domain (blocker target) and a second binding specificity for VEGF (sensor target). In some embodiments, the affinity of the inventive designed repeat domain for the sensor target is higher than the affinity of the repeat domain for the blocker target.

[0229] Thus, in one embodiment, a therapeutic agent described herein is a binding molecule, wherein the binding molecule is an immune cell engager, hi a specific embodiment, the binding molecule is TCE.

[0230] In one embodiment, a recombinant protein of the invention comprises an α-CD3 binding domain N-terminal to a designed repeat domain of the invention. In one embodiment, a recombinant protein of the invention comprises an α-CD3 binding domain C-terminal to a designed repeat domain of the invention. In a preferred embodiment, a recombinant protein of the invention comprises, from N-terminus to C-terminus, a designed repeat domain of the invention, another binding domain or a non-binding domain, and an α-CD3 binding domain. In another preferred embodiment, a recombinant protein of the invention comprises, from N-terminus to C-terminus, a TAA binding domain, an α-CD3 binding domain, and a designed repeat domain of the invention. In a more preferred embodiment, a recombinant protein of the invention comprises, from N-terminus to C-terminus, a designed repeat domain of the invention, a TAA binding domain, and an α-CD3 binding domain. In a particular embodiment, the TAA binding domain has the amino acid sequence of the CD 123 binding domain as set forth in SEQ ID NO:4. In a particular embodiment, the non-binding domain has the amino acid sequence as set forth in SEQ ID NO:5.

[0231] In one embodiment, the recombinant protein of the present invention comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NOs: 15 to 30 and (2) sequences 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%, at least 99%, or at least 100% amino acid sequence identity to any of SEQ ID NOs: 15 to 30. In one embodiment, the recombinant protein comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NOs: 15 to 30 and (2) sequences having at least 80% amino acid sequence identity to any of SEQ ID NOs: 15 to 30.

[0232] In other embodiments, the designed repeat domain is described as comprising a sensor portion and an effector portion, the sensor portion corresponding to one of the first or second binding regions, and the effector portion corresponding to the other binding region of the repeat domain. As a result, in such a construct, the repeat domain of the invention has a first binding specificity for a sensor target and a second binding specificity for an effector target. In some embodiments, the effector target is a molecule that induces an effect on a living cell. In some embodiments, the effector target is a cell surface molecule. In some embodiments, the sensor target can be selected as required depending on the effector target, application, or situation. Based on the selected effector target and sensor target, the designed repeat domain can be produced as described in the present disclosure and can be optionally fused to further components using methods known to those skilled in the art.

[0233] Thus, in one embodiment, the recombinant protein of the invention comprises a designed repeat domain of the invention with a first binding specificity for an effector target and a second binding specificity for a sensor target. As long as the designed repeat domain is bound to the effector target, the effector target remains active. At high concentrations of the sensor target, the binding of the sensor target to the designed repeat domain prevents activation of the effector target by preventing the interaction between the repeat domain and the effector target. In such an embodiment, the designed repeat domain can be used as an off switch, as also depicted in Figures 3B and 4B.

[0234] Nucleic Acids, Vectors and Host Cells In another aspect, the present invention relates to an isolated nucleic acid encoding the amino acid sequence of a designed repeat domain of the invention or an 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.

[0235] The present invention further relates to a vector comprising any of the nucleic acids of the present invention. Thus, 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 in a prokaryotic or eukaryotic host cell of an encoded polypeptide operably linked to the nucleic acid. 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.

[0236] Nucleic acids are well known to those skilled in the art. Nucleic acids were used to produce the designed ankyrin repeat domains or recombinant binding proteins of the invention in E. coli, for example as described in US Pat. No. 7,417,130.

[0237] 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, and a yeast such as Saccharomyces, an insect cell, a mammalian cell such as a Chinese Hamster Ovary (CHO) cell, a Cl 27 mouse cell line, a BHK cell line of Syrian hamster cells, or a Human Embryonic Kidney 293 (HEK 293) cell. In some embodiments, the host cell is a CHO cell or a HEK 293 cell. The host cell can be used, for example, to express a recombinant protein of the present invention.

[0238] composition The present invention further relates to pharmaceutical compositions comprising one or more of the designed repeat domains, recombinant proteins, nucleic acids and / or recombinant expression vectors described herein and a pharma- ceutically acceptable carrier or diluent. The present invention also relates to uses and methods of treatment using the pharmaceutical compositions disclosed herein. The methods and uses encompassed by the present invention are described in more detail below.

[0239] As shown above, the designed repeat domains of the present invention can be used to provide a conditional activation and / or deactivation mechanism (e.g., an on-switch or off-switch mechanism) for a therapeutic agent. Thus, in some embodiments, the designed repeat domain does not substantially alter the biological effect of a therapeutic agent in vivo other than inhibiting the therapeutic agent (the blocker target of the designed repeat domain) as long as the sensor target of the designed repeat domain is absent and substantially bound to the designed repeat domain.

[0240] The pharmaceutical compositions described herein can be prepared using methods known in the art.

[0241] The pharmaceutical composition may include a pharma- ceutically 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.

[0242] 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, drying agents, detergents, diluents, disinfectants, disintegrants, dispersants, dissolution enhancers, dyes, emollients, emulsifiers, emulsion stabilizers, fillers, film formers, seasonings, flavorings, flow enhancers, gelling agents, fabricators, and the like. Any other pharma- ceutically acceptable ingredients may be included, including granules, moisturizers, lubricants, mucoadhesives, ointment bases, ointments, oily vehicles, organic bases, pastel bases, pigments, plasticizers, abrasives, preservatives, sequestering agents, skin-penetrating agents, solubilizers, solvents, stabilizers, suppository bases, surfactants, surface-active substances, suspending agents, sweeteners, therapeutic agents, thickening agents, tonicity agents, toxicity agents, thickening agents, water-absorbency agents, water-miscible cosolvents, softening agents, or wetting agents.See, for example, Handbook of Pharmaceutical Excipients, Third Edition, AH Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated by reference in its entirety.Remington's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980), which is incorporated by reference in its entirety. In one embodiment, the present invention provides a pharmaceutical composition comprising one or more of the following: (i) a designed 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 at least one pharma- ceutically acceptable carrier or diluent.

[0243] Therapeutic Uses and Methods According to the 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.

[0244] In another aspect, the present invention provides a method of treating a medical condition, comprising administering to a patient in need of treating the medical condition a therapeutically 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.

[0245] Further provided is a designed repeat domain, recombinant protein, nucleic acid, or pharmaceutical composition of the invention for use in a method of treating a medical condition.

[0246] 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 invention for the treatment of a disease. For that purpose, the designed repeat domain, the recombinant protein, the nucleic acid or the pharmaceutical composition according to the invention is administered in a therapeutically effective amount to a patient in need thereof.

[0247] In one embodiment, the present invention relates to a method for treating a medical condition, comprising the step of administering to a patient in need of such treatment a therapeutically effective amount of a designed repeat domain, recombinant protein, nucleic acid or pharmaceutical composition of the invention.

[0248] 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 of a medical condition. 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 of a medical condition.

[0249] In one embodiment, the present invention relates to the use of a designed repeat domain, a recombinant protein, a nucleic acid, or a pharmaceutical composition of the invention for the treatment of a medical condition.

[0250] In one embodiment, the invention relates to a process for treating a medical condition using a designed repeat domain, recombinant protein, nucleic acid or pharmaceutical composition of the invention. In one embodiment, the invention relates to a process for the manufacture of a medicament for the treatment 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.

[0251] In one aspect, 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 manufacture of a medicament.

[0252] 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 manufacture of a medicament for the treatment of a medical condition.

[0253] 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.

[0254] In a preferred embodiment, the medical condition is cancer.

[0255] In some embodiments, the subject is a mammal. In preferred embodiments, the subject is a human.

[0256] In some embodiments, a single administration of the designed 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 of the therapeutic agent and the typical administration regimen, will affect the number and frequency of administration.

[0257] The designed repeat domains, recombinant proteins, nucleic acids or pharmaceutical compositions described herein can be used in combination with another therapeutic agent. Each therapeutic agent may be administered simultaneously (e.g., in the same agent or simultaneously), in parallel (i.e., in separate agents, one administered immediately after the other, in any order), or sequentially, in any order. Sequential administration is useful when the therapeutic agents in the combination therapy are in different dosage forms (e.g., one agent is a tablet or capsule and another agent is a sterile liquid) and / or when administered on different administration schedules (e.g., in the case of a pain reliever administered at least daily and a biotherapeutic agent administered less frequently, such as once a week or once every two weeks).

[0258] The invention is not limited to the specific embodiments described in the examples. EXAMPLES

[0259] The starting materials and reagents disclosed below are known to those of ordinary skill in the art, are commercially available, and / or can be prepared using well-known techniques.

[0260] material Chemicals were purchased from Sigma-Aldrich (USA). Oligonucleotides were purchased from Microsynth (Switzerland). DNA polymerases, restriction enzymes, and buffers were purchased from New England Biolabs (USA) or Fermentas / Thermo Fisher Scientific (USA) unless otherwise stated. Inducible E. coli expression strains were used for cloning and protein production, e.g., E. coli XL1-blue (Stratagene, USA) or BL21 (Novagen, USA). NLC chips for SPR measurements were from BioRad (BioRad, USA). HTRF reagents were from Cisbio (Cisbio, France). Pan-T cell isolation kit was from Miltenyi Biotec (Germany). The detection antibody anti-hCD8 was from BD Pharmingen™ (BD, USA) and anti-hCD69 was from BioLegend (BioLegend, USA).

[0261] 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).

[0262] Designed ankyrin repeat protein library Methods for generating designed ankyrin repeat protein libraries are described, for example, in US 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 WO 2021 / 116462). Preferably, such libraries are assembled such that they do not have any of the amino acids C, G, M, N (before a G residue) and P in the randomized positions of the repeat or capping modules.

[0263] Furthermore, such randomized modules in such libraries may 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, KT, 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 of two ankyrin repeats, ankyrin repeat protein libraries can contain randomized loops (with fixed and randomized positions) of variable length (e.g., 1-20 amino acids) inserted into one or more beta turns of the ankyrin repeat domain.

[0264] 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.

[0265] The design of such ankyrin repeat protein libraries can be guided by known structures of ankyrin repeat domains interacting 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.

[0266] Examples of designed ankyrin repeat protein libraries have been described, such as N2C and N3C designed ankyrin repeat protein libraries (U.S. Pat. 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.

[0267] The nomenclature used to define the repeat units and positions within the modules is based on Binz et al. (2004) citation 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) citation above corresponds to position 2 of the ankyrin repeat module of the present disclosure, and consequently, position 33 of the ankyrin repeat module of Binz et al. (2004) citation above corresponds to position 1 of the following ankyrin repeat module of the present disclosure.

[0268] Example 1: Design of 2-in-1 DARPin overview DARPins (designed ankyrin repeat proteins) are repeat proteins that contain one or more internal repeat modules that are identical except for their randomized positions and are flanked by N- and C-terminal capping repeat modules (Binz et al. 2003). As used herein, the term "repeat module" encompasses internal and terminal repeat modules (C-cap and N-cap modules). 27 of the 33 amino acid positions are highly conserved, while the other 6 are less conserved and are mostly involved in the interaction of the ankyrin repeat with its target (Binz et al. 2003). The paratope of a DARPin is formed by a continuous surface formed by these variable positions of the internal repeat and sometimes the capping repeat modules. Thus, the DARPin structure offers an inherent modularity to shorten, extend or fuse the paratope of one DARPin to the paratope of another DARPin.

[0269] As shown in FIG. 1, the first DARPin (1) binding target A and the second DARPin (2) binding target B can be split after a desired number of repeat modules and genetically coupled to complementary fragments of other DARPins showing their respective paratopes or binding surfaces ((4) and (5)). In this way, monodomain "2-in-1 DARPins" (or "SwitchDARPins" (6)) can be generated. Such 2-in-1 DARPins can bind mutually exclusively to two targets A and B, i.e., either target A or target B. In addition, one or several internal repeat modules within a 2-in-1 DARPin can be a mixed (or merged) repeat module (7). A repeat module is called a mixed repeat module if its paratope contains at least one target interaction residue from the corresponding module in the first parent repeat protein and at least one target interaction residue from the corresponding module in the second parent repeat protein. The mutually exclusive binding properties of 2-in-1 DARPins are illustrated in FIG. 2. Due to the proximity or overlap of the two paratopes of 2-in-1 DARPins, the molecule can bind to target A (Figure 2A) or target B (Figure 2B) individually, but cannot bind to both targets simultaneously (Figure 2C), since simultaneous binding of both targets A and B is sterically hindered. This mutually exclusive binding feature consequently allows the design of on- or off-switches for conditional activation or inactivation of drug molecules. This mutually exclusive binding feature also distinguishes 2-in-1 DARPins from other ankyrin repeat protein formats (Mohan, K. et al., 2019, Science, 364(6442); WO 2020 / 190852 A2).

[0270] FIG. 3 shows typical examples of constructs comprising the 2-in-1 DARPin of the invention. These exemplary constructs are also called "Switchdrug DARPins". In the on-switch setting (FIG. 3A), the 2-in-1 DARPin binds mutually exclusively to target A (e.g., a sensor target) or target B (e.g., a drug molecule) and is chemically linked to target B (e.g., a drug molecule). The state of target B (e.g., an active or inactive drug molecule) as part of the Switchdrug DARPin is conditionally regulated by the interaction of the 2-in-1 DARPin with target A, and the binding of target A by the 2-in-1 DARPin results in the release of the bound target B (and thus the active drug molecule). In the exemplary off-switch setting shown in FIG. 3B, the 2-in-1 DARPin is designed to bind to target A (e.g., a sensor target) or target B (e.g., a cell receptor). A downstream activity resulting from the interaction of a 2-in-1 DARPin with target B can be conditionally switched off upon binding of target A to the 2-in-1 DARPin. Such a 2-in-1 DARPin can be further linked to other binding domains or molecules.

[0271] T cell engagers are ideal drug molecules for 2-in-1 DARPin proof-of-concept.

[0272] T cell engagers (TCEs, also called T cell bispecifics) are known to be very potent drugs that recruit T cells to target cells and have shown impressive clinical results. However, they often suffer from severe side effects, e.g., on-target, off-tumor toxicity. 2-in-1 DARPins can be advantageously used to add another layer of safety to the activation of TCEs, to reduce this on-target, off-tumor effect. In the on-switch concept (Figure 4A), the T cell engager comprises a CD3-binding DARPin (target B) and a tumor-associated antigen (TAA)-binding DARPin. A 2-in-1 DARPin is used that can bind and block the paratope of the CD3-binding DARPin, which can also alternatively bind, e.g., to a molecule (represented as target A) that is predominantly present in the tumor microenvironment. The 2-in-1 DARPin is chemically conjugated to the TCE to form the Switchdrug DARPin. Upon binding of target A, the 2-in-1 DARPin releases the CD3-binding domain of the TCE (target B), thus enabling it to bind to CD3, thus resulting in the formation of a trimolecular complex between the T cell, the drug and the target cell.

[0273] In an exemplary off-switch concept (Figure 4B), a 2-in-1 DARPin is used that binds target A (sensor target) and in return binds target B (CD3). The TCE in this case comprises a tumor-associated antigen (TAA)-binding DARPin chemically linked to the 2-in-1 DARPin. For high concentrations of target A, the TCE can be silenced by binding to target A, thereby preventing the binding of TCE to CD3 on T cells.

[0274] Design of 2-in-1 DARPins with mutually exclusive binding specificities for VEGF and TCE α-CD3 binders Based on the on-switch concept shown in Figure 4A, a 2-in-1 DARPin was designed, resulting from the combination of a first VEGF-binding DARPin (SEQ ID NO: 1) with a second DARPin targeting the paratope of the CD3-binding DARPin, also called "α-CD3 DARPin-blocker" (SEQ ID NO: 2). These two parent DARPin sequences are based on previous studies: the VEGF-binder is disclosed in WO2016 / 156596, the α-CD3 DARPin-binder was obtained as disclosed in US Provisional Applications Nos. 63126356 and 63182394.

[0275] 2-in-1 DARPins were designed by combining the N-terminal capping repeat and internal repeat module(s) from a VEGF-binder with the C-terminal internal repeat and capping repeat modules from an α-CD3 DARPin blocker (FIG. 5A-B). A total of 29 constructs were designed by combining different numbers of repeat modules of VEGF-binders and α-CD3 DARPin blockers. The repeat modules were either combined as present in the parent binder (intact) or contained further amino acid modifications compared to the parent binder (mixed or merged repeat modules).

[0276] Eight 2-in-1 DARPins out of the 29 showed binding to the VEGF and α-CD3 DARPin targets and were selected for further characterization as described in the following paragraphs. Of the eight 2-in-1 DARPins shown in Figure 5A, five are N3C domains (i.e. 2-in-1 DARPins 11, 12, 13, 15, 17 corresponding to SEQ ID NOs: 6, 7, 8, 9, 10, respectively) and three are N4C domains (i.e. 2-in-1 DARPins 18, 19, and 20 corresponding to SEQ ID NOs: 11, 12, and 13, respectively) shown in Figure 5B. The residues involved in the binding of a DARPin to its target(s) are known or identifiable to the skilled person and together form a paratope on the DARPin (or define a binding surface). In the 2-in-1 DARPins of the invention, residues are marked as consensus if they occupy the same position in the paratope of the 2-in-1 DARPin compared to the paratopes of the two parent DARPins (i.e. the DAPRins with which the 2-in-1 DARPin is combined). In this case, the consensus residues can be determined by aligning the 2-in-1 DARPin sequences with the parent VEGF-binder and with the α-CD3 DARPin-blocker and comparing the paratope positions viewed from the N-terminus of the VEGF-binder or the C-terminus of the α-CD3 DARPin-blocker (e.g. position N_5 is a consensus residue between the VEGF-binder and the α-CD3 DARPin-blocker for N3C 2-in-1 DARPins 11, 12, 13, 15 and 17 but not for N4C 2-in-1 DARPins 18, 19 and 20, because the N-cap of the α-CD3 DARPin-blocker aligns with internal repeat 1 of N4C 2-in-1 DARPins). In Figures 5A-5B, the paratope residues contained in the 2-in-1 DARPins are further highlighted according to their origin, i.e. in black (from the α-CD3 DARPin blocker) or white (from the VEGF binder), and the paratope positions are also indicated in the repeat numbering line (numbers boxed in black).

[0277] To facilitate the design of 2-in-1 DARPins, especially with regard to the generation of mixed repeat modules, a 2D representation method was developed. Figure 5C shows an exemplary schematic of an N3C DARPin (i.e. a DARPin with an N-cap, three internal repeats and a C-cap). The positions constituting the binding surface of the DARPin are labelled (N-terminal cap, C-terminal cap or internal repeats, numbering per repeat). The orientation of these residues allows them to be organised in a 2D representation, as shown in Figure 5D (the dotted boxes in the 3D view (5C) and in the 2D view (5D) highlight the same stretch of residues). Figure 5D therefore shows, for each of the repeat modules, which positions of the paratope contain framework residues or potential target interaction residues. This position map can be further extended to repeat domains containing additional internal repeat domains, for example the N4C repeat domain contains an additional "repeat 4" module with the same position map as the repeats in N3C. This 2D representation therefore provides an improved visualization of the 2-in-1 DARPins. Figure 5E shows a 2D representation of the VEGF-binding agent, the α-CD3 DARPin blocker and the eight 2-in-1 DARPin designs. Residue coloring is as defined in Figures 5A-5B.

[0278] High-level and soluble expression of an engineered 2-in-1 DARPin Twenty-nine different 2-in-1 DARPin designs were expressed in E. coli cells and purified using the His-tag according to standard protocols. 5 mL of stationary overnight culture (TB, 1% glucose, 50 mg / L ampicillin; 37 °C) was used to inoculate a 150 mL culture (TB, 50 mg / L ampicillin, 37 °C). At an absorbance at 600 nm of 1.0–1.5, the culture was induced with 1 mM IPTG and incubated at 37 °C for 4–5 h with shaking. The culture was centrifuged and the resulting pellet was diluted with 10 mL of TBS. 500The homogenates were resuspended in (50 mM Tris-HCl, 500 mM NaCl, pH 8) and stored at -20°C, then thawed and mixed with 50KU DNase / mL and 1 mg / mL lysozyme and dissolved (sonication or French press). After lysis, the samples were centrifuged and the supernatants were collected and filtered. Imidazole (final concentration 20 mM) was added to the homogenates. Proteins were purified on a benchtop Ni-nitrilotriacetic acid (Ni-NTA) acid column followed by a desalting column (NAP-25) step according to standard protocols and resins known to those skilled in the art. Highly soluble 2-in-1 ankyrin repeat proteins were purified from E. coli cultures (up to 200 mg ankyrin repeat protein per liter of culture) and the purity estimated from 4-12% SDS-PAGE was >95%. These non-formated (or monodomain) 2-in-1 DARPins were biophysically characterized by size-exclusion chromatography, ProteOn surface plasmon resonance (SPR) target affinity assessment, ELISA, target protein competition HTRF experiments, and / or SDS-PAGE.

[0279] Example 2: SPR binding assay of 2-in-1 DARPins An important feature of the 2-in-1 DARPin moiety of the present invention is its affinity for its first and second targets, in this exemplary design the VEGF molecule and the α-CD3 DARPin. Even more importantly, the binding of the 2-in-1 DARPin to the immobilized α-CD3 DARPin target should be impaired in the presence of soluble VEGF, which allows for a switching mechanism.

[0280] Surface plasmon resonance (SPR) assays were used to determine the binding affinity and dissociation rate of the 2-in-1 DARPin to a CD3 binding domain that may typically be included in a TOE molecule such as SEQ ID NO:31.

[0281] All SPR data were generated using a Bio-Rad ProteOn XPR36 instrument with PBS-T (0.005% Tween 20) as the running buffer. New neutravidin sensor chips (NLCs) were initialized with air and tuned according to the Bio-Rad manual.

[0282] Eight 2-in-1 DARPin candidates out of 29 designs initially generated showed binding to VEGF covering a range of affinities, whereas 19 out of 29 constructs showed binding to the CD3 binding domain. Of the 29 constructs, eight showed binding to both VEGF and α-CD3 DARPin (2-in-1 DARPins 11, 12, 13, 15, 17, 18, 19 and 20, corresponding to SEQ ID NOs: 6-13, respectively). SPR data were also generated for the parent VEGF binder of SEQ ID NO: 1 and the α-CD3 DARPin-blocker of SEQ ID NO: 2. Binding was tested against a biotinylated CD3 specific binding domain with SEQ ID NO: 3 and against VEGF165 immobilized on a chip (Figure 6A). Data were generated at 25° C. at five binder concentrations (i.e., multi-trace), 50 nM, 12.5 nM, 3.12 nM, 0.781 nM and 0.195 nM, except for the parent VEGF binder, which was measured only at a single concentration of 50 nM (single trace). SPR traces obtained with both parent binders SEQ ID NO: 1 and 2 are shown in FIGS. 6B-6C. The 2-in-1 DARPins SEQ ID NO: 6-13 showed binding to both VEGF and α-CD3 DARPins with different affinities, as can be observed in FIG. 6D (columns 1 and 2). The results are shown in Table 1 below.

[0283] Further SPR data were generated for 2-in-1 DARPins of SEQ ID NO: 6-13 binding to a biotinylated CD3 specific binding domain with SEQ ID NO: 3 on a chip. Data were generated at a single concentration of 50 nM either in the presence or absence of 130 nM VEGF (pre-incubated with the 2-in-1 DARPin). The right-most column of Figure 6D shows the corresponding SPR traces.

[0284] [Table 1]

[0285] From these eight 2-in-1 DARPins, four were subsequently selected and formatted into a multi-DARPin format (also called Switchdrug DARPins) to constitute switchable T cell engager drugs comprising a TAA-binding domain and a CD3-binding domain as further disclosed below.

[0286] Example 3: Switchdrug DARPin format overview Switchdrug DARPin constructs were cloned according to standard procedures into format 1 ([α-CD3-binder]-[2-in-1DARPin]), format 3 ([2-in-1DARPin]-[Ni2C]-[α-CD3 binder]), format A ([α-TAA binder]-[α-CD3 binder]-[2-in-1DARPin]) and format B ([2-in-1DARPin]-[α-TAA binder]-[α-CD3 binder]), where Ni2C is a non-binding DARPin (SEQ ID NO: 5) and the α-TAA binder is an α-CD123 binding DARPin domain (SEQ ID NO: 4). In all these tested construct formats an N-terminal His 6-tag (SEQ ID NO: 34) was added.

[0287] Switchdrug DARPins were expressed and purified following the same procedure as in Example 1, except that for the Format A and Format B formatted constructs, after IMAC purification, an additional purification step was performed by size exclusion chromatography on an AKTAxpress™ system according to standard protocols and resins known to those skilled in the art. Candidates were biophysically characterized by size exclusion chromatography and SDS-PAGE.

[0288] Example 4: ELISA assay to assess residual binding of Switchdrug DARPin to VEGF The residual binding of the following Switchdrug DARPin constructs to VEGF (biotinylated VEGF) was assessed. Switchdrug DARPin13-A (2-in-1 DARPin13 in format A (SEQ ID NO: 23) with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein). Switchdrug DARPin15-A (2-in-1 DARPin15 in format A (SEQ ID NO: 24) with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein). Switchdrug DARPin17-A (2-in-1 DARPin17 in format A (SEQ ID NO: 25) with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein). Switchdrug DARPin18-A (2-in-1 DARPin18 in format A (SEQ ID NO: 26) with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein). Switchdrug DARPin13-B (2-in-1 DARPin13 in format B (SEQ ID NO: 27) with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein). Switchdrug DARPin15-B (2-in-1 DARPin15 in format B (SEQ ID NO: 28) with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein). Switchdrug DARPin17-B (2-in-1 DARPin17 in format B (SEQ ID NO: 29) with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein). Switchdrug DARPin18-B (2-in-1 DARPin18 in format B (SEQ ID NO: 30) with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein).

[0289] The binding of these constructs to VEGF was compared to the VEGF binding domain of SEQ ID NO:1 (FIG. 7).

[0290] The experimental conditions were as follows: nunc Maxisorp plates were coated with 66 nM neutravidin, washed, blocked with BSA, and incubated with 100 nM biotinylated VEGF. 165 (VEGF) was loaded. A titration of Switchdrug DARPin was then applied, incubated, and washed. Detection of bound his-tagged construct was performed with anti-RGS(his)4 mAb HRP conjugate and detected with TMB substrate.

[0291] Significant differences can be observed for the EC50 of the constructs of format A or B compared to the VEGF binding domain. In general, the binding EC50 of the tested Switchdrug DARPins correlates well with the SPR data shown in FIG. 6D. Thus, these data show that the closed form of the tested Switchdrug DARPins (i.e., without soluble VEGF to compete with the binding of the 2-in-1 DARPin to the α-CD3 DARPin) provides a concentration window that can be exploited for the switch mechanism.

[0292] Example 5: ELISA assay to investigate the switching properties of Switchdrug DARPins The ELISA assay provides an easy and qualitative method to determine binding of Switchdrug DARPin constructs to their targets. Differences in binding of Switchdrug DARPin to scCD3εy in the presence and absence of VEGF were evaluated. Each of the 2-in-1 DARPins 13, 15, 17 or 18 was assembled into Switchdrug DARPin formats 1, 3, A or B for titration experiments. Switchdrug DARPins were mixed with a fixed concentration of VEGF (100 nM) and tested for binding to scCD3εy. Monovalent CD3 binding domain (SEQ ID NO: 3) in the presence and absence of 100 nM VEGF was used as a positive control (Figures 8A-8D). The difference in EC50 of the constructs in the presence and absence of VEGF can be referred to as the "activation window". Comparing Figures 8A and 8B, format 3 appears to be superior in terms of the window of activation for the specific examples of Switchdrug DARPin constructs that can be compared.

[0293] Details of the Switchdrug DARPins tested (FIGS. 8A to 8D) are as follows. Figure 8A Switchdrug DARPin15-1 (2-in-1 DARPin15 in format 1 (SEQ ID NO: 16) with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein). Switchdrug DARPin17-1 (2-in-1 DARPin17 in format 1 (SEQ ID NO: 17) with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein). Switchdrug DARPin18-1 (2-in-1 DARPin18 in format 1 (SEQ ID NO: 18) with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein).

[0294] Format 1 = [α-CD3 binder]-[2-in-1 DARPin] Figure 8B Switchdrug DARPin13-3 (2-in-1 DARPin13 in format 3 (SEQ ID NO: 19) with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein). Switchdrug DARPin15-3 (2-in-1 DARPin15 in format 3 (SEQ ID NO: 20) with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein). Switchdrug DARPin17-3 (2-in-1 DARPin17 in format 3 (SEQ ID NO: 21), with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein). Switchdrug DARPin18-3 (2-in-1 DARPin18 in format 3 (SEQ ID NO: 22) with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein). Format 3 = [2-in-1 DARPin]-[Ni2C]-[α-CD3 binder].

[0295] Figure 8C Switchdrug DARPin17-A (2-in-1 DARPin17 in format A (SEQ ID NO: 25) with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein). Switchdrug DARPin18-A (2-in-1 DARPin18 in format A (SEQ ID NO: 26) with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein). Format A = [α-CD123 binder]-[α-CD3 binder]-[2-in-1 DARPin].

[0296] Figure 8D Switchdrug DARPin17-B (2-in-1 DARPin17 in format B (SEQ ID NO: 29) with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein). Switchdrug DARPin18-B (2-in-1 DARPin18 in format B (SEQ ID NO: 30) with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein). Format B = [2-in-1 DARPin]-[α-CD123 binder]-[α-CD3 binder].

[0297] Switchdrug DARPin 13-3 and Switchdrug DARPin 18-3 show little binding in the absence of VEGF, but strong binding to bscCD3εy in the presence of VEGF. In contrast, the EC50 of the control CD3 binding domain (SEQ ID NO: 3) was not affected by VEGF addition. Formats A and B (containing the α-CD123 binding domain instead of the non-binding Ni2C DARPin domain as in formats 1 and 3) showed similar behavior to that observed for formats 1 and 3 (Figures 8C and 8D). Overall, Switchdrug DARPin 17-B provides the largest activation window (about 178-fold), while at the same time its binding can be fully restored to the CD3 binding domain control upon addition of 100 nM VEGF.

[0298] The experimental conditions were as follows: nunc Maxisorp plates were coated with 66 nM neutravidin, washed, blocked with BSA, and loaded with 20 nM biotinylated scCD3εy. Switchdrug DARPin mixed with VEGF was titrated and preincubated with VEGF for 1 day to establish thermodynamic equilibrium. These samples were loaded onto ELISA plates, incubated, washed, and then bound Switchdrug DARPin was detected as described above.

[0299] Thus, these data support that the presence or absence of 100 nM VEGF can generate an activation window for the binding of the tested Switchdrug DARPins to their CD3 target.

[0300] Next, the effect of VEGF concentration on the binding of Switchdrug DARPins was evaluated. The Switchdrug DARPins 17-1, 17-3 and 18-3 described above were kept at a constant concentration while the concentration of VEGF was titrated. Figure 9A shows that Switchdrug DARPin 17-1, when fixed at a concentration of 10 nM and incubated with titrated amounts of VEGF, can restore binding to scCD3ε in a dose-dependent manner. The results for Switchdrug DARPins 17-3 and 18-3 are similarly presented in Figures 9B and 9C, which show that format 3 of the two constructs containing 2-in-1 DARPin17 is superior compared to format 1, since in the absence of VEGF the constructs show no binding, but binding to CD3 can be regained in a VEGF dose-dependent manner.

[0301] Thus, these data support that the binding of exemplary Switchdrug DARPins to their target CD3 can be dose-dependently regulated by VEGF.

[0302] Example 6: Validation HTRF assay Homogeneous time-resolved fluorescence (HTRF) is a very fast and completely solution-based assay for determining protein interactions. To validate the results from the above ELISA assay, Switchdrug DARPin 13-3, 15-3, 17-3 or 18-3 were tested (details of these constructs are given in Example 5). Switchdrug DARPin (20 nM final concentration) was mixed with 1 nM (final concentration) scCD3εy, 1:200 (final concentration) anti-FLAG-d 2 HTRF antibody-FRET acceptor conjugate (Cisbio) and 1:200 (final concentration) anti-strep-Tb antibody FRET donor conjugate (Cisbio, France). The results of the assay are shown in Figures 10A-10B. Where applicable, VEGF was added at 100 nM (final concentration) (Figure 10C) or titrated from 1 μM in 2.5-fold dilution steps (Figure 10D). The mixture was added to wells of a 384-well plate and incubated for 90 minutes. HTRF was read on a Tecan M1000 with a 340 nm excitation wavelength and a 620±10 nm emission filter for background fluorescence detection and a 665±10 nm emission filter for detecting the fluorescence signal for specific binding. The results shown in Figures 10C-10D confirm that activation windows for Switchdrug DARPin 13-3, 15-3, 17-3 or 18-3 can be observed similar to the results of the ELISA assay in Example 5.

[0303] Thus, this data also supports that binding of Switchdrug DARPin TCEs to their target CD3 can be dose-dependently regulated by VEGF.

[0304] Example 7: Jurkat cell binding assay The following constructs were used in this assay:

[0305] Switchdrug DARPin 17-B (2-in-1 DARPin 17 in format B (SEQ ID NO: 29) with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein).

[0306] Format B control construct ([α-VEGF binder]-[α-CD123 binder]-[α-CD3 binder] of SEQ ID NO: 33 with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein).

[0307] Binding of Switchdrug DARPin 17-B to human CD3 on Jurkat cells was measured by flow cytometry (Flow Cytometer FACS Canto). Titrations of Switchdrug DARPin 17-B or format B control constructs in the presence and absence of VEGF (100 nM or 400 nM) were incubated with 100,000 Jurkat cells per well for 60 min at 4°C. After washing, CD3 binding of SwitchdrugDARPin was detected by anti-penta-His antibody diluted 1:100 (incubated for 30 min at 4°C). Cells were subsequently washed, stained for live / dead (aqua, 1:1000, Thermofisher) and resuspended in Cytofix fixing buffer (BD Biosciences). The median mean fluorescence intensity of Alexa Fluor 488 binding on live cells was measured by flow cytometry and the data were plotted using GraphPad Prism 8 (Figure 11).

[0308] In the absence of VEGF, Switchdrug DARPin 17-B shows minimal binding at higher construct concentrations (>1 μM), but addition of 100 nM VEGF restores binding to levels similar to the format B control construct. Increasing the level of VEGF to 400 nM had no further effect on Jurkat cell binding.

[0309] Example 8: T cell activation / killing assay The specificity and potency of Switchdrug DARPins was assessed in an in vitro short-term T cell activation assay by FACS measuring the CD69 activation marker on CD8+ T cells.

[0310] Details of the Switchdrug DARPins tested are as follows: Figure 12A: Switchdrug DARPin 13-A (2-in-1 DARPin 13 in format A (SEQ ID NO: 23), with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein). Switchdrug DARPin 15-A (2-in-1 DARPin 15 in format A (SEQ ID NO: 24) with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein). Switchdrug DARPin 17-A (2-in-1 DARPin 17 in format A (SEQ ID NO: 25) with His tag (SEQ ID NO: 34) fused to the N-terminus of the protein. Switchdrug DARPin 18-A (2-in-1 DARPin 18 in format A (SEQ ID NO: 26) with His tag (SEQ ID NO: 34) fused to the N-terminus of the protein. Format A = [α-CD123 binder]-[α-C33 binder]-[2-in-1 DARPin].

[0311] Control Control TCE ([α-CD123 binder]-[α-CD3 binder] of SEQ ID NO: 31 with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein), Format A control construct ([α-CD123 binder]-[α-CD3 binder]-[α-VEGF binder] of SEQ ID NO: 32 with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein).

[0312] Figure 12B: Switchdrug DARPin 13-B (2-in-1 DARPin 13 in format B (SEQ ID NO: 27) with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein). Switchdrug DARPin 15-B (2-in-1 DARPin 15 in format B (SEQ ID NO: 28) with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein). Switchdrug DARPin 17-B 2-in-1 DARPin 17 in format B (SEQ ID NO: 29), with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein. Switchdrug DARPin 18-B (2-in-1 DARPin 18 in format B (SEQ ID NO: 30) with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein). Format B = [2-in-1 DARPin]-[α-CD123 binder]-[α-CD3 binder].

[0313] Control Control TCE ([α-CD123 binder]-[α-CD3 binder] of SEQ ID NO: 31 with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein), Format B control construct ([α-VEGF binder]-[α-CD123 binder]-[α-CD3 binder] of SEQ ID NO: 33 with a His tag (SEQ ID NO: 34) fused to the N-terminus of the protein).

[0314] 20'000 target tumor cells (Molm-13 cells, expressing CD123) and 100'000 effector T cells (pan T cells from healthy blood donors) were co-incubated with serial dilutions of Switchdrug DARPin samples in the presence of 600 pM human serum albumin for 48 h at 37°C (effector to target cell ratio 5:1). All Switchdrug DARPin samples and controls were pre-incubated in the presence or absence of VEGF for at least 48 h at +4°C. After 48 h, cells were washed and stained with 1:5'000 Live / Dead FITC (Thermo Fisher), 1:250 mouse anti-human CD8 Pacific Blue (BD), and 1:250 mouse anti-hCD 69-APC from BioLegend (BioLegend, US) antibodies for 30 min at 4°C. After washing and fixation, cells were analyzed on a Flow Cytometer Attune Nxt. T cell activation was assessed by measuring CD69 positive cells on live / dead negative and CD8 positive gated T cells. FACS data was analyzed using FlowJo software and data were plotted using GraphPad Prism8. Assays were performed in duplicate with pan T cells from two individual human donors. As shown in Figures 12 A-12B, Switchdrug DARPins 13-A, 13-B, 15-A, 15-B, 17-A, 17-B, 18-A and 18-B were tested and compared to control TCE and respective format A or B control constructs. For both formats, Switchdrug DARPins including 2-in-1 DARPin 17 showed the best activation window (i.e. difference between Switchdrug 50 in the absence and presence of 100 nM VEGF), with a 75-fold maximum window observed for EDPin 17-B (Figure 12B). The format B control construct did not reach the potency of the control TCE even in the absence of VEGF. Thus, the addition of a second binding moiety to the N-terminus of the control TCE reduced potency.Therefore, Switchdrug DARPins could maximally be expected to reach the potency of the Format B control construct in the presence of VEGF, which was the case for Switchdrug DARPins 15-B or 17-B. For Switchdrug DARPins 13-B or 18-B, the addition of 100 nM VEGF was not sufficient to fully activate the construct.

[0315] Example 9: Preparation and characterization of additional 2-in-1 DARPins Further 2-in-1 DARPins according to the invention were produced and characterized. Unless otherwise stated, the experiments described in this section were carried out essentially according to the procedures described in Examples 1-6.

[0316] 9.1 Overview Starting from four different target-specific parent DARPins, 2-in-1 DARPins were designed and generated. The results are shown by five representative functional 2-in-1 DARPins. Based on the parent DARPins, these 2-in-1 DARPins can be classified into two different families, as detailed in Tables 2 and 3.

[0317] [Table 2]

[0318] [Table 3]

[0319] A generally applicable process is described herein below in section 9.1, which can optionally be used to facilitate the design and characterization of engineered repeat domains of the invention, such as 2-in-1 DARPins.

[0320] To facilitate the design of the designed repeat domains of the invention, one or both parent repeat domains can be subjected to profiling to determine whether the parent repeat domain is suitable to be linked at the N-terminus and / or C-terminus with another parent repeat domain (or a fragment thereof) to form a designed repeat domain with dual binding specificity, in which the binding to two targets is mutually exclusive. Such profiling provides an optimal starting point for the subsequent combination of the parent repeat domains (or fragments thereof). This profiling comprises a series of systematic assays in which variants of the parent repeat domain of interest are generated and evaluated. Each of the generated variants is tested for its ability to bind to the target to determine whether the N-terminal capping module, the C-terminal capping module, and / or the internal repeat module(s) adjacent to the N-terminal or C-terminal capping module are important for target binding. This repeat binding evaluation can be carried out, for example, by ELISA. An exemplary threshold for classifying mutants as mutants that have lost target binding or mutants that have maintained target binding is an EC50 of approximately 300 nM, as described further below (i.e. a higher EC50 value means that the mutant has lost binding, a lower EC50 value means that the mutant has maintained binding). Such profiling of the parent repeat domain may include one or more steps, which are described further below (steps 1-4) and are also shown in FIG. 13 for the parent DARPin. FIG. 13 shows only the steps for the evaluation of the N-terminal side of the parent DARPin. The steps for the evaluation of the C-terminal side of the parent DARPin are similar and follow the same logic. The steps described below and shown in FIG. 13 are exemplary and are not intended to be limiting.

[0321] Step 1. The terminal capping module of the parent repeat domain (i.e. either the N-terminal or C-terminal capping module) is replaced with the corresponding non-templated terminal capping module (such as SEQ ID NO: 36 or 38) and the resulting mutants are tested for whether target binding is maintained or lost. This allows to determine whether the terminal capping module of the parent repeat domain is involved in target binding. If target binding is lost, the terminal capping module is involved in target binding and can perform step 2 (i.e., a version of step 2 when binding is lost in step 1). If target binding is maintained, the terminal capping module does not participate in target binding and step 2 (i.e., the version of step 2 where binding was maintained in step 1) can be performed.

[0322] This step 1 (and subsequently steps 2, 3 and / or 4 below) can be carried out separately for each end of the parent DARPin of interest.

[0323] Step 2 (the result of step 1 is loss of target binding): The terminal capping module of the parent repeat domain replaced in step 1 is replaced with a group of three connected repeat modules, which include (from the inside towards the capping module) (1) a non-templated internal repeat module (such as SEQ ID NO: 89), (2) a non-templated internal repeat module into which the paratope residues of the replaced capping module are introduced, and (3) a non-templated terminal capping module, and the resulting mutants are tested for whether target binding is maintained or lost. Alternatively, the replacement can be performed with a group of two connected repeat modules, which include (from the inside towards the capping module) (1) a non-templated internal repeat module into which the paratope residues of the replaced capping module are introduced, and (2) a non-templated terminal capping module. This makes it possible to determine whether target binding can be maintained if the paratope residues of the terminal capping module of the parent repeat domain are present in the designed internal repeat module that replaces the capping module in the designed repeat domain. If target binding is lost, the considered terminal capping module and its paratope residues are essential for target binding and the parent repeat domain cannot be linked to another repeat domain at this terminal side; If target binding is maintained, the considered terminal side of the parent repeat domain is compatible to be linked with another (compatible) parent repeat domain. To do so, the considered terminal capping module can be removed and the paratope residues of the terminal capping module must be present in an adjacent internal repeat module that replaces the capping module in the designed repeat domain (this internal repeat module comes from a second parent repeat domain or from a newly designed internal repeat module).

[0324] Step 2 (result of step 1 is maintained target binding): The terminal capping module of the parent repeat domain replaced in step 1 is now replaced with a group of two connected repeat modules comprising (from the inside towards the capping module) (1) a non-templated internal repeat module and (2) a non-templated terminal capping module, and the resulting mutants are tested for whether target binding is maintained or lost. This makes it possible to determine whether target binding can be maintained when the terminal capping module of the parent repeat domain is removed and the adjacent internal repeat module is combined with an internal repeat module of another repeat domain. If target binding is lost, the parent repeat domain cannot be linked to another repeat domain at the considered end (extension by additional internal repeat module(s) impairs binding); If target binding is maintained, the parent repeat domain is compatible to be linked to another (compatible) parent repeat domain at the considered end, and steps 3 and 4 can optionally be performed to further evaluate whether the outermost internal repeat module of the parent repeat domain (i.e. the internal repeat module located adjacent to the considered end capping module) is involved in target binding.

[0325] Step 3. Both the terminal capping module of the parent repeat domain replaced in step 1 and the adjacent internal repeat module are now replaced with a template-free terminal capping module into which the paratope residues of the replaced internal repeat module are introduced, and the resulting mutants are tested for whether target binding is maintained or lost. This makes it possible to determine whether the adjacent internal repeat module must be retained to maintain target binding, or whether its paratope residues can be removed if they are present in the terminal capping module that occupies its place. If target binding is lost, the considered flanking internal repeat modules are involved in target binding and should be maintained as part of the designed repeat domain of the invention; If target binding is maintained, the considered adjacent internal repeat module can be converted into a terminal capping module in the designed repeat domain of the invention (by having the paratope residues of the internal repeat module in the terminal capping module).

[0326] Step 4. Both the terminal capping module and the adjacent internal repeat module of the parent repeat domain replaced in step 1 are now replaced with a non-templated terminal capping module and the resulting mutants are tested for whether target binding is maintained or lost. This makes it possible to determine whether target binding can be maintained when the terminal capping module and the adjacent internal repeat module of the parent repeat domain are removed and replaced with another repeat module. If target binding is lost, the considered flanking internal repeat modules are involved in target binding and should be maintained as part of the designed repeat domain of the invention. If target binding is maintained, the considered neighboring internal repeat modules are not involved in target binding and can be omitted in the designed repeat domain of the present invention.

[0327] It is not strictly necessary to respect the order of the above steps, since one or more or all steps may be carried out in parallel, by generating and evaluating several or all variants in parallel. Moreover, each of the above steps may be carried out individually or in combination with one or more other steps to evaluate the properties of the parent repeat domains.

[0328] The results of the evaluation steps performed can be used to inform the design of engineered repeat domains of the present invention.

[0329] For the purposes of identifying and characterizing designed repeat domains of the invention, the repeat domains generated for the purposes of generating designed repeat domains of the invention are assessed for their target binding properties using techniques known in the art and / or as described herein. Properties of such generated repeat domains assessed for this purpose include binding affinity to both targets and mutually exclusive characteristics of binding to the first and second targets.

[0330] The 2D paratope representations of the 2-in-1 DARPins described in this Example 9 and their respective parent DARPins are shown in FIG.

[0331] 9.2 Family 1 DARPin parent number 1 (binding to MBP) and parent number 2 (binding to APH) have been described previously and correspond respectively to "off7" in Binz et al. 2004 (loc.Cit) and "AR 3b" in Amstutz, P, et al., 2005, Journal of Biological Chemistry 280.26, 24715-24722: These DARPins were prepared according to standard procedures known to those skilled in the art.

[0332] The profiling methodology described in Section 9.1 was carried out. Relevant information for parents 1 and 2 is summarized in Table 4.

[0333] [Table 4]

[0334] where X means that the repeat module cannot be modified or extended; Y means that the repeat module type can be changed (e.g. converted from an internal repeat module to a terminal capping module); Z means that the repeat module can be removed; N is an N-terminal capping module; C is a C-terminal capping module; and IR1, IR2 and IR3 are the first, second and third internal repeat modules from the N-terminus, respectively.

[0335] Based on this profiling, eleven 2-in-1 DARPin designs were generated containing parental DARPin number 1 at the C-terminus and parental DARPin number 2 at the N-terminus (second main step mentioned in section 9.1). 2-in-1 DARPins containing between 5 (Table 5) and 10 (Table 6) internal repeat modules were generated.

[0336] [Table 5]

[0337] [Table 6]

[0338] In Tables 5 and 6, bold and underlined typeface is used to indicate the origin of the repeat module; IR3 / IR1 represents the mixed repeat module obtained from IR3 of parent 2 and IR1 of parent 1; Rt represents the template internal repeat module (unlinked). An example of such a template internal repeat module is SEQ ID NO:89.

[0339] Characterization of these 2-in-1 designed repeat domains is shown for two representative constructs, 2-in-1 DARPin 21 (SEQ ID NO: 90) and 2-in-1 DARPin 22 (SEQ ID NO: 91), each with dual binding specificity for APH and MBP. Expression of the 2-in-1 DARPins was performed as described in Example 1.

[0340] SEC analysis Samples (2 mg / mL) were analyzed on a GE Superdex 2005 / 150 Increase GL column on an Agilent 1200 HPLC system at a flow rate of 0.5 mL / min in PBS. The results are shown in Figure 14A.

[0341] Binding affinity (SPR) The binding affinity of 2-in-1 DARPin 21 and 2-in-1 DARPin 22 to APH or MBP was determined independently by SPR analysis as described in Example 2. The target molecules (APH or MBP) were coated on a HC200M chip (coating density: APH 550 Rd, MBP 800 Rd). Samples were diluted in 1 / 3 steps with PBS-T and run at 300 nM, 100 nM, 33.3 nM, 11.1 nM, 3.7 nM and 0 nM. The SPR curves are shown in Figure 14B and the KD values ​​are given in Table 7. The binding affinity of 2-in-1 DARPin21 and 2-in-1 DARPin22 to their respective targets is maintained compared to the respective affinity of the parent DARPins.

[0342] [Table 7]

[0343] The aminoglycoside phosphotransferase (APH) used in this assay corresponds to SEQ ID NO:95 and the maltose binding protein (MBP) used in this assay corresponds to SEQ ID NO:96.

[0344] competitive binding (SPR) To assess the mutual binding exclusivity of 2-in-1 DARPin 21 and 2-in-1 DARPin 22 towards APH and MBP, two competitive SPR assays were performed (Comp-SPR1 and Comp-SPR2).

[0345] For Comp-SPR1, APH was coated on the chip and 80 nM of the 2-in-1 DARPin was pre-incubated with various concentrations of soluble MBP (2500, 833, 277, 92, 31 or 0 nM) for 40 min. MBP acts as a competitor for the binding of APH. Samples were then injected onto the chip and binding to APH was measured (Figure 14C, parts 1) and 3)). The response at the end of the injection (maximum SPR signal) was measured and plotted against the concentration of MBP (Figure 14C parts 2) and 4)). Figure 14C parts 1) and 2) show the results for 2-in-1 DARPin 21 and Figure 14C parts 3) and 4) show the results for 2-in-1 DARPin 22.

[0346] Comp-SPR2 corresponds to the inverse setup of Comp-SPR1, i.e. in Comp-SPR2 MBP was coated on the chip and 80 nM of 2-in-1 DARPins were pre-incubated for 40 min with different concentrations of soluble APH (2500, 833, 277, 92, 31 or 0 nM). APH acts as a competitor for the binding of MBP. Samples were then injected onto the chip and binding to MBP was measured (Figure 14D, parts 1) and 3)). The response at the end of the injection (maximum SPR signal) was measured and plotted against the concentration of APH (Figure 14D parts 2) and 4)). Figure 14D parts 1) and 2) show the results for 2-in-1 DARPin 21 and Figure 14D parts 3) and 4) show the results for 2-in-1 DARPin 22. The IC50 values ​​obtained from the two competitive SPR assays are shown in Table 8.

[0347] [Table 8]

[0348] In each case, increasing concentrations of competitors decrease binding to the coated targets, as shown by the drop in SPR signal at the end of the injection (maximum SPR signal). Thus, binding of 2-in-1 DARPins 21 and 22 to the first and second targets (MBP and APH) is mutually exclusive.

[0349] Competitive binding (ELISA) Alternatively, the mutual binding exclusivity of the 2-in-1 DARPins of the invention can be measured by an ELISA-based competitive binding assay. Nunc Maxisorp plates were first coated with APH. 2-in-1 DARPin 21 and 2-in-1 DARPin 22 at a final concentration of 100 nM were exposed to 1 / 3 serial dilutions of MBP (40000, 13333, 4444, 1481, 494, 165, 54.9, 18.3, 6.1, 2.0, or 0 nM) for 1 hour. A standard readout was then performed. The results are shown in Figure 14E. A dose response can be observed.

[0350] Binding affinity (ELISA) Binding to the targets MBP or APH was tested separately for 2-in-1 DARPin 21 and 2-in-1 DARPin 22 in a standard ELISA assay. Parental DAPRins no. 1 and 2 were used as positive controls. The assay was performed as described in Example 4. The results are shown in FIG. 14F, where plot 1) represents the binding of the samples to MBP and plot 2) represents the binding of the samples to APH.

[0351] 9.3 Family 2 DARPin parent number 3 (binding to scCD3εy) as well as the scCD3εy target protein itself are described in WO2022129428. These DARPins were performed according to standard procedures known to those skilled in the art.

[0352] The profiling methods described in section 9.1 were performed for both parent DARPins no. 3 and no. 4. Relevant information is summarized in Tables 9 and 10.

[0353] [Table 9]

[0354] [Table 10]

[0355] In Tables 9 and 10, X means that the repeat module cannot be modified or extended; Y means that the repeat module type can be changed (e.g. converted from an internal repeat module to a terminal capping module); N is an N-terminal capping module; C is a C-terminal capping module; and IR1, IR2 and IR3 are the first, second and third internal repeat modules from the N-terminus, respectively.

[0356] Based on this profiling, ten 2-in-1 designed repeat domains were generated (second main step mentioned in section 9.1) containing parent DARPin number 3 (or part thereof) at the C-terminus and parent DARPin number 4 (or part thereof) at the N-terminus. 2-in-1 DARPins containing between 6 (Table 11) and 10 (Table 12) internal repeat modules were generated.

[0357] [Table 11] Bold and underlined typeface is used to indicate the origin of the repeat modules, where C / N represents a mixed repeat module derived from C of parent 4 and N of parent 3.

[0358] [Table 12] Here, Rt represents the template internal repeat module (unlinked). An example of such a template internal repeat module is SEQ ID NO: 89.

[0359] Characterization of these 2-in-1 designed repeat domains is shown for three representative constructs: 2-in-1 DARPin 23 (SEQ ID NO: 92), 2-in-1 DARPin 24 (SEQ ID NO: 93) and 2-in-1 DARPin 25 (SEQ ID NO: 94), which have dual binding specificity for scCD3εy and Target A. Expression of the 2-in-1 DARPins was performed as described in Example 1.

[0360] SEC analysis was performed as in section 9.2, and the results are shown in Figure 15A.

[0361] Binding affinity (SPR) The binding affinity of 2-in-1 DARPin 23, 2-in-1 DARPin 24 and 2-in-1 DARPin 25 for scCD3εy or TagretA was determined independently by SPR analysis as described in Example 2. Biotinylated target molecules (bio-scCD3εy or bio-TargetA) were coated on a NAD chip (coating density: 460 Rd for bio-scCD3εy and 560 Rd for bio-TargetA). Samples were diluted in 1 / 3 steps in PBS-T and run at 333.33 nM, 111.11 nM, 37.00 nM, 12.34 nM, 4.11 nM and 0 nM. The SPR curves are shown in Figure 15B and the KD values ​​are given in Table 13. The binding affinity of 2-in-1 DARPin 23, 2-in-1 DARPin 24 and 2-in-1 DARPin 25 for each of the targets is maintained compared to the respective affinity of the parent DARPins.

[0362] [Table 13]

[0363] competitive binding (SPR) The mutual binding exclusivity of the 2-in-1 DARPin 23 for scCD3εy and TargetA was assessed in competitive SPR assays (similar to Comp-SPR1 and Comp-SPR2 in section 9.2).

[0364] Biotinylated scCD3εy was coated on a NAD chip and 50 nM of 2-in-1 DARPin was pre-incubated with various concentrations of soluble TargetA (150, 50, 16.7, 5.5, 1.85 or 0 nM) for 4.5 h. TargetA acts as a competitor for the binding of scCD3εy. Samples were then injected onto the chip and binding to scCD3εy was measured (Figure 15C part 1)). The response at the end of the injection (maximum SPR signal) was measured and plotted against the concentration of TargetA (Figure 15C part 2)). The IC50 values ​​obtained from the above competitive SPR assay are shown in Table 14.

[0365] [Table 14]

[0366] Increasing the concentration of competitor reduces binding to the coated target as shown by the drop in the SPR signal at the end of the injection (maximum SPR signal). Thus, binding of 2-in-1 DARPin 23 to the first and second targets (scCD3εγ and Target A) is mutually exclusive.

[0367] Competitive binding (HTRF) Alternatively, the mutual binding exclusivity of the 2-in-1 DARPins of the invention can be measured by a competitive binding assay based on HTRF. In this assay, the binding of the 2-in-1 DARPins to bio-scCD3εy in the presence of different concentrations of non-biotinylated Target A competitor is evaluated. The HTRF assay was essentially performed as described in Example 6. The following parameters were used: 1 nM bio-scCD3εy, 3 nM of the 2-in-1 DARPin, titration of the Target A competitor at 300, 100, 33.333, 11.111, 3.704, 1.235, 0.412, 0.137, 0.046, 0.015 and 0 nM. The curve obtained from this HTRF assay is shown in Figure 15D. Based on this data it can be concluded that in the presence of increasing concentrations of a competitor (here Target A), binding to the second target (here scCD3εy) is hindered and therefore binding of the 2-in-1 DARPin to the first or second target is mutually exclusive.

[0368] Binding affinity (ELISA) Binding to the targets scCD3εy or TargetA was tested separately for 2-in-1 DARPin 23, 2-in-1 DARPin 24 and 2-in-1 DARPin 25 in a standard ELISA assay. Parental DAPRins no. 3 and 4 were used as positive controls. The assay was performed as described in Example 4. The results are shown in FIG. 15E, where plot 1) represents the binding of the samples to TargetA and plot 2) represents the binding of the samples to scCD3εy.

[0369] In conclusion, the examples demonstrate that designed repeat domains with a first binding specificity to a first target and a second binding specificity to a second target, where the binding of the designed repeat domain to the first and second targets is mutually exclusive, can be generated using many different repeat domains with different target specificities as parent repeat domains. Thus, the examples provide a generally applicable method of generating such designed repeat domains and establish a general concept of designed repeat domains with such novel properties.

[0370] This specification is most fully understood in light of the teachings of the references cited within the specification. The embodiments within this specification provide examples of embodiments of the invention and should not be construed as limiting the scope of the invention. Those skilled 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 the material incorporated by reference contradicts or is inconsistent with this specification, this specification takes precedence over any such material. The citation of a reference herein is not an admission that such reference is prior art to the present invention.

[0371] 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 which equivalents are intended to be encompassed by the following claims.

Claims

1. 1. A method for producing a designed repeat domain, the method comprising: (i) providing a first repeat domain and a second repeat domain, wherein the first repeat domain has binding specificity for a first target and the second repeat domain has binding specificity for a second target; (ii) linking a part of the first repeat domain with a part of the second repeat domain by covalently combining at least one repeat module of the first repeat domain with at least one repeat module of the second repeat domain to form a designed repeat domain with dual binding specificity; Including, wherein said designed repeat domain has binding specificity for said first target and binding specificity for said second target, and binding of said designed repeat domain to said first and second targets is mutually exclusive.

2. 2. The method of claim 1, wherein said step of covalently combining at least one repeat module of the first repeat domain with at least one repeat module of the second repeat domain comprises a step of covalently connecting said at least one repeat module of the first repeat domain with said at least one repeat module of the second repeat domain.

3. 3. The method of claim 2, wherein said at least one repeat module of the first repeat domain and said at least one repeat module of the second repeat domain are covalently connected by a linker, said linker preferably being a peptide linker or a non-linked repeat module.

4. 2. The method of claim 1, wherein said step of covalently combining at least one repeat module of the first repeat domain with at least one repeat module of the second repeat domain comprises covalently combining said at least one repeat module of the first repeat domain with said at least one repeat module of the second repeat domain.

5. The method according to any one of claims 1 to 4, wherein the designed repeat domain comprises an N-terminal and / or a C-terminal capping module and at least one internal repeat module.

6. The designed repeat domain is －５ Dissociation constant (K Ｄ ) and / or the designed repeat domain binds to the first target at －５ Dissociation constant (K Ｄ 6. The method of claim 1, wherein the second target is bound by a second nucleotide.

7. 7. The method according to any one of claims 1 to 6, wherein the first repeat domain is an ankyrin repeat domain, the second repeat domain is an ankyrin repeat domain and / or the designed repeat domain is an ankyrin repeat domain.

8. A designed repeat domain obtainable by the method according to any one of claims 1 to 7.

9. A designed repeat domain having a first binding specificity for a first target and a second binding specificity for a second target, wherein binding of said designed repeat domain to said first and second targets is mutually exclusive.

10. The designed repeat domain is －５ Dissociation constant (K Ｄ ) and / or the designed repeat domain binds to the first target at －５ Dissociation constant (K Ｄ 10. The designed repeat domain of claim 9 , which binds to the second target at

11. 11. The designed repeat domain according to any one of claims 9 to 10, wherein said designed repeat domain comprises an N-terminal and / or a C-terminal capping module and at least one internal repeat module.

12. The designed repeat domain according to any one of claims 9 to 11, wherein said designed repeat domain is an ankyrin repeat domain.

13. The designed repeat domain of any one of claims 9 to 12, wherein said designed repeat domain comprises at least one internal repeat module comprising a sequence selected from SEQ ID NOs: 75 to 81.

14. 14. The designed repeat domain of any one of claims 9 to 13, wherein the designed repeat domain comprises: (i) an N-terminal capping module comprising a sequence selected from the group consisting of (1) SEQ ID NOs: 35, 36, 47-63, 83-85, and 97-100, (2) any of SEQ ID NOs: 35, 36, 47, 53, 59, 60, 83, 84, in which up to 9 amino acids have been replaced by other amino acids; and / or (ii) a C-terminal capping module comprising a sequence selected from the group consisting of (1) SEQ ID NOs: 37, 38, and 64-74, and (2) any of SEQ ID NOs: 37, 38, 69, in which up to 9 amino acids have been replaced by other amino acids; and / or (iii) at least one internal repeat module comprising a sequence selected from the group consisting of (1) SEQ ID NOs: 40-46, and (2) any of SEQ ID NOs: 40-46, in which up to 9 amino acids have been replaced by other amino acids.

15. A recombinant protein comprising a designed repeat domain according to any one of claims 8 to 14.

16. The protein of claim 15 , wherein the protein further comprises a therapeutic agent, the therapeutic agent being preferably a binding molecule.

17. An isolated nucleic acid encoding a designed repeat domain according to any one of claims 8 to 14, or a protein according to any one of claims 15 to 16.

18. A pharmaceutical composition comprising: (i) a designed repeat domain according to any one of claims 8 to 14; (ii) a recombinant protein according to any one of claims 15 to 16; and / or (iii) 20. A pharmaceutical composition comprising the nucleic acid of claim 17 and, optionally, one or more pharma- ceutically acceptable carriers or diluents.

19. 19. A method for treating a medical condition, comprising administering to a patient in need of treatment a therapeutically effective amount of a designed repeat domain of any one of claims 8-14, a recombinant protein of any one of claims 15-16, a nucleic acid of claim 17, or a pharmaceutical composition of claim 18.

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