Recombinant CD3 binding proteins and their uses

Abstract

The present invention relates to recombinant binding proteins comprising a designed ankyrin repeat domain with binding specificity for CD3. In addition, the present invention relates to nucleic acids encoding such binding proteins, pharmaceutical compositions comprising such binding proteins or nucleic acids, and the use of such binding proteins, nucleic acids or pharmaceutical compositions in methods for the local activation of T cells in diseases, as well as methods for the treatment of diseases such as infections or cancer in mammals, including humans.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Patent Application No. 63 / 126,356 filed on 16 December 2020, European Patent No. 20216705 filed on 22 December 2020, and U.S. Provisional Patent Application No. 63 / 182,394 filed on 30 April 2021. The disclosures of these patent applications are incorporated herein by reference in their entirety for all purposes.

[0002] The present invention relates to a recombinant binding protein comprising a designed ankyrin repeat domain having binding specificity to CD3. In addition, the present invention relates to nucleic acids encoding such binding proteins, pharmaceutical compositions comprising such binding proteins or nucleic acids, and the use of such binding proteins, nucleic acids or pharmaceutical compositions in methods for disease localization activation of T cells, and methods for treating diseases such as infectious diseases or cancer in mammals, including humans. [Background technology]

[0003] T cells play a crucial role in adaptive immune responses, specifically recognizing heterologous proteins in infected or cancer cells. This recognition occurs via T cell receptors (TCRs) that bind to heterologous protein antigens presented by the major histocompatibility complex (MHC). TCR ligands typically bind relatively weakly, but even a small number of ligands are sufficient to fully activate T cells (Birnbaum et al, Proc Natl Acad Sci USA;111(49):17576-81(2014); Davis et al, Annu Rev Immunol;16:523-44(1998)). T cell receptors do not signal themselves, but non-covalently bind to multi-subunit signaling molecules, typically containing CD3εγ and CD3εδ heterodimers and CD3ζζ homodimers, which collectively form the TCR / CD3 complex. Due to its crucial role in the immune adaptive response, the multiprotein TCR / CD3 complex has attracted significant attention for the development of novel therapeutic tools in cancer immunotherapy. Among these tools are T-cell engagers.

[0004] T cell engagers (TCEs) are proteins that induce T cell activation by simultaneously binding to target antigens on tumor cells and the TCR / CD3 complex on T cells, for example, by bypassing normal TCR-MHC interactions (Ellerman, Methods; 154:102-117 (2019)). An example of a TCE is the BiTE® molecule (Amgen).

[0005] Currently, T cell engagers typically take the form of bispecific antibodies or antibody fragments directed against the constant component of the TCR / CD3 complex (e.g., CD3) and tumor-associated antigens (TAAs). However, these T-cell binding therapeutic tools present several drawbacks, such as high manufacturing costs and the inability to target multiple disease-related antigens. Furthermore, they carry the risk of causing certain serious side effects, including cytokine release syndrome (CRS) (Shimabukuro-Vornhagen et al, J Immunother Cancer; 6(1):56(2018); Labrjin et al, Nat Rev Drug Discov; 18(8):585-608(2019)) and on-target / off-tumor toxicity (Weiner et al, Cancer Res.; 55(20):4586-4593(1995); Weiner et al, Cancer Immunol.Immunother.; 42(3); 141-150(1996)). For example, EGFR-targeted TCEs have been tested in non-human primates, providing evidence of safety regarding binding to normal tissues expressing low levels of the antigen (Lutterbuese et al, Proc Natl Acad Sci). USA;107(28):12605-10(2010)). Furthermore, it has been shown that high-affinity CD3-binding agents of HER-targeted TCEs preferentially distribute to secondary lymphoid tissues and reduce systemic exposure (Mandikian et al, Mol. Cancer Ther.;17(4):776 LP-785(2018)). Antibody-based T cell engagers often show more than 1000-fold higher affinity for CD3 compared to innate TTCR-MHC interactions ((Wu et al, Pharmacol Ther;182:161-75(2018); International Publication No. 2014 / 167022; Junntila et al, Cancer Res.;19:5561-71(2014); Yang et al, J Immunol Aug.;15(137):1097-100(1986)).This high affinity correlates with lower efficiency in terms of T cell activation and tumor cell death (Bortoletto et al, Eur.J.Immunol.32;11:3102-3107(2002);Ellerman,Methods;154:102-117(2019);Mandikian et al,Mol.Cancer Ther.;17(4):776 LP-785(2018);Vafa et al,Frontiers in Oncology;10:446(2020)).

[0006] Therefore, there is still a need for novel CD3-specific binding proteins with beneficial properties suitable for use in T-cell engager formats, as well as therapeutic approaches for treating diseases, including cancer and infectious diseases, that benefit from CD3-specific binding. [Overview of the project]

[0007] The present invention provides recombinant binding proteins comprising a designed ankyrin repeat domain having binding specificity to CD3. Further provided are one or more binding agents having binding specificity to disease-related antigens (DAAs), such as infection-related antigens (IAAs), preferably virus-related antigens (VAAs), or tumor-related antigens (TAAs), preferably an ankyrin repeat domain (these promote DAA-dependent activation of T cells by the binding protein) and such binding proteins linked to them. In addition, the present invention provides nucleic acids encoding such binding proteins and pharmaceutical compositions comprising such binding proteins or nucleic acids. The present invention also provides the use of such binding proteins, nucleic acids, or pharmaceutical compositions in methods for localization activation of T cells, such as tumor localization or infection localization activation, and in methods for treating diseases such as infectious diseases, preferably viral infections or cancer, in mammals including humans.

[0008] The applicant has conducted dedicated research to design, generate, and select CD3-specific binding domains with beneficial properties suitable for use in T cell engager pharmaceuticals (see below). Combining a complex screening process with several rounds of affinity maturation with rational design, four CD3-specific ankyrin repeat proteins were engineered: DARPin® protein #1 (SEQ ID NO: 1), DARPin® protein #2 (SEQ ID NO: 2), DARPin® protein #3 (SEQ ID NO: 3), and DARPin® protein #4 (SEQ ID NO: 4). All of these recombinant binding proteins contain engineered ankyrin repeat domains with binding specificity to CD3, exhibiting different binding affinities to CD3 and / or T cells. The binding proteins have binding affinities to CD3 and / or T cells that are high enough to achieve targeted T cell activation, while simultaneously having relatively low affinities to reduce the risk of adverse effects (e.g., those caused by excessive T cell activation). These recombinant binding proteins have been found to be suitable for ligation to one or more DAA-specific binders to form functional TCE molecules containing multispecific T cell engagers (TCEs). Therefore, the binding proteins of the present invention may further comprise binders having binding specificity to disease-associated antigens. Such binding proteins in TCE format can bind to the immune system, more specifically to T cells, in a localized and targeted manner depending on the presence of each DAA on disease-associated cells such as tumor cells or infected cells. The binding proteins of the present invention, comprising an ankyrin repeat domain with binding specificity to CD3 (SEQ ID NOs: 1, 2, 3, or 4 or their variants), therefore represent a "toolbox" of CD3-specific binding proteins used, in particular, in the generation of TCE drug candidates and in methods for treating diseases in humans involving efficient T cell activation while avoiding or reducing T cell hyperstimulation and / or adverse effects.

[0009] In one embodiment, the present invention provides a recombinant binding protein comprising an ankyrin repeat domain having binding specificity to CD3, wherein the ankyrin repeat domain comprises an ankyrin repeat module having an amino acid sequence selected from the group consisting of sequences in which up to nine amino acids in any of (1) SEQ ID NOs. 5-9 and (2) SEQ ID NOs. 5-9 are substituted with other amino acids. In one embodiment, the ankyrin repeat module is a first ankyrin repeat module, and the ankyrin repeat domain further comprises a second ankyrin repeat module having an amino acid sequence selected from the group consisting of sequences in which up to nine amino acids in any of (1) SEQ ID NOs. 5-9 and (2) SEQ ID NOs. 5-9 are substituted with other amino acids. In a particular embodiment, the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of sequences in which up to nine amino acids in (1) SEQ ID NOs. 5 and (2) SEQ ID NOs. 5 are substituted with other amino acids, and the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of sequences in which up to nine amino acids in (1) Sequence 6 and (2) SEQ ID NOs. 6 are substituted with other amino acids. In another specific embodiment, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to nine amino acids in (1) SEQ ID NO: 7 and (2) SEQ ID NO: 7 are substituted with other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to nine amino acids in (1) SEQ ID NO: 6 and (2) SEQ ID NO: 6 are substituted with other amino acids. In yet another specific embodiment, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to nine amino acids in (1) SEQ ID NO: 7 and (2) SEQ ID NO: 7 are substituted with other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to nine amino acids in (1) SEQ ID NO: 8 and (2) SEQ ID NO: 8 are substituted with other amino acids.In further specific embodiments, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) Sequence ID No. 7 and (2) Sequence ID No. 7 in which up to nine amino acids are substituted by other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) Sequence ID No. 9 and (2) Sequence ID No. 9 in which up to nine amino acids are substituted by other amino acids. In a preferred embodiment, the first ankyrin repeat module is located at the N-terminus of the second ankyrin repeat module within the ankyrin repeat domain.

[0010] In one embodiment, the present invention provides a recombinant binding protein comprising an ankyrin repeat domain having binding specificity to CD3, wherein the ankyrin repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity with any one of SEQ ID NOs: 1 to 4, wherein the second-to-last A in SEQ ID NOs: 1 to 4 is optionally substituted with L, and / or the last A in SEQ ID NOs: 1 to 4 is optionally substituted with N.

[0011] In one embodiment, any of the binding proteins is 10 -7 Dissociation constant less than M (K D ) binds to human CD3 in PBS. In further embodiments, any of the binding proteins have an EC2 level in the range of 2 to 900 nM, preferably about 5 to about 400 nM. 50 It binds to human CD3 on T cells. In a further embodiment, the ankyrin repeat domain contained in the binding protein, which has binding specificity to CD3, has a melting temperature higher than 65°C, preferably higher than 70°C (T m ) has. In further embodiments, the ankyrin repeat domain having binding specificity to CD3 contained in the binding protein has an aggregation onset temperature (T) higher than 70°C, preferably higher than 75°C. agg ) has. In one particular embodiment, the ankyrin repeat domain having binding specificity to CD3 comprises an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 1, and the binding protein is 10-7 dissociation constant (K) less than M D binds to human CD3 in PBS and / or has an EC in the range of 2 - 900 nM, preferably about 5 - about 400 nM 50 binds to human CD3 on T cells. In a particular embodiment, the ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 1, and the ankyrin repeat domain has a melting temperature (T m ) higher than 75 °C, preferably higher than 80 °C, and / or the ankyrin repeat domain has an aggregation onset temperature (T agg ) higher than 75 °C, preferably higher than 80 °C, and / or the binding protein has a dissociation constant (K -7 less than M D ) binds to human CD3 in PBS and / or the binding protein has an EC in the range of 2 - 900 nM, preferably about 5 - about 400 nM 50 binds to human CD3 on T cells. In another particular embodiment, the ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 2, and the binding protein has a dissociation constant (K -7 less than M, preferably or about 5×10 -8 dissociation constant (K) less than M D ) binds to human CD3 in PBS and / or has an EC in the range of 2 - 900 nM, preferably about 250 - about 400 nM 50 binds to human CD3 on T cells. In another particular embodiment, the ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 2, and the ankyrin repeat domain has a melting temperature (T m ) higher than 72 °C, preferably higher than 77 °C, and / or the ankyrin repeat domain has an aggregation onset temperature (T agg [[ID=P=27]]) higher than 70 °C, preferably higher than 75 °C, and / or the binding protein has a dissociation constant (K -7 less than M, preferably or about 5×10 -8 dissociation constant (K) less than M D) binds to human CD3 in PBS and / or the binding protein has an EC in the range of 2 to 900 nM, preferably about 250 to about 400 nM. 50 It binds to human CD3 on T cells. In an alternative specific embodiment, the ankyrin repeat domain having binding specificity to CD3 comprises an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 3, and the binding protein is 10 -7 Less than M, preferably or about 2 × 10 -8 Dissociation constant less than M (K D ) binds to human CD3 in PBS and / or has an EC in the range of 2 to 900 nM, preferably about 50 to about 100 nM 50 It binds to human CD3 on T cells. In another specific embodiment, the ankyrin repeat domain having binding specificity to CD3 comprises an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 3, and the ankyrin repeat domain has a melting temperature higher than 65°C, preferably higher than 70°C (T m ) and / or the ankyrin repeat domain has an aggregation onset temperature (T) higher than 70°C, preferably higher than 75°C. agg ) has and / or the binding protein is 10 -7 Less than M, preferably or about 2 × 10 -8 Dissociation constant less than M (K D ) binds to human CD3 in PBS, and / or the binding protein has an EC of 2 to 900 nM, preferably in the range of about 50 to about 100 nM. 50 It binds to human CD3 on T cells. In another alternative embodiment, the ankyrin repeat domain having binding specificity to CD3 comprises an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 4, and the binding protein is 10 -7 Less than M, preferably or about 10 -8 Dissociation constant less than M (K D ) binds to human CD3 in PBS and / or has an EC in the range of 2 to 900 nM, preferably about 5 to about 150 nM 50It binds to human CD3 on T cells. In another specific embodiment, the ankyrin repeat domain having binding specificity to CD3 comprises an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 4, and the ankyrin repeat domain has a melting temperature higher than 73°C, preferably higher than 78°C (T m ) and / or the ankyrin repeat domain has an aggregation onset temperature (T) higher than 70°C, preferably higher than 75°C. agg ) has and / or the binding protein is 10 -7 Less than M, preferably or about 10 -8 Dissociation constant less than M (K D ) binds to human CD3 in PBS, and / or the binding protein has an EC of 2 to 900 nM, preferably about 5 to about 15 nM. 50 It then binds to human CD3 on T cells.

[0012] In another embodiment, the present invention provides a recombinant binding protein comprising an ankyrin repeat domain having binding specificity to CD3, wherein the binding protein further comprises a binder having binding specificity to a disease-related antigen. In one embodiment, the binder is a designed ankyrin repeat domain having binding specificity to a disease-related antigen. In a further embodiment, the disease-related antigen is an infection-related antigen (IAA), preferably a virus-related antigen (VAA). In another embodiment, the disease-related antigen is a tumor-related antigen (TAA). In one embodiment of the present invention, the binder having binding specificity to a disease-related antigen is covalently bonded or fused to the designed ankyrin repeat domain having binding specificity to CD3. In a particular embodiment, the binder having binding specificity to a disease-related antigen is covalently bonded to the ankyrin repeat domain having binding specificity to CD3 using a peptide linker, preferably a high-proline-threonine peptide linker. In one embodiment, the amino acid sequence of the peptide linker has a length of 1 to 50 amino acids, preferably 6 to 38 amino acids. In one preferred embodiment, the binder having binding specificity to a disease-associated antigen is located at the N-terminus of the ankyrin repeat domain having binding specificity to CD3 within the binding protein.

[0013] In another embodiment, the present invention provides a recombinant binding protein comprising an ankyrin repeat domain having binding specificity to CD3, wherein the binding protein further comprises a half-life extension portion. In one embodiment, the half-life extension portion comprises a binder having binding specificity to human serum albumin. In one embodiment, the binder having binding specificity to human serum albumin is a designed ankyrin repeat domain having binding specificity to human serum albumin. In a particular embodiment, the binder having binding specificity to human serum albumin is a designed ankyrin repeat domain having binding specificity to human serum albumin, comprising any one amino acid sequence of SEQ ID NOs. 28 to 30, preferably the amino acid sequence of SEQ ID NO. 29. In one embodiment, the half-life extension portion is located at the N-terminus of the ankyrin repeat domain having binding specificity to CD3 within the binding protein.

[0014] In another aspect, the present invention provides a nucleic acid encoding the recombinant binding protein of the present invention, and a pharmaceutical composition comprising the binding protein or nucleic acid of the present invention and optionally a pharmaceutically acceptable carrier and / or diluent.

[0015] In another embodiment, the present invention provides a binding protein, a nucleic acid, or a pharmaceutical composition for use in a method of treating a medical condition. In one embodiment, the medical condition is an infectious disease, preferably a viral infection. In another embodiment, the medical condition is cancer.

[0016] In another embodiment, the present invention provides a method for tumor localization activation of T cells in a mammal, preferably a human, comprising administering to the mammal a binding protein of the present invention, a nucleic acid of the present invention, or a pharmaceutical composition of the present invention. In a particular embodiment, such a method comprises administering a binding protein of the present invention, further comprising a binding agent having binding specificity to a tumor-associated antigen, to a mammal, including a human patient having a tumor, to induce localized T cell activation in tumor tissue.

[0017] In another embodiment, the present invention provides a method for localizing and activating T cells in a mammal, preferably a human, comprising administering to the mammal a binding protein of the present invention, a nucleic acid of the present invention, or a pharmaceutical composition of the present invention. In a particular embodiment, such a method comprises administering a binding protein of the present invention, further comprising a binding agent having binding specificity to a virus-associated antigen, to a mammal, including a human patient with a viral infection, to bring about localized T cell activation in the infected tissue.

[0018] In another embodiment, the present invention relates to a method for treating a medical condition in a human patient, the method comprising administering to the patient a therapeutically effective amount of the recombinant binding protein of the present invention, the nucleic acid of the present invention, or the pharmaceutical composition of the present invention. In a particular embodiment, such a method comprises administering to the patient a therapeutically effective amount of the binding protein of the present invention, a nucleic acid encoding such a binding protein of the present invention, or a pharmaceutical composition comprising such a binding protein of the present invention, further comprising a binder having binding specificity to a disease-associated antigen. In one embodiment, the medical condition is cancer. In another embodiment, the medical condition is an infectious disease, preferably a viral infection.

[0019] In one particular embodiment, the medical condition is cancer, and the cancer or tumor tissue comprises cells expressing or presenting a tumor-associated antigen, and the binder having binding specificity to the disease-associated antigen binds to the tumor-associated antigen expressed or presented in the cells. In one particular embodiment, the disease-associated antigen is the extracellular domain of a cell surface protein expressed or overexpressed in the cancer or tumor tissue. In another particular embodiment, the binder having binding specificity to the tumor-associated antigen binds to a peptide-MHC complex, the peptide being derived from a protein expressed in tumor cells (e.g., NY-0ESO-1 or MAGE-A3). In one particular embodiment, the MHC is MHC class I. In one embodiment, the cancer is selected from carcinoma, sarcoma, myeloma, leukemia, lymphoma, and mixed-type cancer.

[0020] In another specific embodiment, the medical condition is an infectious disease, preferably a viral infection, and the infected tissue comprises cells expressing or presenting an infection-related antigen, preferably a virus-related antigen, and the binder having binding specificity to the disease-related antigen binds to the infection-related antigen expressed or presented in the cells. In another specific embodiment, the disease-related antigen is the extracellular domain of a viral protein expressed or overexpressed in the infected tissue. In yet another specific embodiment, the binder having binding specificity to the virus-related antigen binds to a peptide-MHC complex, the peptide being derived from a protein of an infectious agent, such as a bacterial or viral infectious agent, preferably a viral infectious agent (e.g., HBcAg or EBNA-1). In another specific embodiment, the MHC is MHC class I.

[0021] The present invention further provides a kit comprising the recombinant binding protein of the present invention, the nucleic acid of the present invention, or the pharmaceutical composition of the present invention. The present invention further provides a method for producing the recombinant binding protein of the present invention, comprising the steps of (i) expressing the recombinant binding protein in a suitable host cell (eukaryotic or prokaryotic cell) and (ii) purifying the recombinant binding protein (for example, by chromatography).

[0022] Based on the disclosures provided herein, those skilled in the art will recognize many equivalents to the particular embodiments of the invention described herein, or can confirm them by routine experimentation alone. Such equivalents are intended to be encompassed by the following embodiment (E). A recombinant binding protein comprising an ankyrin repeat domain having binding specificity to E1.CD3, wherein the ankyrin repeat domain further comprises a second ankyrin repeat module having an amino acid sequence selected from the group consisting of sequences in which up to nine amino acids in any of (1) SEQ ID NOs. 5-9 and (2) SEQ ID NOs. 5-9 are substituted with other amino acids. E2. The binding protein according to E1, wherein the ankyrin repeat module is a first ankyrin repeat module, and the ankyrin repeat domain further comprises a second ankyrin repeat module having an amino acid sequence selected from the group consisting of sequences in which up to nine amino acids in any of (1) SEQ ID NOs. 5-9 and (2) SEQ ID NOs. 5-9 are replaced by other amino acids. E3. The binding protein according to E2, wherein the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) Sequence ID No. 5 and (2) Sequence ID No. 5 in which up to nine amino acids are substituted by other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) Sequence ID No. 6 and (2) Sequence ID No. 6 in which up to nine amino acids are substituted by other amino acids. E4. The binding protein according to E2, wherein the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) Sequence ID No. 7 and (2) Sequence ID No. 7 in which up to nine amino acids are substituted by other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) Sequence ID No. 6 and (2) Sequence ID No. 6 in which up to nine amino acids are substituted by other amino acids. E5. The binding protein described in E2, wherein the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) Sequence ID No. 7 and (2) Sequence ID No. 7 in which up to nine amino acids are substituted by other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) Sequence ID No. 8 and (2) Sequence ID No. 8 in which up to nine amino acids are substituted by other amino acids. E6. The binding protein described in E2, wherein the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) Sequence ID No. 7 and (2) Sequence ID No. 7 in which up to nine amino acids are substituted by other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) Sequence ID No. 9 and (2) Sequence ID No. 9 in which up to nine amino acids are substituted by other amino acids. E7. A binding protein according to any one of E2 to E6, wherein the first ankyrin repeat module is located at the N-terminus of the second ankyrin repeat module within the ankyrin repeat domain. A recombinant binding protein comprising an ankyrin repeat domain having binding specificity to E8.CD3, wherein the ankyrin repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity with any one of SEQ ID NOs: 1 to 4, wherein the second-to-last A in SEQ ID NOs: 1 to 4 is optionally substituted with L, and / or the last A in SEQ ID NOs: 1 to 4 is optionally substituted with N. E9. Binding protein, 10 -7 Dissociation constant less than M (K D A binding protein described in one of E1 to E8 that binds to human CD3 in PBS. E10. The binding protein has an EC in the range of 2 to 900 nM. 50 A binding protein described in one of E1 to E9 that binds to human CD3 on T cells. The binding protein according to any one of E1 to E10, wherein the ankyrin repeat domain having binding specificity to E11.CD3 contains an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 1. The binding protein according to any one of E1 to E10, wherein the ankyrin repeat domain having binding specificity to E12.CD3 contains an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 2. E13. The binding protein is approximately 5 × 10 -8 Dissociation constant less than M (K DThe binding protein described in E12 binds to human CD3 in PBS. E14. The binding protein has an EC in the range of approximately 250 to 400 nM. 50 A binding protein described in E12 or E13 that binds to human CD3 on T cells. The binding protein according to any one of E1 to E10, wherein the ankyrin repeat domain having binding specificity to E15.CD3 contains an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 3. E16. The binding protein is approximately 2 × 10 -8 Dissociation constant less than M (K D The binding protein described in E15 binds to human CD3 in PBS. E17. The binding protein has an EC in the range of approximately 50 to 100 nM. 50 A binding protein described in E15 or E16 that binds to human CD3 on T cells. The binding protein according to any one of E1 to E10, wherein the ankyrin repeat domain having binding specificity to E18.CD3 contains an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 4. E19. The binding protein is approximately 10 -8 Dissociation constant less than M (K D The binding protein described in E18 binds to human CD3 in PBS. E20. The binding protein has an EC value in the range of approximately 5 to 15 nM. 50 A binding protein described in E18 or E19 that binds to human CD3 on T cells. E21. The binding protein according to any one of E1 to E20, further comprising a binder having binding specificity to a disease-related antigen. E22. The binding protein described in E21, wherein the binding agent is an ankyrin repeat domain having binding specificity to disease-related antigens. E23. The binding protein according to E21 or E22, wherein the disease-related antigen is an infection-related antigen (IAA), preferably a virus-related antigen (VAA), or a tumor-related antigen (TAA). E24. The binding protein according to any one of E21 to E23, wherein the binding agent having binding specificity to a disease-related antigen is located at the N-terminus of the ankyrin repeat domain having binding specificity to CD3 within the binding protein. E25. A binding protein according to any one of E21 to E24, wherein the binding agent having binding specificity to a disease-related antigen is covalently bound to the ankyrin repeat domain having binding specificity to CD3 within the binding protein. E26. The binding protein described in E25, wherein the peptide linker is a high-proline-threonine peptide linker. E27. The binding protein according to E25 or E26, wherein the amino acid sequence of the peptide linker has a length of 1 to 50 amino acids, preferably 6 to 38 amino acids. E28. The binding protein according to any one of E1 to E27, wherein the binding protein further comprises a half-life extension portion. E29. The binding protein according to E28, wherein the half-life extension portion contains a binder that has binding specificity to human serum albumin. E30. The binding protein according to E29, wherein the binding agent having binding specificity to human serum albumin is an ankyrin repeat domain containing any one of the amino acid sequences of SEQ ID NOs. 28 to 30, preferably the amino acid sequence of SEQ ID NO. 29. E31. The binding protein according to any one of E28 to E30, wherein the half-life extension portion is located at the N-terminus of the ankyrin repeat domain that has binding specificity to CD3 within the binding protein. E32. A nucleic acid that codes for one of the binding proteins listed in E1 to E31. A pharmaceutical composition comprising any one of E33.E1 to E31, a binding protein or the nucleic acid described in E32, and optionally a pharmaceutically acceptable carrier and / or diluent. E34. A binding protein according to any one of E1 to E31, a nucleic acid according to E32, or a pharmaceutical composition according to E33, for use in a method of treating a medical condition. E35. The binding protein described in E34, wherein the medical condition is an infection, preferably a viral infection, or cancer. E36. A method for localized tumor activation of T cells in a mammal, including a human, comprising the step of administering to the mammal a binding protein described in any one of E1 to E31, a nucleic acid described in E32, or a pharmaceutical composition described in E33. E37. A method for local activation of T cells in a mammal, including a human, comprising the step of administering to the mammal a binding protein described in any one of E1 to E31, a nucleic acid described in E32, or a pharmaceutical composition described in E33. E38. A method for treating a medical condition, comprising the step of administering to a patient in need of treatment for the medical condition a therapeutically effective amount of a binding protein described in any one of E1 to E31, a nucleic acid described in E32, or a pharmaceutical composition described in E33. E39. The method according to E38, wherein the medical condition is an infectious disease, preferably a viral infection, or cancer. [Brief explanation of the drawing]

[0023] [Figure 1] This is an SDS-PAGE gel analysis of purified ankyrin repeat proteins with binding specificity to human CD3, DARPin® protein #1, DARPin® protein #2, DARPin® protein #3, and DARPin® protein #4. M represents the protein size marker (reduced SDS-PAGE, NuPAGE 4-12%, Bis Tris (invitrogen) gel; 5 μg / lane; MES buffer; instant blue staining). It shows the molecular weight (kDa) of the marker protein. Lane 1: Protein size marker; Lane 2: Purified DARPin® protein #1; Lane 3: Purified DARPin® protein #2; Lane 4: Purified DARPin® protein #3; Lane 5: Purified DARPin® protein #4. [Figure 2A]This study evaluates the stability of exemplary designed ankyrin repeat proteins using UV spectroscopy. DARPin® proteins #1-4 were diluted 1:1 in TBS pH 8.0 (50 mM Tris, 500 mM NaCl), thoroughly mixed, and then subjected to UV-VIS (ultraviolet-visible) measurement using Nanodrop. Samples were evaluated at -70°C, and samples subjected to thermal stress at 50°C for 18 days were also evaluated. No absorption was observed at 320 nm (an indicator of precipitation). There was no difference in the spectra between the reference sample and the stressed sample. [Figure 2B] This study evaluates the stability of exemplary designed ankyrin repeat proteins using UV spectroscopy. DARPin® proteins #1-4 were diluted 1:1 in TBS pH 8.0 (50 mM Tris, 500 mM NaCl), thoroughly mixed, and then subjected to UV-VIS (ultraviolet-visible) measurement using Nanodrop. Samples were evaluated at -70°C, and samples subjected to thermal stress at 50°C for 18 days were also evaluated. No absorption was observed at 320 nm (an indicator of precipitation). There was no difference in the spectra between the reference sample and the stressed sample. [Figure 2C] This study evaluates the stability of exemplary designed ankyrin repeat proteins using UV spectroscopy. DARPin® proteins #1-4 were diluted 1:1 in TBS pH 8.0 (50 mM Tris, 500 mM NaCl), thoroughly mixed, and then subjected to UV-VIS (ultraviolet-visible) measurement using Nanodrop. Samples were evaluated at -70°C, and samples subjected to thermal stress at 50°C for 18 days were also evaluated. No absorption was observed at 320 nm (an indicator of precipitation). There was no difference in the spectra between the reference sample and the stressed sample. [Figure 2D]This study evaluates the stability of exemplary designed ankyrin repeat proteins using UV spectroscopy. DARPin® proteins #1-4 were diluted 1:1 in TBS pH 8.0 (50 mM Tris, 500 mM NaCl), thoroughly mixed, and then subjected to UV-VIS (ultraviolet-visible) measurement using Nanodrop. Samples were evaluated at -70°C, and samples subjected to thermal stress at 50°C for 18 days were also evaluated. No absorption was observed at 320 nm (an indicator of precipitation). There was no difference in the spectra between the reference sample and the stressed sample. [Figure 3] This is a stability assessment of an exemplary designed ankyrin repeat protein using reduced SDS-PAGE; 10% Bis-Tris gel; 5 μg / lane; MES buffer; and Instant Blue staining. Expected MW: 14.5 kDa. Lane 1, M: Stained marker, showing molecular weight (kDa) of the marker protein; Lanes 2 and 3: DARPin® protein #1 at -70°C vs. sample subjected to 18 days of heat stress at 50°C; Lanes 4 and 5: DARPin® protein #2 at -70°C vs. sample subjected to 18 days of heat stress at 50°C; Lanes 6 and 7: DARPin® protein #3 at -70°C vs. sample subjected to 18 days of heat stress at 50°C; Lanes 8 and 9: DARPin® protein #4 at -70°C vs. sample subjected to 18 days of heat stress at 50°C. [Figure 4A] This is a stability evaluation of exemplary designed ankyrin repeat proteins using size exclusion chromatography (SEC). DARPin® #1-4 protein samples were diluted to 2 mg / ml and scanned on a GE Superdex 200 150 / 5 Increased column in TBS pH 8.0 (50 mM Tris, 500 mM NaCl). Traces were analyzed at 280 nm. Black: Reference sample stored at -70°C; Gray: Sample subjected to heat stress and incubated at 50°C for 18 days. These curves largely overlap. [Figure 4B]This is a stability evaluation of exemplary designed ankyrin repeat proteins using size exclusion chromatography (SEC). DARPin® #1-4 protein samples were diluted to 2 mg / ml and scanned on a GE Superdex 200 150 / 5 Increased column in TBS pH 8.0 (50 mM Tris, 500 mM NaCl). Traces were analyzed at 280 nm. Black: Reference sample stored at -70°C; Gray: Sample subjected to heat stress and incubated at 50°C for 18 days. These curves largely overlap. [Figure 4C] This is a stability evaluation of exemplary designed ankyrin repeat proteins using size exclusion chromatography (SEC). DARPin® #1-4 protein samples were diluted to 2 mg / ml and scanned on a GE Superdex 200 150 / 5 Increased column in TBS pH 8.0 (50 mM Tris, 500 mM NaCl). Traces were analyzed at 280 nm. Black: Reference sample stored at -70°C; Gray: Sample subjected to heat stress and incubated at 50°C for 18 days. These curves largely overlap. [Figure 4D] This is a stability evaluation of exemplary designed ankyrin repeat proteins using size exclusion chromatography (SEC). DARPin® #1-4 protein samples were diluted to 2 mg / ml and scanned on a GE Superdex 200 150 / 5 Increased column in TBS pH 8.0 (50 mM Tris, 500 mM NaCl). Traces were analyzed at 280 nm. Black: Reference sample stored at -70°C; Gray: Sample subjected to heat stress and incubated at 50°C for 18 days. These curves largely overlap. [Figure 5A]This study evaluates the thermal stability of exemplary designed ankyrin repeat proteins using circular dichroism (CD) spectroscopy. Samples of DARPin® protein #1-4 (A-D, respectively) were diluted to 2 μM in TBS pH 8.0 (50 mM Tris, 500 mM NaCl). Protein unfolding (forward) and refolding (reverse) were monitored. All proteins showed reversible unfolding at Tm above 65°C (indicated by dotted lines in the graph). [Figure 5B] This study evaluates the thermal stability of exemplary designed ankyrin repeat proteins using circular dichroism (CD) spectroscopy. Samples of DARPin® protein #1-4 (A-D, respectively) were diluted to 2 μM in TBS pH 8.0 (50 mM Tris, 500 mM NaCl). Protein unfolding (forward) and refolding (reverse) were monitored. All proteins showed reversible unfolding at Tm above 65°C (indicated by dotted lines in the graph). [Figure 5C] This study evaluates the thermal stability of exemplary designed ankyrin repeat proteins using circular dichroism (CD) spectroscopy. Samples of DARPin® protein #1-4 (A-D, respectively) were diluted to 2 μM in TBS pH 8.0 (50 mM Tris, 500 mM NaCl). Protein unfolding (forward) and refolding (reverse) were monitored. All proteins showed reversible unfolding at Tm above 65°C (indicated by dotted lines in the graph). [Figure 5D] This study evaluates the thermal stability of exemplary designed ankyrin repeat proteins using circular dichroism (CD) spectroscopy. Samples of DARPin® protein #1-4 (A-D, respectively) were diluted to 2 μM in TBS pH 8.0 (50 mM Tris, 500 mM NaCl). Protein unfolding (forward) and refolding (reverse) were monitored. All proteins showed reversible unfolding at Tm above 65°C (indicated by dotted lines in the graph). [Figure 6A]This study determined the aggregation onset temperature (Tagg) by dynamic light scattering (DLS). Samples of DARPin® protein #1-4 were diluted to 1 mg / ml in TBS pH 8.0 (50 mM Tris, 500 mM NaCl), and the Tag was determined by DLS (5 repeats, A1, A2, A3, A4, A5). All four ankyrin repeat proteins tested showed high Tag levels above 80°C. [Figure 6B] This study determined the aggregation onset temperature (Tagg) by dynamic light scattering (DLS). Samples of DARPin® protein #1-4 were diluted to 1 mg / ml in TBS pH 8.0 (50 mM Tris, 500 mM NaCl), and the Tag was determined by DLS (5 repeats, A1, A2, A3, A4, A5). All four ankyrin repeat proteins tested showed high Tag levels above 80°C. [Figure 6C] This study determined the aggregation onset temperature (Tagg) by dynamic light scattering (DLS). Samples of DARPin® protein #1-4 were diluted to 1 mg / ml in TBS pH 8.0 (50 mM Tris, 500 mM NaCl), and the Tag was determined by DLS (5 repeats, A1, A2, A3, A4, A5). All four ankyrin repeat proteins tested showed high Tag levels above 80°C. [Figure 6D] This study determined the aggregation onset temperature (Tagg) by dynamic light scattering (DLS). Samples of DARPin® protein #1-4 were diluted to 1 mg / ml in TBS pH 8.0 (50 mM Tris, 500 mM NaCl), and the Tag was determined by DLS (5 repeats, A1, A2, A3, A4, A5). All four ankyrin repeat proteins tested showed high Tag levels above 80°C. [Figure 7A]This is a pharmacokinetic analysis of exemplary CD3-specific binding proteins in female BALB / c mice. The figure shows the group-mean serum concentration-time profiles of DARPin® protein #5, DARPin® protein #6, DARPin® protein #7, and DARPin® protein #8 in female BALB / c mice after a single intravenous bolus administration of 1 mg / kg (mean ± maximum / min, N=3 per group). [Figure 7B] This is a surface plasmon resonance (SPR) analysis of binding proteins that bind to human CD3, exemplified by DARPin® protein #3. Purified ankyrin repeat proteins at various concentrations (8, 16, 32, and 64 nM) were applied to GLC chips with immobilized human CD3 for on-rate and off-rate measurements. The resulting SPR trace analysis was used to determine the ankyrin repeat protein-CD3 interaction. RU: resonance units; s: time (seconds). [Figure 8A] This shows the CD3 binding of the exemplified binding proteins to T cells. Binding to CD3 on Pan-T cells was evaluated using Mirrorball. Benchmark control molecules (known benchmark T cell engagers, TCE1 with binding specificity to TAA2 and TCE2 with binding specificity to TAA3) and selected ankyrin repeat proteins are shown, along with DARPin® protein A, DARPin® protein B, DARPin® protein C, and DARPin® protein D, with or without (HLE) half-life extension (Figure A) (Figure B). The half-life extension protein (B) shows similar binding to the corresponding non-HLE molecule shown in (A). Pan-T cells from five different donors were tested, and one representative donor is shown here. (*Negative controls: designed ankyrin repeat proteins with and without half-life extension, and with binding specificity only to TAA2 and TAA3, respectively). [Figure 8B]This shows the CD3 binding of the exemplified binding proteins to T cells. Binding to CD3 on Pan-T cells was evaluated using Mirrorball. Benchmark control molecules (known benchmark T cell engagers, TCE1 with binding specificity to TAA2 and TCE2 with binding specificity to TAA3) and selected ankyrin repeat proteins are shown, along with DARPin® protein A, DARPin® protein B, DARPin® protein C, and DARPin® protein D, with or without (HLE) half-life extension (Figure A) (Figure B). The half-life extension protein (B) shows similar binding to the corresponding non-HLE molecule shown in (A). Pan-T cells from five different donors were tested, and one representative donor is shown here. (*Negative controls: designed ankyrin repeat proteins with and without half-life extension, and with binding specificity only to TAA2 and TAA3, respectively). [Figure 9A]This is short-term target cell death (LDH). Pan-T cells (as effector cells (E)) and TAA2 / TAA3-expressing tumor cells (as target cells (T)) were incubated in a 5:1 E:T ratio, and tumor cell death was evaluated by LDH release in the supernatant after 48 hours of co-culture in the presence of serial dilutions of the indicated molecules. The benchmark control molecule (a known benchmark T cell engager, TCE1 with binding specificity to TAA2) and selected ankyrin repeat proteins, DARPin® protein A, DARPin® protein B, DARPin® protein C, and DARPin® protein D with or without half-life extension (HLE) (Figure A) are shown. (A) For proteins without half-life extension, tumor cell death induced by DARPin® protein B and DARPin® protein C is comparable to the benchmark molecule, but DARPin® protein D and DARPin® protein A show lower potency in cytotoxicity. (B) Proteins with extended half-lives show a 4- to 70-fold decrease in potency compared to the corresponding non-HLE molecules shown in (A). Pan-T cells from five different donors were tested, and one representative donor is shown here. (*Negative control: engineered ankyrin repeat proteins with binding specificity to TAA2 and TAA3, respectively, with or without extended half-lives). [Figure 9B]This is short-term target cell death (LDH). Pan-T cells (as effector cells (E)) and TAA2 / TAA3-expressing tumor cells (as target cells (T)) were incubated in a 5:1 E:T ratio, and tumor cell death was evaluated by LDH release in the supernatant after 48 hours of co-culture in the presence of serial dilutions of the indicated molecules. The benchmark control molecule (a known benchmark T cell engager, TCE1 with binding specificity to TAA2) and selected ankyrin repeat proteins, DARPin® protein A, DARPin® protein B, DARPin® protein C, and DARPin® protein D with or without half-life extension (HLE) (Figure A) are shown. (A) For proteins without half-life extension, tumor cell death induced by DARPin® protein B and DARPin® protein C is comparable to the benchmark molecule, but DARPin® protein D and DARPin® protein A show lower potency in cytotoxicity. (B) Proteins with extended half-lives show a 4- to 70-fold decrease in potency compared to the corresponding non-HLE molecules shown in (A). Pan-T cells from five different donors were tested, and one representative donor is shown here. (*Negative control: engineered ankyrin repeat proteins with binding specificity to TAA2 and TAA3, respectively, with or without extended half-lives). [Figure 10A]This is short-term T cell activation measured by the activation marker CD25. Pan-T cells and TAA2 / TAA3-expressing tumor cells were incubated in a 1:1 E:T ratio, and T cell activation was evaluated by FACS after 24 hours of co-culture in the presence of serial dilutions of the indicated molecules. Activated T cells were gated as viable CD8+ / CD25+ cells. Benchmark control molecules (known benchmark T cell engagers, TCE1 with binding specificity to TAA2) and selected ankyrin repeat proteins, DARPin® protein A, DARPin® protein B, DARPin® protein C, and DARPin® protein D with or without (HLE) half-life extension (HLE) (Figure A) are shown. (A) T cell activation induced by DARPin® protein B and DARPin® protein C without half-life extension is comparable to the benchmark molecule, while DARPin® protein D and DARPin® protein A show lower potency. (B) Half-life extension proteins show a 4- to 100-fold decrease in potency compared to the corresponding non-HLE molecules shown in (A). Pan-T cells from seven different donors were tested, and one representative donor is shown here. (*Negative control: engineered ankyrin repeat proteins with binding specificity only to TAA2 and TAA3, respectively, with or without half-life extension). [Figure 10B]This is short-term T cell activation measured by the activation marker CD25. Pan-T cells and TAA2 / TAA3-expressing tumor cells were incubated in a 1:1 E:T ratio, and T cell activation was evaluated by FACS after 24 hours of co-culture in the presence of serial dilutions of the indicated molecules. Activated T cells were gated as viable CD8+ / CD25+ cells. Benchmark control molecules (known benchmark T cell engagers, TCE1 with binding specificity to TAA2) and selected ankyrin repeat proteins, DARPin® protein A, DARPin® protein B, DARPin® protein C, and DARPin® protein D with or without (HLE) half-life extension (HLE) (Figure A) are shown. (A) T cell activation induced by DARPin® protein B and DARPin® protein C without half-life extension is comparable to the benchmark molecule, while DARPin® protein D and DARPin® protein A show lower potency. (B) Half-life extension proteins show a 4- to 100-fold decrease in potency compared to the corresponding non-HLE molecules shown in (A). Pan-T cells from seven different donors were tested, and one representative donor is shown here. (*Negative control: engineered ankyrin repeat proteins with binding specificity only to TAA2 and TAA3, respectively, with or without half-life extension). [Figure 11A]This is short-term T cell activation measured by IFNγ secretion. Pan-T and TAA2 / TAA3-expressing tumor cells were incubated in a 1:1 E:T ratio. After co-culturing for 24 hours in the presence of serial dilutions of the indicated molecules, IFNγ secretion in the culture supernatant was analyzed by ELISA. The benchmark control molecules (a known benchmark T cell engager, TCE1 with binding specificity to TAA2) and selected ankyrin repeat proteins, DARPin® protein A, DARPin® protein B, DARPin® protein C, and DARPin® protein D with or without half-life extension (HLE) (Figure A) are shown. (A) T cell activation induced by DARPin® protein B and DARPin® protein C without half-life extension is comparable to that of the benchmark molecule, while DARPin® protein D and v118 DARPin® protein A show lower potency. (B) The half-life extension proteins show a 3- to 20-fold decrease in potency compared to the corresponding non-HLE molecules shown in (A). Pan-T cells from four different donors were tested, and one representative donor is shown here. (*Negative control: an engineered ankyrin repeat protein with binding specificity to TAA2 and TAA3 only, with or without half-life extension). [Figure 11B]This is short-term T cell activation measured by IFNγ secretion. Pan-T and TAA2 / TAA3-expressing tumor cells were incubated in a 1:1 E:T ratio. After co-culturing for 24 hours in the presence of serial dilutions of the indicated molecules, IFNγ secretion in the culture supernatant was analyzed by ELISA. The benchmark control molecules (a known benchmark T cell engager, TCE1 with binding specificity to TAA2) and selected ankyrin repeat proteins, DARPin® protein A, DARPin® protein B, DARPin® protein C, and DARPin® protein D with or without half-life extension (HLE) (Figure A) are shown. (A) T cell activation induced by DARPin® protein B and DARPin® protein C without half-life extension is comparable to that of the benchmark molecule, while DARPin® protein D and v118 DARPin® protein A show lower potency. (B) The half-life extension proteins show a 3- to 20-fold decrease in potency compared to the corresponding non-HLE molecules shown in (A). Pan-T cells from four different donors were tested, and one representative donor is shown here. (*Negative control: an engineered ankyrin repeat protein with binding specificity to TAA2 and TAA3 only, with or without half-life extension). [Figure 12]This study focused on long-term tumor cell death. Pan-T cells and TAA2 / TAA3-expressing tumor cells were incubated at a 5:1 E:T ratio, and T cell death was assessed by IncuCyte in co-culture for 6 days in the presence of serial dilutions of the indicated molecules. Tumor cell death was calculated as the ratio between the area under the curve under Annexin V staining and cell proliferation. Two benchmark control molecules (known benchmark T cell engagers, TCE1 with binding specificity to TAA2 and TCE2 with binding specificity to TAA3) and selected ankyrin repeat proteins, DARPin® protein A, DARPin® protein B, DARPin® protein C, and DARPin® protein D, with or without half-life extension (HLE), are shown. The data show that, regardless of half-life extension, most of the tested proteins exhibit potent and specific tumor cell death comparable to the benchmark molecules. Only the lower affinity DARPin® protein A showed a significant decrease in cell death efficacy. The concentrations, from left to right, were 2 nM for the benchmark and test proteins without the HSA-binding domain, followed by a 1 / 10 dilution, and 20 nM for the test protein containing the HSA-binding domain, followed by a 1 / 10 dilution. Pan-T cells from five different donors were used, with one representative donor shown here. [Figure 13A]This study involved long-term T cell activation. Pan-T cells and TAA2 / TAA3-expressing tumor cells were incubated in a 1:1 E:T ratio, and T cell activation was evaluated by FACS after 5 days of co-culture in serial dilutions of the indicated molecules. Activated T cells were gated as viable CD8+ / CD25+ cells. Two benchmark control molecules (known benchmark T cell engagers, TCE1 with binding specificity to TAA2 and TCE2 with binding specificity to TAA3) and selected ankyrin repeat proteins, DARPin® protein A, DARPin® protein B, DARPin® protein C, and DARPin® protein D, with or without HLE (HLE), are shown. (A) T cell activation induced by DARPin® protein B and DARPin® protein C without half-life extension is comparable to the benchmark molecule, but DARPin® protein A and DARPin® protein D show a >100-fold decrease in potency. (B) Half-life extension proteins show a >10-fold decrease in potency compared to the corresponding non-HLE molecule shown in (A). Pan-T cells from two different donors were used, with one representative donor shown here. [Figure 13B]This study involved long-term T cell activation. Pan-T cells and TAA2 / TAA3-expressing tumor cells were incubated in a 1:1 E:T ratio, and T cell activation was evaluated by FACS after 5 days of co-culture in serial dilutions of the indicated molecules. Activated T cells were gated as viable CD8+ / CD25+ cells. Two benchmark control molecules (known benchmark T cell engagers, TCE1 with binding specificity to TAA2 and TCE2 with binding specificity to TAA3) and selected ankyrin repeat proteins, DARPin® protein A, DARPin® protein B, DARPin® protein C, and DARPin® protein D, with or without HLE (HLE), are shown. (A) T cell activation induced by DARPin® protein B and DARPin® protein C without half-life extension is comparable to the benchmark molecule, but DARPin® protein A and DARPin® protein D show a >100-fold decrease in potency. (B) Half-life extension proteins show a >10-fold decrease in potency compared to the corresponding non-HLE molecule shown in (A). Pan-T cells from two different donors were used, with one representative donor shown here. [Figure 14A]This study involved long-term T cell proliferation. Pan-T cells and TAA2 / TAA3-expressing tumor cells were incubated in a 1:1 E:T ratio, and T cell proliferation was evaluated by FACS after 5 days of co-culture in the presence of serial dilutions of the indicated molecules. Proliferating T cells were gated as CellTrace Violet-positive cells showing at least one cell division. Two benchmark control molecules (known benchmark T cell engagers, TCE1 with binding specificity to TAA2 and TCE2 with binding specificity to TAA3) and selected ankyrin repeat proteins, DARPin® protein A, DARPin® protein B, DARPin® protein C, and DARPin® protein D, with or without HLE (HLE), are shown. (A) T cell proliferation induced by DARPin® protein B and DARPin® protein C without half-life extension is comparable to the benchmark molecule, but DARPin® protein A and DARPin® protein D show a >100-fold decrease in potency. (B) Half-life extended DARPin® proteins show a >30-fold decrease in potency compared to the corresponding non-HLE molecule shown in (A). Pan-T cells from two different donors were used, with one representative donor shown here. [Figure 14B]This study involved long-term T cell proliferation. Pan-T cells and TAA2 / TAA3-expressing tumor cells were incubated in a 1:1 E:T ratio, and T cell proliferation was evaluated by FACS after 5 days of co-culture in the presence of serial dilutions of the indicated molecules. Proliferating T cells were gated as CellTrace Violet-positive cells showing at least one cell division. Two benchmark control molecules (known benchmark T cell engagers, TCE1 with binding specificity to TAA2 and TCE2 with binding specificity to TAA3) and selected ankyrin repeat proteins, DARPin® protein A, DARPin® protein B, DARPin® protein C, and DARPin® protein D, with or without HLE (HLE), are shown. (A) T cell proliferation induced by DARPin® protein B and DARPin® protein C without half-life extension is comparable to the benchmark molecule, but DARPin® protein A and DARPin® protein D show a >100-fold decrease in potency. (B) Half-life extended DARPin® proteins show a >30-fold decrease in potency compared to the corresponding non-HLE molecule shown in (A). Pan-T cells from two different donors were used, with one representative donor shown here. [Figure 15A] This describes the time course of tumor growth in mice after injection and treatment of tumor cells. Treatment was carried out using a vehicle, benchmark TAA3 T cell engager, DARPin® protein #E, DARPin® protein #F, DARPin® protein #G, and DARPin® protein #H, as shown and described in Example 10. [Figure 15B] This describes the time course of tumor growth in mice after injection and treatment of tumor cells. Treatment was carried out using a vehicle, benchmark TAA3 T cell engager, DARPin® protein #E, DARPin® protein #F, DARPin® protein #G, and DARPin® protein #H, as shown and described in Example 10. [Figure 15C] This describes the time course of tumor growth in mice after injection and treatment of tumor cells. Treatment was carried out using a vehicle, benchmark TAA3 T cell engager, DARPin® protein #E, DARPin® protein #F, DARPin® protein #G, and DARPin® protein #H, as shown and described in Example 10. [Figure 16A] (A) This shows tumor growth over time in mice injected intraperitoneally with hPBMCs (n=5 mice per donor / 2 hPBMC donors used). TAA2 / TAA3 / TAA4 expressing tumor cells were subcutaneously transplanted into xenografts and treated with PBS 1× (black circles), 0.5 mg / kg of multispecific TCE format DARPin® protein #3 (black squares), or 0.5 mg / kg of benchmark TAA3 T cell engager (black triangles). Treatment was carried out as described in Example 10. Data are shown as mean + SEM. (B) This shows the evaluation of tumor volume 17 days after tumor cell xenografting. [Figure 16B] (A) This shows tumor growth over time in mice injected intraperitoneally with hPBMCs (n=5 mice per donor / 2 hPBMC donors used). TAA2 / TAA3 / TAA4 expressing tumor cells were subcutaneously transplanted into xenografts and treated with PBS 1× (black circles), 0.5 mg / kg of multispecific TCE format DARPin® protein #3 (black squares), or 0.5 mg / kg of benchmark TAA3 T cell engager (black triangles). Treatment was carried out as described in Example 10. Data are shown as mean + SEM. (B) This shows the evaluation of tumor volume 17 days after tumor cell xenografting. [Modes for carrying out the invention]

[0024] As disclosed and illustrated herein, this disclosure provides ankyrin repeat proteins that specifically target CD3. A library of designed ankyrin repeat proteins (International Publication No. 2002 / 020565, Binz et al., Nat. Biotechnol. 22, 575-582, 2004; Stumppet et al., Drug Discov. Today, 13, 695-701, 2008) can be used to select target-specific designed ankyrin repeat domains that bind to their targets with high affinity. Such target-specific designed ankyrin repeat domains can then be used as valuable components of recombinant binding proteins for treating diseases. Designed ankyrin repeat proteins represent a class of binding molecules that have the potential to overcome the limitations of monoclonal antibodies and thus enable novel therapeutic approaches. Such ankyrin repeat proteins may comprise a single designed ankyrin repeat domain or a combination of two or more designed ankyrin repeat domains having the same or different target specificity (Stumpp et al., Drug Discov. Today). (13,695-701, 2008; U.S. Patent No. 9,458,211). Ankyrin repeat proteins, containing only a single designed ankyrin repeat domain, are small proteins (14 kDa) that can be selected to bind to a given target protein with high affinity and specificity. These properties, and the ability to combine two, three, four or more designed ankyrin repeat domains in a single protein, resulting in binding proteins with two, three, four or more different specificities, make designed ankyrin repeat proteins ideal agonist, antagonist, and / or inhibitory drug candidates, enabling novel drug design, including the design of novel T cell engager drug molecules, for example. Furthermore, such ankyrin repeat proteins can be manipulated to carry various effector functions, such as cytotoxic agents or half-life extenders, enabling entirely new drug formats. In summary, designed ankyrin repeat proteins represent the next generation of protein therapies with the potential to surpass existing antibody drugs.

[0025] DARPin® is a trademark owned by Molecular Partners AG, Switzerland.

[0026] In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to CD3, and the ankyrin repeat domain comprises an ankyrin repeat module having an amino acid sequence selected from the group consisting of sequences in which up to 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid in either (1) SEQ ID NOs. 5-9 and (2) SEQ ID NOs. 5-9 are substituted with other amino acids. Accordingly, in one embodiment, the ankyrin repeat module further comprises a second ankyrin repeat module having an amino acid sequence selected from the group consisting of sequences in which up to 9 amino acids in either (1) SEQ ID NOs. 5-9 and (2) SEQ ID NOs. 5-9 are substituted with other amino acids. In one embodiment, the ankyrin repeat module further comprises a second ankyrin repeat module having an amino acid sequence selected from the group consisting of sequences in which up to nine amino acids in either (1) SEQ ID NOs. 5-9 and (2) SEQ ID NOs. 5-9 are substituted by other amino acids. In one embodiment, the ankyrin repeat module further comprises a second ankyrin repeat module having an amino acid sequence selected from the group consisting of sequences in which up to nine amino acids in either (1) SEQ ID NOs. 5-9 and (2) SEQ ID NOs. 5-9 are substituted by other amino acids. In one embodiment, the ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to one amino acid in either (1) SEQ ID NOs. 5-9 and (2) SEQ ID NOs. 5-9 are substituted by other amino acids. In one embodiment, all of the 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid substitutions occur at the framework positions of the ankyrin repeat module. In one embodiment, the ankyrin repeat module includes an amino acid sequence selected from the group consisting of SEQ ID NOs. 5-9.

[0027] In one embodiment, the ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 5 or a sequence in which one or two amino acids in SEQ ID NO: 5 are substituted with other amino acids. In one embodiment, the ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 6 or a sequence in which one or two amino acids in SEQ ID NO: 6 are substituted with other amino acids. In one embodiment, the ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 7 or a sequence in which one or two amino acids in SEQ ID NO: 7 are substituted with other amino acids. In one embodiment, the ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 8 or a sequence in which one or two amino acids in SEQ ID NO: 8 are substituted with other amino acids. In one embodiment, the ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 9 or a sequence in which one or two amino acids in SEQ ID NO: 9 are substituted with other amino acids. In one embodiment, the ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 5. In one embodiment, the ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 6. In one embodiment, the ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 7. In one embodiment, the ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 8. In another embodiment, the ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 9.

[0028] In one embodiment, the ankyrin repeat domain includes a first ankyrin repeat module and a second ankyrin repeat module. In a preferred embodiment, the first ankyrin repeat module is located at the N-terminus of the second ankyrin repeat module within the ankyrin repeat domain. In one embodiment, the first and second ankyrin repeat modules each further include a second ankyrin repeat module having an amino acid sequence selected from the group consisting of sequences in which up to nine amino acids in any of (1) SEQ ID NOs. 5-9 and (2) SEQ ID NOs. 5-9 are substituted with other amino acids. Accordingly, in one embodiment, the ankyrin repeat domain includes a first ankyrin repeat module having an amino acid sequence selected from the group consisting of sequences in which up to one amino acid is substituted by another amino acid, which has an amino acid sequence selected from the group consisting of sequences in which up to nine amino acids in (1) SEQ ID NOs. 5 to 9 and (2) SEQ ID NOs. 5 to 9 are substituted by other amino acids, and further includes a second ankyrin repeat module having an amino acid sequence selected from the group consisting of sequences in which up to nine amino acids in either (1) SEQ ID NOs. 5 to 9 and (2) SEQ ID NOs. 5 to 9 are substituted by other amino acids.

[0029] In one particular embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to nine, or up to eight, or up to seven, or up to six, or up to five, or up to four, or up to three, or up to two, or up to one amino acid in (1) SEQ ID NO: 5 and (2) SEQ ID NO: 5 is substituted with other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to nine, or up to eight, or up to seven, or up to six, or up to five, or up to four, or up to three, or up to two, or up to one amino acid in (1) SEQ ID NO: 6 and (2) SEQ ID NO: 6 is substituted with other amino acids.

[0030] Accordingly, in one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to nine amino acids in (1) SEQ ID NO: 5 and (2) SEQ ID NO: 5 are substituted with other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to two amino acids in (1) SEQ ID NO: 6 and (2) SEQ ID NO: 6 are substituted with other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to six amino acids in (1) SEQ ID NO: 5 and (2) SEQ ID NO: 5 are substituted with other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to two amino acids in (1) SEQ ID NO: 6 and (2) SEQ ID NO: 6 are substituted with other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to five amino acids in (1) SEQ ID NO: 5 and (2) SEQ ID NO: 5 are substituted with other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to nine, or up to eight, or up to seven, or up to six, or up to five, or up to four, or up to three, or up to one amino acid in (1) SEQ ID NO: 6 and (2) SEQ ID NO: 6 are substituted with other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to four amino acids in (1) SEQ ID NO: 5 and (2) SEQ ID NO: 5 are substituted with other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to nine, or up to eight, or up to seven, or up to six, or up to five, or up to four, or up to three, or up to two, or up to one amino acid in (1) SEQ ID NO: 6 and (2) SEQ ID NO: 6 are substituted with other amino acids.In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to three amino acids in (1) SEQ ID NO: 5 and (2) SEQ ID NO: 5 are substituted with other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to nine, or up to eight, or up to seven, or up to six, or up to five, or up to four, or up to three, or up to two, or up to one amino acid in (1) SEQ ID NO: 6 and (2) SEQ ID NO: 6 are substituted with other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to two amino acids in (1) SEQ ID NO: 5 and (2) SEQ ID NO: 5 are substituted with other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to two amino acids in (1) SEQ ID NO: 6 and (2) SEQ ID NO: 6 are substituted with other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) Sequence ID No. 5 and (2) Sequence ID No. 5 in which one amino acid is replaced by another amino acid, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) Sequence ID No. 6 and (2) Sequence ID No. 6 in which one amino acid is replaced by another amino acid. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes the amino acid sequence of Sequence ID No. 5, and the second ankyrin repeat module includes the amino acid sequence of Sequence ID No. 6.

[0031] In a preferred embodiment, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to three, up to two, or up to one amino acid in (1) SEQ ID NO: 5 and (2) SEQ ID NO: 5 are substituted with other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to three, up to two, or up to one amino acid in (1) SEQ ID NO: 6 and (2) SEQ ID NO: 6 are substituted with other amino acids, and the first ankyrin repeat module is located at the N-terminus of the second ankyrin repeat module within the ankyrin repeat domain. Furthermore, in a more preferred embodiment, the first ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 5, and the second ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 6, and the first ankyrin repeat module is located at the N-terminus of the second ankyrin repeat module within the ankyrin repeat domain.

[0032] In another specific embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to nine, or up to eight, or up to seven, or up to six, or up to five, or up to four, or up to three, or up to two, or up to one amino acid in (1) SEQ ID NO: 7 and (2) SEQ ID NO: 7 is substituted by other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to nine, or up to eight, or up to seven, or up to six, or up to five, or up to four, or up to three, or up to two, or up to one amino acid in (1) SEQ ID NO: 6 and (2) SEQ ID NO: 6 is substituted by other amino acids.

[0033] Accordingly, in one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to nine amino acids in (1) SEQ ID NO: 7 and (2) SEQ ID NO: 7 are substituted with other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to nine amino acids in (1) SEQ ID NO: 6 and (2) SEQ ID NO: 6 are substituted with other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to six amino acids in (1) SEQ ID NO: 7 and (2) SEQ ID NO: 7 are substituted with other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to six amino acids in (1) SEQ ID NO: 6 and (2) SEQ ID NO: 6 are substituted with other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to five amino acids in (1) SEQ ID NO: 7 and (2) SEQ ID NO: 7 are substituted with other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to five amino acids in (1) SEQ ID NO: 6 and (2) SEQ ID NO: 6 are substituted with other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to four amino acids in (1) SEQ ID NO: 7 and (2) SEQ ID NO: 7 are substituted with other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to four amino acids in (1) SEQ ID NO: 6 and (2) SEQ ID NO: 6 are substituted with other amino acids.In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to three amino acids in (1) SEQ ID NO: 7 and (2) SEQ ID NO: 7 are substituted with other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to three amino acids in (1) SEQ ID NO: 6 and (2) SEQ ID NO: 6 are substituted with other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to two amino acids in (1) SEQ ID NO: 7 and (2) SEQ ID NO: 7 are substituted with other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to two amino acids in (1) SEQ ID NO: 6 and (2) SEQ ID NO: 6 are substituted with other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) sequences of sequence numbers 81, 87 and (2) sequences of sequence number 7 in which one amino acid is substituted by another amino acid, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) sequences of sequence number 6 and (2) sequences of sequence number 6 in which one amino acid is substituted by another amino acid. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes the amino acid sequence of sequence number 7, and the second ankyrin repeat module includes the amino acid sequence of sequence number 6.

[0034] In one preferred embodiment, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 7 and (2) sequences in which up to three, up to two, or up to one amino acid in SEQ ID NO: 7 is substituted by other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 6 and (2) sequences in which up to three, up to two, or up to one amino acid in SEQ ID NO: 6 is substituted by other amino acids, and the first ankyrin repeat module is located at the N-terminus of the second ankyrin repeat module within the ankyrin repeat domain. Furthermore, in a more preferred embodiment, the first ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 7, and the second ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 6, and the first ankyrin repeat module is located at the N-terminus of the second ankyrin repeat module within the ankyrin repeat domain.

[0035] In another specific embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to nine, or up to eight, or up to seven, or up to six, or up to five, or up to four, or up to three, or up to two, or up to one amino acid in (1) SEQ ID NO: 7 and (2) SEQ ID NO: 7 is substituted by other amino acids, and the second ankyrin repeat module includes a sequence in which up to nine, or up to eight, or up to seven, or up to six, or up to five, or up to four, or up to three, or up to two, or up to one amino acid in (1) SEQ ID NO: 8 and (2) SEQ ID NO: 8 is substituted by other amino acids.

[0036] Therefore, in one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to nine amino acids in (1) SEQ ID NO: 7 and (2) SEQ ID NO: 7 are substituted with other amino acids, and the second ankyrin repeat module includes a sequence in which up to nine amino acids in (1) SEQ ID NO: 8 and (2) SEQ ID NO: 8 are substituted with other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to six amino acids in (1) SEQ ID NO: 7 and (2) SEQ ID NO: 7 are substituted with other amino acids, and the second ankyrin repeat module includes a sequence in which up to six amino acids in (1) SEQ ID NO: 8 and (2) SEQ ID NO: 8 are substituted with other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to five amino acids in (1) SEQ ID NO: 7 and (2) SEQ ID NO: 7 are substituted with other amino acids, and the second ankyrin repeat module includes a sequence in which up to five amino acids in (1) SEQ ID NO: 8 and (2) SEQ ID NO: 8 are substituted with other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to four amino acids in (1) SEQ ID NO: 7 and (2) SEQ ID NO: 7 are substituted with other amino acids, and the second ankyrin repeat module includes a sequence in which up to four amino acids in (1) SEQ ID NO: 8 and (2) SEQ ID NO: 8 are substituted with other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) Sequence ID No. 7 and (2) Sequence ID No. 7 in which up to three amino acids are substituted by other amino acids, and the second ankyrin repeat module includes (1) Sequence ID No. 8 and (2) Sequence ID No. 8 in which up to three amino acids are substituted by other amino acids.In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to two amino acids in (1) SEQ ID NO: 7 and (2) SEQ ID NO: 7 are substituted with other amino acids, and the second ankyrin repeat module includes a sequence in which up to two amino acids in (1) SEQ ID NO: 8 and (2) SEQ ID NO: 8 are substituted with other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which one amino acid in (1) SEQ ID NO: 7 and (2) SEQ ID NO: 7 is substituted with another amino acid, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which one amino acid in (1) SEQ ID NO: 8 and (2) SEQ ID NO: 8 is substituted with another amino acid. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 7, and the second ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 8.

[0037] In one preferred embodiment, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 7 and (2) sequences in which up to three, up to two, or up to one amino acid in SEQ ID NO: 7 is substituted by other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 8 and (2) sequences in which up to three, up to two, or up to one amino acid in SEQ ID NO: 8 is substituted by other amino acids, and the first ankyrin repeat module is located at the N-terminus of the second ankyrin repeat module within the ankyrin repeat domain. Furthermore, in a more preferred embodiment, the first ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 7, and the second ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 8, and the first ankyrin repeat module is located at the N-terminus of the second ankyrin repeat module within the ankyrin repeat domain.

[0038] In another specific embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to nine, or up to eight, or up to seven, or up to six, or up to five, or up to four, or up to three, or up to two, or up to one amino acid of (1) SEQ ID NO: 7 and (2) SEQ ID NO: 7 are substituted by other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to nine, or up to eight, or up to seven, or up to six, or up to five, or up to four, or up to three, or up to two, or up to one amino acid of (1) SEQ ID NO: 9 and (2) SEQ ID NO: 9 are substituted by other amino acids.

[0039] Accordingly, in one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to nine amino acids of (1) SEQ ID NO: 7 and (2) SEQ ID NO: 7 are substituted with other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to nine amino acids of (1) SEQ ID NO: 9 and (2) SEQ ID NO: 9 are substituted with other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to six amino acids of (1) SEQ ID NO: 7 and (2) SEQ ID NO: 7 are substituted with other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to six amino acids of (1) SEQ ID NO: 9 and (2) SEQ ID NO: 9 are substituted with other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to five amino acids of (1) SEQ ID NO: 7 and (2) SEQ ID NO: 7 are substituted with other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to five amino acids of (1) SEQ ID NO: 9 and (2) SEQ ID NO: 9 are substituted with other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to four amino acids of (1) SEQ ID NO: 7 and (2) SEQ ID NO: 7 are substituted with other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to four amino acids of (1) SEQ ID NO: 9 and (2) SEQ ID NO: 9 are substituted with other amino acids.In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to three amino acids of (1) SEQ ID NO: 7 and (2) SEQ ID NO: 7 are substituted with other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to three amino acids of (1) SEQ ID NO: 9 and (2) SEQ ID NO: 9 are substituted with other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to two amino acids of (1) SEQ ID NO: 7 and (2) SEQ ID NO: 7 are substituted with other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of sequences in which up to two amino acids of (1) SEQ ID NO: 9 and (2) SEQ ID NO: 9 are substituted with other amino acids. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) Sequence ID No. 7 and (2) sequences in which one amino acid in Sequence ID No. 7 is substituted with another amino acid, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) Sequence ID No. 9 and (2) sequences in which one amino acid in Sequence ID No. 9 is substituted with another amino acid. In one embodiment, in such an ankyrin repeat domain, the first ankyrin repeat module includes the amino acid sequence of Sequence ID No. 7, and the second ankyrin repeat module includes the amino acid sequence of Sequence ID No. 9.

[0040] In a preferred embodiment, the first ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 7 and (2) sequences in which up to three, up to two, or up to one amino acid of SEQ ID NO: 7 is substituted by other amino acids, and the second ankyrin repeat module includes an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 9 and (2) sequences in which up to three, up to two, or up to one amino acid of SEQ ID NO: 9 is substituted by other amino acids, and the first ankyrin repeat module is located at the N-terminus of the second ankyrin repeat module within the ankyrin repeat domain. Furthermore, in a more preferred embodiment, the first ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 7, and the second ankyrin repeat module includes the amino acid sequence of SEQ ID NO: 9, and the first ankyrin repeat module is located at the N-terminus of the second ankyrin repeat module within the ankyrin repeat domain.

[0041] In a preferred embodiment, all such amino acid substitutions of the ankyrin repeat module as described and referenced herein occur at the framework position of the ankyrin repeat module, and typically the overall structure of the module is not affected by the substitutions. Embodiments of such substitutions at the framework position shall apply to all embodiments, regardless of whether such substitutions are explicitly described.

[0042] In one preferred embodiment, all of the amino acid substitutions of the ankyrin repeat module as described and referenced herein occur at the framework positions of the ankyrin repeat module of SEQ ID NOs. 5-9 and at positions other than 3, 4, 6, 14, and 15, preferably at positions other than 2, 3, 4, 5, 6, 14, and 15, and typically the overall structure of the module is unaffected by the substitutions.

[0043] In another embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to CD3, and the ankyrin repeat domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% amino acid sequence identity with any one of SEQ ID NOs: 1 to 4, wherein the second-to-last A in SEQ ID NOs: 1 to 4 is optionally substituted with L, and / or the last A in SEQ ID NOs: 1 to 4 is optionally substituted with N.

[0044] Therefore, in one embodiment, the ankyrin repeat domain includes an amino acid sequence having at least 80% amino acid sequence identity with any one of SEQ ID NOs: 1 to 4. In another embodiment, the ankyrin repeat domain includes an amino acid sequence having at least 90% amino acid sequence identity with any one of SEQ ID NOs: 1 to 4. In yet another embodiment, the ankyrin repeat domain includes an amino acid sequence having at least 93% amino acid sequence identity with any one of SEQ ID NOs: 1 to 4, and the ankyrin repeat domain includes an amino acid sequence having at least 95% amino acid sequence identity with any one of SEQ ID NOs: 1 to 4. In yet another embodiment, the ankyrin repeat domain includes an amino acid sequence having at least 98% amino acid sequence identity with any one of SEQ ID NOs: 1 to 4, and the ankyrin repeat domain includes the amino acid sequence of any one of SEQ ID NOs: 1 to 4. Therefore, in one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to CD3, the ankyrin repeat domain comprising an amino acid sequence selected from SEQ ID NOs: 1-4, wherein the second-to-last A in SEQ ID NOs: 1-4 is optionally substituted with L, and / or the last A in SEQ ID NOs: 1-4 is optionally substituted with N.

[0045] In one embodiment, the ankyrin repeat domain includes an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO: 1, wherein the second-to-last A in SEQ ID NO: 1 is optionally substituted with L, and / or the last A in SEQ ID NO: 1 is optionally substituted with N. Thus, in one embodiment, the ankyrin repeat domain includes an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 1. In another embodiment, the ankyrin repeat domain includes an amino acid sequence having at least 90% amino acid sequence identity with SEQ ID NO: 1. In another embodiment, the ankyrin repeat domain includes an amino acid sequence having at least 93% amino acid sequence identity with SEQ ID NO: 1, and in a further embodiment, the ankyrin repeat domain includes an amino acid sequence having at least 95% amino acid sequence identity with SEQ ID NO: 1. In one embodiment, the ankyrin repeat domain includes an amino acid sequence having at least 98% amino acid sequence identity with SEQ ID NO: 1, and in one embodiment, the ankyrin repeat domain includes the amino acid sequence of SEQ ID NO: 1. Therefore, in one embodiment, the recombinant binding protein includes an ankyrin repeat domain that has binding specificity to CD3, and the ankyrin repeat domain includes the amino acid sequence of SEQ ID NO: 1.

[0046] In one embodiment, the ankyrin repeat domain includes an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO: 2, wherein the second-to-last A in SEQ ID NO: 2 is optionally substituted with L, and / or the last A in SEQ ID NO: 2 is optionally substituted with N. Thus, in one embodiment, the ankyrin repeat domain includes an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 2. In one embodiment, the ankyrin repeat domain includes an amino acid sequence having at least 90% amino acid sequence identity with SEQ ID NO: 2. In another embodiment, the ankyrin repeat domain includes an amino acid sequence having at least 93% amino acid sequence identity with SEQ ID NO: 2, and in a further embodiment, the ankyrin repeat domain includes an amino acid sequence having at least 95% amino acid sequence identity with SEQ ID NO: 2. In one embodiment, the ankyrin repeat domain includes an amino acid sequence having at least 98% amino acid sequence identity with SEQ ID NO: 2, and in one embodiment, the ankyrin repeat domain includes the amino acid sequence of SEQ ID NO: 2. Therefore, in one embodiment, the recombinant binding protein includes an ankyrin repeat domain that has binding specificity to CD3, and the ankyrin repeat domain includes the amino acid sequence of SEQ ID NO: 2.

[0047] In one embodiment, the ankyrin repeat domain includes an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO: 3, wherein the second-to-last A in SEQ ID NO: 3 is optionally substituted with L, and / or the last A in SEQ ID NO: 3 is optionally substituted with N. Thus, in one embodiment, the ankyrin repeat domain includes an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 3. In one embodiment, the ankyrin repeat domain includes an amino acid sequence having at least 90% amino acid sequence identity with SEQ ID NO: 3. In another embodiment, the ankyrin repeat domain includes an amino acid sequence having at least 93% amino acid sequence identity with SEQ ID NO: 3, and in a further embodiment, the ankyrin repeat domain includes an amino acid sequence having at least 95% amino acid sequence identity with SEQ ID NO: 3. In one embodiment, the ankyrin repeat domain includes an amino acid sequence having at least 98% amino acid sequence identity with SEQ ID NO: 3, and in one embodiment, the ankyrin repeat domain includes the amino acid sequence of SEQ ID NO: 3. Therefore, in one embodiment, the recombinant binding protein includes an ankyrin repeat domain that has binding specificity to CD3, and the ankyrin repeat domain includes the amino acid sequence of SEQ ID NO: 3.

[0048] In one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO: 4, wherein the second-to-last A in SEQ ID NO: 4 is optionally substituted with L, and / or the last A in SEQ ID NO: 4 is optionally substituted with N. Thus, in one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 4, the binding protein according to any one of E1 to E10. In one embodiment, the ankyrin repeat domain comprises an amino acid sequence having at least 90% amino acid sequence identity with SEQ ID NO: 4. In another embodiment, the ankyrin repeat domain includes an amino acid sequence having at least 93% amino acid sequence identity with SEQ ID NO: 4, and in a further embodiment, the ankyrin repeat domain includes an amino acid sequence having at least 95% amino acid sequence identity with SEQ ID NO: 4. In one embodiment, the ankyrin repeat domain includes an amino acid sequence having at least 98% amino acid sequence identity with SEQ ID NO: 4, and in one embodiment, the ankyrin repeat domain includes the amino acid sequence of SEQ ID NO: 4. Therefore, in one embodiment, the recombinant binding protein includes an ankyrin repeat domain that has binding specificity to CD3, and the ankyrin repeat domain includes the amino acid sequence of SEQ ID NO: 4.

[0049] In one embodiment, the recombinant binding protein includes an ankyrin repeat domain that has binding specificity to CD3, and the potential interaction residue of the ankyrin repeat domain is identical to the corresponding position in any one of the ankyrin repeat domains of SEQ ID NOs: 1-4.

[0050] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to CD3, and the binding protein has an EC of 900, 700, 500, 400, 300, 200, 150, 100, 70, 60, 50, 40, 30, 20, 15, or less than 10 nM. 50 It then binds to human CD3 on T cells. Therefore, in one embodiment, the binding protein has an EC of less than 900 nM. 50 In another embodiment, the binding protein has an EC of less than 700 nM. 50 In another embodiment, the binding protein has an EC of less than 500 nM. 50 In another embodiment, the binding protein has an EC of less than 400 nM. 50 In another embodiment, the binding protein has an EC of less than 300 nM; 50 In another embodiment, the binding protein has an EC of less than 200 nM. 50 In another embodiment, the binding protein has an EC of less than 100 nM. 50 In another embodiment, the binding protein has an EC of less than 70 nM. 50 In another embodiment, the binding protein has an EC of less than 60 nM. 50 In another embodiment, the binding protein has an EC of less than 50 nM. 50 In another embodiment, the binding protein has an EC of less than 40 nM. 50 In another embodiment, the binding protein has an EC of less than 30 nM. 50 It binds to T cells; in another embodiment, the binding protein has an EC of less than 20 nM 50 It binds to T cells; in another embodiment, the binding protein has an EC of less than 15 nM 50 It binds to T cells; in a further embodiment, the binding protein has an EC of less than 0 nM 50 It then binds to T cells.

[0051] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to CD3, and the binding protein has an EC in the range of 2 to 900 nM. 50 It binds to human CD3 on T cells. In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to CD3, and the binding protein has an EC in the range of about 5 to about 400 nM. 50 It binds to human CD3 on T cells. In a further embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to CD3, and the binding protein has an EC in the range of about 250 to about 400 nM. 50 In another embodiment, the binding protein binds to human CD3 on T cells; in another embodiment, the binding protein has an EC in the range of about 50 to about 100 nM. 50 In a further embodiment, the binding protein has an EC2 level in the range of about 5 to about 15 nM. 50 It then binds to human CD3 on T cells.

[0052] CD3 binding (EC) of the recombinant binding protein of the present invention, which has binding specificity to CD3, on T cells by using Mirrorball laser scanning imaging cytometry. 50 A typical and preferred determination of ) is described in Example 5 (using primary human T cells). Therefore, in one embodiment, the CD binding (EC) of the recombinant binding protein of the present invention is determined. 50 The ) is determined on primary human T cells by Mirrorball laser scanning imaging cytometry, as described in Example 5.

[0053] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to CD3, and the binding protein has an EC in the range of 2 to 900 nM. 50The binding protein binds to human CD3 on T cells, and the binding protein contains an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% amino acid sequence identity with any one of SEQ ID NOs: 1-4, wherein the second-to-last A in SEQ ID NOs: 1-4 is optionally substituted with L, and / or the last A in SEQ ID NOs: 1-4 is optionally substituted with N. Therefore, in one embodiment, the binding protein has an EC in the range of 2-900 nM. 50 The ankyrin repeat domain binds to human CD3 on T cells, and the ankyrin repeat domain contains an amino acid sequence having at least 80% amino acid sequence identity with any one of SEQ ID NOs: 1-4. In one embodiment, the binding protein has an EC in the range of 2-900 nM. 50 The ankyrin repeat domain binds to human CD3 on T cells, and the ankyrin repeat domain contains an amino acid sequence having at least 90% amino acid sequence identity with any one of SEQ ID NOs: 1-4. In another embodiment, the binding protein has an EC range of 2-900 nM. 50 The ankyrin repeat domain binds to human CD3 on T cells, and the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity with any one of SEQ ID NOs: 1-4. In a further embodiment, the binding protein has an EC2 range of 2-900 nM. 50 The ankyrin repeat domain binds to human CD3 on T cells, and the ankyrin repeat domain contains an amino acid sequence having at least 95% amino acid sequence identity with any one of SEQ ID NOs: 1-4. In one embodiment, the binding protein has an EC in the range of 2-900 nM. 50 The ankyrin repeat domain binds to human CD3 on T cells, and the ankyrin repeat domain comprises an amino acid sequence having at least 98% amino acid sequence identity with any one of SEQ ID NOs: 1-4. In one embodiment, the binding protein has an EC2 range of 2-900 nM. 50It binds to human CD3 on T cells, and the ankyrin repeat domain contains any one of the amino acid sequences of SEQ ID NO: 1-4. Therefore, in one embodiment, the recombinant binding protein contains an ankyrin repeat domain having binding specificity for CD3, and the binding protein has an EC in the range of 2-900 nM 50 It binds to human CD3 on T cells, and the ankyrin repeat domain contains any one of the amino acid sequences of SEQ ID NO: 1-4, wherein A at the second position from the end of SEQ ID NO: 1-4 is optionally replaced by L, and / or A at the last position of SEQ ID NO: 1-4 is optionally replaced by N.

[0054] In one embodiment, the recombinant binding protein contains an ankyrin repeat domain having binding specificity for CD3, and the binding protein has an EC in the range of about 250 to about 400 nM 50 It binds to human CD3 on T cells, and the binding protein contains an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with any one of SEQ ID NO: 2, wherein A at the second position from the end of SEQ ID NO: 2 is optionally replaced by L, and / or A at the last position of SEQ ID NO: 2 is optionally replaced by N. Therefore, in one embodiment, the binding protein has an EC in the range of 250-400 nM 50 It binds to human CD3 on T cells, and the ankyrin repeat domain contains an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 2. In one embodiment, the binding protein has an EC in the range of 250-400 nM 50 It binds to human CD3 on T cells, and the ankyrin repeat domain contains an amino acid sequence having at least 90% amino acid sequence identity with SEQ ID NO: 2. In another embodiment, the binding protein has an EC in the range of 250-400 nM 50The ankyrin repeat domain binds to human CD3 on T cells, and the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity with SEQ ID NO: 2. In further embodiments, the binding protein has an EC in the range of 250-400 nM. 50 The ankyrin repeat domain binds to human CD3 on T cells, and the ankyrin repeat domain contains an amino acid sequence having at least 95% amino acid sequence identity with SEQ ID NO: 2. In one embodiment, the binding protein has an EC in the range of 250-400 nM. 50 The ankyrin repeat domain binds to human CD3 on T cells, and the ankyrin repeat domain contains an amino acid sequence having at least 98% amino acid sequence identity with SEQ ID NO: 2. In one embodiment, the binding protein has an EC in the range of 250-400 nM. 50 The ankyrin repeat domain binds to human CD3 on T cells, and the ankyrin repeat domain contains the amino acid sequence of SEQ ID NO: 2. Therefore, in one embodiment, the recombinant binding protein contains an ankyrin repeat domain that has binding specificity to CD3, and the binding protein has an EC in the range of about 250-400 nM. 50 It binds to human CD3 on T cells, and the ankyrin repeat domain contains the amino acid sequence of SEQ ID NO: 2, wherein the second-to-last A in SEQ ID NO: 2 is optionally substituted with L, and / or the last A in SEQ ID NO: 2 is optionally substituted with N.

[0055] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to CD3, and the binding protein has an EC in the range of about 50 to about 100 nM. 50It binds to human CD3 on T cells, and the binding protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO: 3, wherein the A at the second - last position of SEQ ID NO: 3 is optionally substituted by L, and / or the A at the last position of SEQ ID NO: 3 is optionally substituted by N. Thus, in one embodiment, the binding protein has an EC in the range of 50 - 100 nM 50 It binds to human CD3 on T cells, and the ankyrin - repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 3. In one embodiment, the binding protein has an EC in the range of 50 - 100 nM 50 It binds to human CD3 on T cells, and the ankyrin - repeat domain comprises an amino acid sequence having at least 90% amino acid sequence identity with SEQ ID NO: 3. In another embodiment, the binding protein has an EC in the range of 50 - 100 nM 50 It binds to human CD3 on T cells, and the ankyrin - repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity with SEQ ID NO: 3. In a further embodiment, the binding protein has an EC in the range of 50 - 100 nM 50 It binds to human CD3 on T cells, and the ankyrin - repeat domain comprises an amino acid sequence having at least 95% amino acid sequence identity with SEQ ID NO: 3. In one embodiment, the binding protein has an EC in the range of 50 - 100 nM 50 It binds to human CD3 on T cells, and the ankyrin - repeat domain comprises an amino acid sequence having at least 98% amino acid sequence identity with SEQ ID NO: 3. In one embodiment, the binding protein has an EC in the range of 50 - 100 nM 50 It binds to human CD3 on T cells, and the ankyrin - repeat domain comprises the amino acid sequence of SEQ ID NO: 3. Thus, in one embodiment, the recombinant binding protein comprises an ankyrin - repeat domain having binding specificity for CD3, and the binding protein has an EC in the range of about 50 - about 100 nM 50It binds to human CD3 on T cells, and the ankyrin repeat domain contains the amino acid sequence of SEQ ID NO: 3, wherein the second-to-last A in SEQ ID NO: 3 is optionally substituted with L, and / or the last A in SEQ ID NO: 3 is optionally substituted with N.

[0056] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to CD3, and the binding protein has an EC in the range of about 5 to about 15 nM. 50 The binding protein binds to human CD3 on T cells, and the binding protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO: 4, wherein the second-to-last A in SEQ ID NO: 4 is optionally substituted with L, and / or the last A in SEQ ID NO: 4 is optionally substituted with N. Thus, in one embodiment, the binding protein has an EC in the range of 5-15 nM. 50 The ankyrin repeat domain binds to human CD3 on T cells, and the ankyrin repeat domain contains an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 4. In one embodiment, the binding protein has an EC in the range of 5-15 nM. 50 The ankyrin repeat domain binds to human CD3 on T cells, and the ankyrin repeat domain contains an amino acid sequence having at least 90% amino acid sequence identity with SEQ ID NO: 4. In another embodiment, the binding protein has an EC in the range of 5-15 nM. 50 The ankyrin repeat domain binds to human CD3 on T cells, and the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity with SEQ ID NO: 4, and in further embodiments, the binding protein has an EC in the range of 5-15 nM. 50 The ankyrin repeat domain binds to human CD3 on T cells, and the ankyrin repeat domain contains an amino acid sequence having at least 95% amino acid sequence identity with SEQ ID NO: 4. In one embodiment, the binding protein has an EC in the range of 5-15 nM. 50The ankyrin repeat domain binds to human CD3 on T cells, and the ankyrin repeat domain contains an amino acid sequence having at least 98% amino acid sequence identity with SEQ ID NO: 4. In one embodiment, the binding protein has an EC ratio in the range of 5-15 nM. 50 The ankyrin repeat domain binds to human CD3 on T cells, and the ankyrin repeat domain contains the amino acid sequence of SEQ ID NO: 4. Therefore, in one embodiment, the recombinant binding protein contains an ankyrin repeat domain that has binding specificity to CD3, and the binding protein has an EC in the range of about 5 to about 15 nM. 50 It binds to human CD33 on T cells, and the ankyrin repeat domain contains the amino acid sequence of SEQ ID NO: 4, wherein the second-to-last A in SEQ ID NO: 4 is optionally substituted with L, and / or the last A in SEQ ID NO: 4 is optionally substituted with N.

[0057] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to CD3, and the ankyrin repeat domain is 10 -6 Less than M, or 10 -7 Less than M, or 5 × 10 -8 Less than M, or 2 × 10 -8 Less than M, or 10 -8 Dissociation constant less than M (K D ) binds to human CD3 in PBS. Therefore, in one embodiment, the binding protein is approximately 10 -6 Dissociation constant less than M (K D ) binds to human CD3 in PBS. In another embodiment, the polypeptide is about 10 -7 Dissociation constant less than M (K D ) binds to human CD3 in PBS. In another embodiment, the ankyrin repeat domain is 5x10 -8 Dissociation constant less than M (K D ) binds to human CD3 in PBS, and in a further embodiment, the binding is 2 × 10 -8 Dissociation constant less than M (K D ) binds to human CD3 in PBS. In another embodiment, the binding protein is approximately 10 -8 Dissociation constant less than M (KD It binds to human CD3 in PBS.

[0058] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to CD3, and the ankyrin repeat domain is 10 -6 Dissociation constant less than M (K D ) binds to human CD3 in PBS, and the ankyrin repeat domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% amino acid sequence identity with any one of SEQ ID NOs: 1-4, wherein the second-to-last A in SEQ ID NOs: 1-4 is optionally substituted with L, and / or the last A in SEQ ID NOs: 1-4 is optionally substituted with N. In another embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to CD3, wherein the ankyrin repeat domain comprises 10 -7 Dissociation constant less than M (K D ) binds to CD3 in PBS, and the ankyrin repeat domain contains an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% amino acid sequence identity with any one of SEQ ID NOs: 1-4, wherein the second-to-last A in SEQ ID NOs: 1-4 is optionally substituted with L, and / or the last A in SEQ ID NOs: 1-4 is optionally substituted with N. Therefore, in one embodiment, the binding protein is 10 -7 Dissociation constant less than M (K D ) binds to CD3 in PBS, and the ankyrin repeat domain contains an amino acid sequence having at least 80% amino acid sequence identity with any one of SEQ ID NOs: 1 to 4. In one embodiment, the binding protein is 10 -7 Dissociation constant less than M (K D) binds to CD3 in PBS, and the ankyrin repeat domain contains an amino acid sequence having at least 90% amino acid sequence identity with any one of SEQ ID NOs: 1-4. In another embodiment, the binding protein is 10 -7 Dissociation constant less than M (K D ) binds to CD3 in PBS, and the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity with any one of SEQ ID NOs: 1 to 4, and in a further embodiment, the binding protein is 10 -7 Dissociation constant less than M (K D ) binds to CD3 in PBS, and the ankyrin repeat domain contains an amino acid sequence having at least 95% amino acid sequence identity with any one of SEQ ID NOs: 1 to 4. In one embodiment, the binding protein is 10 -7 Dissociation constant less than M (K D ) binds to CD3 in PBS, and the ankyrin repeat domain contains an amino acid sequence having at least 98% amino acid sequence identity with any one of SEQ ID NOs: 1 to 4, and in one embodiment, the binding protein is 10 -7 Dissociation constant less than M (K D ) binds to CD3 in PBS, and the ankyrin repeat domain contains one of the amino acid sequences of SEQ ID NOs: 1 to 4. Therefore, in one embodiment, the recombinant binding protein contains an ankyrin repeat domain that has binding specificity to CD3, and the ankyrin repeat domain is approximately 10 -7 M or a dissociation constant less than (K) D ) binds to human CD3 in PBS, and the ankyrin repeat domain contains one of the amino acid sequences of SEQ ID NOs: 1-4, wherein the second-to-last A in SEQ ID NOs: 1-4 is optionally substituted with L, and / or the last A in SEQ ID NOs: 1-4 is optionally substituted with N.

[0059] In one embodiment, the recombinant binding protein includes an ankyrin repeat domain that has binding specificity to CD3, and the ankyrin repeat domain is approximately 5 × 10 -8 M or a dissociation constant less than (K) D) binds to human CD3 in PBS, and the ankyrin repeat domain contains an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% amino acid sequence identity with any one of SEQ ID NOs: 1-4, wherein the second-to-last A in SEQ ID NOs: 1-4 is optionally substituted with L, and / or the last A in SEQ ID NOs: 1-4 is optionally substituted with N. Thus, in one embodiment, the binding protein has approximately 5 × 10⁻¹⁶ -8 M or a dissociation constant less than (K) D The ankyrin repeat domain binds to human CD3 in PBS, and the ankyrin repeat domain contains an amino acid sequence having at least 80% amino acid sequence identity with any one of SEQ ID NOs: 1 to 4. In one embodiment, the binding protein is approximately 5 × 10⁻¹⁴ -8 Dissociation constant less than M (K D ) binds to human CD3 in PBS, and the ankyrin repeat domain contains an amino acid sequence having at least 90% amino acid sequence identity with any one of SEQ ID NOs: 1-4. In another embodiment, the binding protein is approximately 5 × 10 -8 Dissociation constant less than M (K D ) binds to human CD3 in PBS, and the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity with any one of SEQ ID NOs: 1-4, and in a further embodiment, the binding protein comprises approximately 5 × 10 -8 Dissociation constant less than M (K D ) binds to human CD3 in PBS, and the ankyrin repeat domain contains an amino acid sequence having at least 95% amino acid sequence identity with any one of SEQ ID NOs: 1 to 4. In one embodiment, the binding protein is approximately 5 × 10 -8 The ankyrin repeat domain binds to human CD3 in PBS with a dissociation constant (KD) of less than M, and the ankyrin repeat domain contains an amino acid sequence having at least 98% amino acid sequence identity with one of sequence numbers 1-4, and in one embodiment, the binding protein is approximately 5 × 10⁻¹⁶ -8The ankyrin repeat domain binds to human CD3 in PBS with a dissociation constant (KD) of less than M, and the ankyrin repeat domain contains one of the amino acid sequences of SEQ ID NOs: 1 to 4. Therefore, in one embodiment, the recombinant binding protein contains an ankyrin repeat domain that has binding specificity to CD3, and the ankyrin repeat domain is approximately 5 × 10⁻¹⁶. -8 M or a dissociation constant less than (K) D ) binds to human CD3 in PBS, and the ankyrin repeat domain contains one of the amino acid sequences of SEQ ID NOs: 1-4, wherein the second-to-last A in SEQ ID NOs: 1-4 is optionally substituted with L, and / or the last A in SEQ ID NOs: 1-4 is optionally substituted with N.

[0060] In one embodiment, the recombinant binding protein includes an ankyrin repeat domain that has binding specificity to CD3, and the ankyrin repeat domain is approximately 2 × 10⁻⁶ -8 M or a dissociation constant less than (K) D ) binds to human CD3 in PBS, and the ankyrin repeat domain contains an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, and 100% amino acid sequence identity with either SEQ ID NOs: 3 or 4, wherein the second-to-last A in SEQ ID NOs: 1-4 is optionally substituted with L, and / or the last A in SEQ ID NOs: 3 or 4 is optionally substituted with N. Thus, in one embodiment, the binding protein is approximately 2 × 10⁻⁶ -8 M or a dissociation constant less than (K) D The ankyrin repeat domain binds to human CD3 in PBS, and the ankyrin repeat domain contains an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 3 or 4. In one embodiment, the binding protein is approximately 2 × 10⁻¹⁶ -8 Dissociation constant less than M (K D) binds to human CD3 in PBS, and the ankyrin repeat domain contains an amino acid sequence having at least 90% amino acid sequence identity with SEQ ID NO: 3 or 4. In another embodiment, the binding protein has a dissociation constant (K) less than about 2 × 10⁻⁸ M. D ) binds to human CD3 in PBS, and the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity with either SEQ ID NO: 3 or 4, and in a further embodiment, the binding protein comprises approximately 2 × 10 -8 Dissociation constant less than M (K D ) binds to human CD3 in PBS, and the ankyrin repeat domain contains an amino acid sequence having at least 95% amino acid sequence identity with SEQ ID NO: 3 or 4. In one embodiment, the binding protein is approximately 2 × 10 -8 Dissociation constant less than M (K D ) binds to human CD3 in PBS, and the ankyrin repeat domain contains an amino acid sequence having at least 98% amino acid sequence identity with SEQ ID NO: 3 or 4, and in one embodiment, the binding protein is approximately 2 × 10 -8 Dissociation constant less than M (K D ) binds to human CD3 in PBS, and the ankyrin repeat domain contains the amino acid sequence of SEQ ID NO: 3 or 4. Therefore, in one embodiment, the recombinant binding protein contains an ankyrin repeat domain that has binding specificity to CD3, and the binding protein is approximately 2 × 10⁻⁶ -8 M or a dissociation constant less than (K) D ) binds to human CD3 in PBS, and the ankyrin repeat domain comprises the amino acid sequence of SEQ ID NO: 3 or 4, wherein the second-to-last A in SEQ ID NO: 3 or 4 is optionally substituted with L, and / or the last A in SEQ ID NO: 3 or 4 is optionally substituted with N.

[0061] In one embodiment, the recombinant binding protein comprises an ankyrin repeat domain having binding specificity to CD3, and the ankyrin repeat domain comprises about 10 -8 M or a dissociation constant less than (K) D) binds to human CD3 in PBS, and the ankyrin repeat domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO: 4, wherein the second-to-last A in SEQ ID NO: 4 is optionally substituted with L, and / or the last A in SEQ ID NO: 4 is optionally substituted with N. Thus, in one embodiment, the binding protein is approximately 10 -8 M or a dissociation constant less than (K) D ) binds to human CD3 in PBS, and the ankyrin repeat domain contains an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 4. In one embodiment, the binding protein is 10 -8 Dissociation constant less than M (K D ) binds to human CD3 in PBS, and the ankyrin repeat domain contains an amino acid sequence having at least 90% amino acid sequence identity with SEQ ID NO: 4. In another embodiment, the binding protein is approximately 10 -8 Dissociation constant less than M (K D ) binds to human CD3 in PBS, and the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity with SEQ ID NO: 4, and in further embodiments, the binding protein comprises about 10 -8 Dissociation constant less than M (K D ) binds to human CD3 in PBS, and the ankyrin repeat domain contains an amino acid sequence having at least 95% amino acid sequence identity with SEQ ID NO: 4. In one embodiment, the binding protein is about 10 -8 Dissociation constant less than M (K D ) binds to human CD3 in PBS, and the ankyrin repeat domain contains an amino acid sequence having at least 98% amino acid sequence identity with SEQ ID NO: 4, and in one embodiment, the binding protein is about 10 -8 Dissociation constant less than M (K D) binds to human CD3 in PBS, and the ankyrin repeat domain contains the amino acid sequence of SEQ ID NO: 4. Therefore, in one embodiment, the recombinant binding protein contains an ankyrin repeat domain that has binding specificity to CD3, and the binding protein is approximately 10 -8 M or a dissociation constant less than (K) D ) binds to human CD3 in PBS, and the ankyrin repeat domain contains the amino acid sequence of SEQ ID NO: 4, wherein the second-to-last A in SEQ ID NO: 4 is optionally substituted with L, and / or the last A in SEQ ID NO: 4 is optionally substituted with N.

[0062] The dissociation constant (K) of the recombinant binding protein of the present invention having binding specificity to CD3 D A typical and preferred determination of the binding specificity of the recombinant binding protein of the present invention to CD3 by surface plasmon resonance (SPR) analysis is described in Example 4. Thus, in one embodiment, the binding specificity of the recombinant binding protein of the present invention to CD3 is determined in PBS by surface plasmon resonance (SPR), as described in Example 4.

[0063] In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain having binding specificity to CD3, wherein the ankyrin repeat domain has a melting temperature (T) of 65°C, 68°C, 70°C, 72°C, 75°C, 78°C, 80°C, approximately 75°C, approximately 80°C, approximately 82°C, approximately 85°C, 65°C to 95°C, 70°C to 90°C, or 72°C to 88°C. m ) has. Therefore, in one embodiment, the ankyrin repeat domain has a melting temperature (T) higher than 65°C. m ) has. In another embodiment, the ankyrin repeat domain has a melting temperature (T) higher than 70°C. m ) has. In another embodiment, the ankyrin repeat domain has a melting temperature (T) higher than 75°C. m) has. In another embodiment, the ankyrin repeat domain has a melting temperature (T) higher than 80°C. m ) has. In another embodiment, the ankyrin repeat domain has a melting temperature of 65°C to 95°C (T m ) has. In another embodiment, the ankyrin repeat domain has a melting temperature of 72°C to 88°C (T m ) has. In one embodiment, the recombinant binding protein of the present invention comprises an ankyrin repeat domain having binding specificity to CD3, wherein the ankyrin repeat domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with any one of SEQ ID NOs: 1 to 4, wherein the second to last A in SEQ ID NOs: 1 to 4 is optionally substituted with L, and / or the last A in SEQ ID NOs: 1 to 4 is optionally substituted with N, wherein the ankyrin repeat domain has a melting temperature higher than 65°C (T m ) has. In one embodiment, the ankyrin repeat domain has a melting temperature (T) higher than 68°C. m ) has. In another embodiment, the ankyrin repeat domain has a melting temperature (T) higher than 70°C. m ) has. In another embodiment, the ankyrin repeat domain has a melting temperature (T) higher than 72°C. m ) has. In another embodiment, the ankyrin repeat domain has a melting temperature (T) higher than 75°C. m ) has. In another embodiment, the ankyrin repeat domain has a melting temperature (T) higher than 78°C. m ) has. In further embodiments, the ankyrin repeat domain has a melting temperature (T) higher than 80°C. m) has. In one embodiment, the recombinant binding protein of the present invention comprises an ankyrin repeat domain having binding specificity to CD3, wherein the ankyrin repeat domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO: 1, wherein the second-to-last A in SEQ ID NO: 1 is optionally substituted with L, and / or the last A in SEQ ID NO: 1 is optionally substituted with N, wherein the ankyrin repeat domain has a melting temperature higher than 75°C (T m ) has. In one embodiment, the ankyrin repeat domain has a melting temperature (T) higher than 78°C. m ) has. In one embodiment, the ankyrin repeat domain has a melting temperature (T) higher than 80°C. m ) has. In one embodiment, the ankyrin repeat domain has a melting temperature of 75°C to 95°C (T m ) has. In one embodiment, the ankyrin repeat domain has a melting temperature of about 85°C (T m ) has.

[0064] In one embodiment, the recombinant binding protein of the present invention comprises an ankyrin repeat domain having binding specificity to CD3, wherein the ankyrin repeat domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO: 2, wherein the second-to-last A in SEQ ID NO: 2 is optionally substituted with L, and / or the last A in SEQ ID NO: 2 is optionally substituted with N, and the ankyrin repeat domain has a melting temperature higher than 72°C (T m ) has. In one embodiment, the ankyrin repeat domain has a melting temperature (T) higher than 75°C. m ) has. In one embodiment, the ankyrin repeat domain has a melting temperature (T) higher than 77°C. m) has. In one embodiment, the ankyrin repeat domain has a melting temperature of 75°C to 92°C (T m ) has. In one embodiment, the ankyrin repeat domain has a melting temperature of about 82°C (T m ) has.

[0065] In one embodiment, the recombinant binding protein of the present invention comprises an ankyrin repeat domain having binding specificity to CD3, wherein the ankyrin repeat domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO: 3, wherein the second-to-last A in SEQ ID NO: 3 is optionally substituted with L, and / or the last A in SEQ ID NO: 3 is optionally substituted with N, and the ankyrin repeat domain has a melting temperature higher than 65°C (T m ) has. In one embodiment, the ankyrin repeat domain has a melting temperature (T) higher than 68°C. m ) has. In one embodiment, the ankyrin repeat domain has a melting temperature (T) higher than 70°C. m ) has. In one embodiment, the ankyrin repeat domain has a melting temperature of 65°C to 85°C (T m ) has. In one embodiment, the ankyrin repeat domain has a melting temperature of about 75°C (T m ) has.

[0066] In one embodiment, the recombinant binding protein of the present invention comprises an ankyrin repeat domain having binding specificity to CD3, wherein the ankyrin repeat domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO: 4, wherein the second-to-last A in SEQ ID NO: 4 is optionally substituted with L, and / or the last A in SEQ ID NO: 4 is optionally substituted with N, and the ankyrin repeat domain has a melting temperature higher than 73°C (T m ) has. In one embodiment, the ankyrin repeat domain has a melting temperature (T) higher than 75°C. m ) has. In one embodiment, the ankyrin repeat domain has a melting temperature (T) higher than 78°C. m ) has. In one embodiment, the ankyrin repeat domain has a melting temperature of 73°C to 93°C (T m ) has. In one embodiment, the ankyrin repeat domain has a melting temperature of about 83°C (T m ) has.

[0067] The melting temperature (T) of the recombinant binding protein or ankyrin repeat domain of the present invention having binding specificity to CD3, as determined by circular dichroism (CD) spectroscopy. m A typical and preferred determination of the melting temperature (T) of the recombinant binding protein or ankyrin repeat domain of the present invention is described in Example 2. Therefore, in one embodiment, the melting temperature (T) of the recombinant binding protein or ankyrin repeat domain of the present invention is determined. m The melting temperature (T) of the recombinant binding protein or ankyrin repeat domain of the present invention is determined by circular dichroism (CD) spectroscopy in TBS pH 8.0 (50 mM Tris, 500 mM NaCl). In one embodiment, the melting temperature (T) of the recombinant binding protein or ankyrin repeat domain of the present invention is determined by circular dichroism (CD) spectroscopy. m The ) is determined by circular dichroism (CD) spectroscopy in TBS pH 8.0 (50 mM Tris, 500 mM NaCl), as described in Example 2.

[0068] In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain having binding specificity to CD3, wherein the ankyrin repeat domain has an aggregation onset temperature (T) of 65°C, 68°C, 70°C, 72°C, 75°C, 78°C, 80°C, 82°C, 85°C, 75°C to 100°C, 70°C to 95°C, or 75°C to 95°C. agg ) has. Therefore, in one embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) higher than 65°C. agg ) has. In another embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) higher than 70°C. agg ) has. In another embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) higher than 75°C. agg ) has. In another embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) higher than 80°C. agg ) has. In another embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) of 75°C to 100°C. agg ) has. In another embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) of 70°C to 95°C. agg ) has.

[0069] In one embodiment, the recombinant binding protein of the present invention comprises an ankyrin repeat domain having binding specificity to CD3, wherein the ankyrin repeat domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with any one of SEQ ID NOs: 1-4, wherein the second-to-last A in SEQ ID NOs: 1-4 is optionally substituted with L, and / or the last A in SEQ ID NOs: 1-4 is optionally substituted with N, wherein the ankyrin repeat domain has an aggregation onset temperature (T) higher than 65°C. agg ) has. In one embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) higher than 68°C. agg) has. In another embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) higher than 70°C. agg ) has. In another embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) higher than 72°C. agg ) has. In another embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) higher than 75°C. agg ) has. In another embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) higher than 78°C. agg ) has. In another embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) higher than 80°C. agg ) has.

[0070] In one embodiment, the recombinant binding protein of the present invention comprises an ankyrin repeat domain having binding specificity to CD3, wherein the ankyrin repeat domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO: 1, wherein the second-to-last A in SEQ ID NO: 1 is optionally substituted with L, and / or the last A in SEQ ID NO: 1 is optionally substituted with N, wherein the ankyrin repeat domain has an aggregation onset temperature (T) higher than 70°C. agg ) has. In one embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) higher than 75°C. agg ) has. In one embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) higher than 80°C. agg ) has. In one embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) higher than 85°C. agg ) has. In one embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) of 75°C to 100°C. agg ) has.

[0071] In one embodiment, the recombinant binding protein of the present invention comprises an ankyrin repeat domain having binding specificity to CD3, wherein the ankyrin repeat domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO: 2, wherein the second-to-last A in SEQ ID NO: 2 is optionally substituted with L, and / or the last A in SEQ ID NO: 2 is optionally substituted with N, wherein the ankyrin repeat domain has an aggregation onset temperature (T) higher than 65°C. agg ) has. In one embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) higher than 70°C. agg ) has. In one embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) higher than 75°C. agg ) has. In one embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) higher than 80°C. agg ) has. In one embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) of 70°C to 95°C. agg ) has.

[0072] In one embodiment, the recombinant binding protein of the present invention comprises an ankyrin repeat domain having binding specificity to CD3, wherein the ankyrin repeat domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO: 3, wherein the second-to-last A in SEQ ID NO: 3 is optionally substituted with L, and / or the last A in SEQ ID NO: 3 is optionally substituted with N, and the ankyrin repeat domain has an aggregation onset temperature (T) higher than 65°C. agg ) has. In one embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) higher than 70°C. agg ) has. In one embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) higher than 75°C.agg ) has. In one embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) higher than 80°C. agg ) has. In one embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) of 70°C to 95°C. agg ) has.

[0073] In one embodiment, the recombinant binding protein of the present invention comprises an ankyrin repeat domain having binding specificity to CD3, wherein the ankyrin repeat domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO: 4, wherein the second-to-last A in SEQ ID NO: 4 is optionally substituted with L, and / or the last A in SEQ ID NO: 4 is optionally substituted with N, and the ankyrin repeat domain has an aggregation onset temperature (T) higher than 65°C. agg ) has. In one embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) higher than 70°C. agg ) has. In one embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) higher than 75°C. agg ) has. In one embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) higher than 80°C. agg ) has. In one embodiment, the ankyrin repeat domain has an aggregation onset temperature (T) of 70°C to 95°C. agg ) has.

[0074] Dynamic light scattering (DLS) analysis determined the aggregation initiation temperature (T) of the recombinant binding protein or ankyrin repeat domain of the present invention that has binding specificity to CD3. agg A typical and preferred determination of the condensation temperature (T) of the recombinant binding protein or ankyrin repeat domain of the present invention is described in Example 2. Thus, in one embodiment, the condensation temperature (T) of the recombinant binding protein or ankyrin repeat domain of the present invention is determined. mThe aggregation onset temperature (T) of the recombinant binding protein or ankyrin repeat domain of the present invention is determined by dynamic light scattering (DLS) analysis in TBS pH 8.0 (50 mM Tris, 500 mM NaCl). In one embodiment, the aggregation onset temperature (T) of the recombinant binding protein or ankyrin repeat domain of the present invention is determined. agg The ) is determined by dynamic light scattering (DLS) analysis in TBS pH 8.0 (50 mM Tris, 500 mM NaCl), as described in Example 2.

[0075] The repeat domains of recombinant binding proteins disclosed herein, preferably ankyrin repeat domains, preferably include an N-terminal and / or C-terminal capping module (hereinafter also referred to as capping repeat or capping unit). The capping module is located at the N-terminal and / or C-terminal of the ankyrin repeat domain and typically forms a close tertiary interaction (i.e., a tertiary structural interaction) with the ankyrin repeat module, thereby providing a cap that laterally shields the hydrophobic core of the ankyrin repeat domain from exposure to solvents. The N-terminal and / or C-terminal capping module may originate from a capping unit or other structural units found in naturally occurring repeat proteins adjacent to the repeat unit. Examples of capping sequences are described in International Publications 2002 / 020565 and 2012 / 069655, U.S. Patent Application Publication 20130296221, and Interlandi et al., J Mol Biol. 2008 Jan 18;375(3):837-54. Examples of N-terminal capping modules (i.e., N-terminal capping iterations) are sequence numbers 11-16, and examples of C-terminal capping modules (i.e., C-terminal capping iterations) are sequence numbers 17-26.

[0076] In an exemplary embodiment, the N-terminal capping module includes one of the amino acid sequences of SEQ ID NOs: 10-15, where up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in one of SEQ ID NOs: 10-15 may be replaced by any amino acid.

[0077] In an exemplary embodiment, the C-terminal capping module comprises one amino acid sequence from SEQ ID NOs. 17 to 25, where up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in one of SEQ ID NOs. 17 to 25 may be replaced by any amino acid.

[0078] Advantageously, in some embodiments, it has been found that by altering certain amino acid residues in the N-terminal capping module and / or C-terminal capping module of the designed ankyrin repeat domain herein, improvements in pharmacokinetic properties, including an extended terminal phase half-life of the designed ankyrin repeat domain and the recombinant binding protein containing the designed ankyrin repeat domain, can be obtained. The altered amino acid residues are mostly surface-exposed residues. Preferably, the altered amino acid residues are the amino acid residues at positions 8 and 15 of the N-terminal capping module, where the amino acid at position 8 is Q and the amino acid at position 15 is L, with position numbers corresponding to the positions in SEQ ID NO: 17, and the amino acid residues at positions 14 and 18 of the C-terminal capping module, where the amino acid at position 14 is R and the amino acid at position 18 is Q, with position numbers corresponding to the positions in SEQ ID NO: 17.

[0079] For example, an N-terminal capping module having altered amino acid residues can include the following sequence: DLGxxLLQAAxxGQLDxVRxLxxxGADVNA (SEQ ID NO: 16), where "x" represents any amino acid.

[0080] For example, a C-terminal capping module with altered amino acid residues can include the following sequence: xDxxGxTPADxAARxGHQxIAxVLQxAA (SEQ ID NO: 26), where "x" represents any amino acid.

[0081] Therefore, in one embodiment, the ankyrin repeat domain having binding specificity to CD3 of the present invention includes an N-terminal capping module having the amino acid sequence of SEQ ID NO: 16, where "x" represents any amino acid. Alternatively or additionally, the ankyrin repeat domain having binding specificity to CD3 of the present invention may include a C-terminal capping module having the amino acid sequence of SEQ ID NO: 26, where "x" represents any amino acid.

[0082] In certain embodiments, the ankyrin repeat domain having CD3 binding specificity in the present invention comprises one of the amino acid sequences of SEQ ID NOs: 1 to 4, wherein amino acid E at position 8 of any one of SEQ ID NOs: 1 to 4 is substituted with Q, and amino acid D at position 15 of any one of SEQ ID NOs: 1 to 3 is substituted with L. Alternatively or additionally, the ankyrin repeat domain having CD3 binding specificity in the present invention comprises one of the amino acid sequences of SEQ ID NOs: 1 to 4, wherein amino acid K at position 110 of any one of SEQ ID NOs: 1 to 4 is substituted with R, and amino acid E at position 114 of SEQ ID NOs: 1 and 2, or amino acid R of SEQ ID NO: 4, is substituted with Q.

[0083] Furthermore, the CD3-binding domain of the present invention may optionally further include a "G", "S", or "GS" sequence at its N-terminus. Thus, in some embodiments, the CD3-binding domain provided herein comprises (i) an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 1-4, and further comprises G, S, or GS at its N-terminus. In an exemplary embodiment, the CD3-binding domain comprises an amino acid sequence that is at least one of SEQ ID NOs: 1-4, and further comprises G, S, or GS at its N-terminus. In exemplary embodiments, the CD3-binding domain comprises one of the amino acid sequences of SEQ ID NOs: 1-4, and further comprises G, S, or GS at its N-terminus.

[0084] In one embodiment, the binding protein of the present invention further comprises a binder having binding specificity to a disease-related antigen.

[0085] Accordingly, in a particular embodiment, the recombinant binding protein of the present invention comprises an ankyrin repeat domain having binding specificity to CD3, as more specifically described in any aspect and embodiment of this specification, and further comprises a binder having binding specificity to a disease-related antigen. In a preferred embodiment, the binder having binding specificity to a disease-related antigen is located at the N-terminus of the ankyrin repeat domain having binding specificity to CD3 within the binding protein.

[0086] In one embodiment, the disease-associated antigen is a tumor-associated antigen (TAA). Exemplary examples of tumor-associated antigens include, but are not limited to, proteins expressed on the cell surface of tumor cells, such as HER2 (also referred herein as TAA1), CD123 (also referred herein as TAA2), CD33 (also referred herein as TAA3), and CD70 (also referred herein as TAA4), or peptide-MHC complexes, wherein the peptide is derived from a protein expressed in tumor cells (e.g., NY-ESO-1 or MAGE-A3), and the MHC is MHC class I. In another embodiment, the disease-associated antigen is an infection-associated antigen, preferably a virus-associated antigen (VAA). Exemplary examples of virus-associated antigens include, but are not limited to, a peptide-MHCC complex, wherein the peptide is derived from a viral infectious agent (e.g., HBcAg or EBNA-1), and the MHC is MHC class I. In one embodiment, the binder having binding specificity to a disease-related antigen is physically attached to the ankyrin repeat domain having binding specificity to CD3 by linking, conjugating, fusing, or other means. In one embodiment, the binder having binding specificity to a disease-related antigen is covalently bound to the ankyrin repeat domain having binding specificity to CD3. In one embodiment, the binder having binding specificity to a disease-related antigen is covalently bound to the ankyrin repeat domain having binding specificity to CD3 using a peptide linker. In one embodiment, the amino acid sequence of the peptide linker has a length of 1 to 50 amino acids, preferably 6 to 38 amino acids. In one embodiment, the peptide linker is a high-proline-threonine peptide linker. In one embodiment, the peptide linker is one of the high-proline-threonine peptide linkers of SEQ ID NOs. 31 and 42-46. In one embodiment, the binder having binding specificity to a disease-related antigen is covalently bound to the ankyrin repeat domain having binding specificity to CD3 using one of the high-proline-threonine peptide linkers 31 and 42-46.In one preferred embodiment, the binder having binding specificity to a disease-related antigen is a designed ankyrin repeat domain having binding specificity to a disease-related antigen.

[0087] In one embodiment, the recombinant binding protein of the present invention comprises an ankyrin repeat domain having binding specificity to CD3 as more specifically described in any aspect or embodiment of this specification, and further comprises two ankyrin repeat domains having binding specificity to a disease-related antigen as more specifically described in any aspect or embodiment of this specification. In a preferred embodiment, the ankyrin repeat domain having binding specificity to a disease-related antigen is located at the N-terminus of the ankyrin repeat domain having binding specificity to CD3 within the binding protein.

[0088] In one embodiment, the recombinant binding protein further includes a half-life extension portion.

[0089] Accordingly, in certain embodiments, the recombinant binding protein of the present invention comprises an ankyrin repeat domain having binding specificity to CD3 as more specifically described in any aspect and embodiment of this specification, and further comprises a half-life extension portion. In one embodiment, the half-life extension portion is located at the N-terminus of the ankyrin repeat domain having binding specificity to CD3 within the binding protein.

[0090] In one embodiment, the half-life extension portion includes a binder having binding specificity to human serum albumin.

[0091] In one embodiment, the half-life extension portion is a designed ankyrin-binding domain having binding specificity to human serum albumin. In one embodiment, the designed ankyrin-binding domain having binding specificity to human serum albumin includes an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with any one of SEQ ID NOs: 28-30. Accordingly, in one embodiment, the recombinant binding protein of the present invention includes an ankyrin repeat domain having binding specificity to CD3 as described more specifically in any aspect or embodiment of this specification, and further includes an ankyrin-binding domain having binding specificity to human serum albumin, having an amino acid sequence having at least 80% amino acid sequence identity with any one of SEQ ID NOs: 28-30. In one embodiment, in humans, the ankyrin repeat domain having binding specificity to human serum albumin includes an amino acid sequence having at least 90% amino acid sequence identity with any one of SEQ ID NOs: 28-30. In another embodiment, the ankyrin repeat domain having binding specificity to human serum albumin includes an amino acid sequence having at least 93% amino acid sequence identity with any one of SEQ ID NOs: 28-30, and in a further embodiment, the ankyrin repeat domain having binding specificity to human serum albumin includes an amino acid sequence having at least 95% amino acid sequence identity with any one of SEQ ID NOs: 28-30. In one embodiment, in humans, the ankyrin repeat domain having binding specificity to human serum albumin includes an amino acid sequence having at least 98% amino acid sequence identity with any one of SEQ ID NOs: 28-30, and in a further embodiment, the ankyrin repeat domain having binding specificity to human serum albumin includes an amino acid sequence of any one of SEQ ID NOs: 28-30.Accordingly, in one embodiment, the recombinant binding protein of the present invention comprises an ankyrin repeat domain having binding specificity to CD3, and further comprises an ankyrin repeat domain having binding specificity to human serum albumin, wherein the ankyrin repeat domain having binding specificity to CD3 comprises one amino acid sequence of any one of SEQ ID NOs: 1 to 4, and the ankyrin repeat domain having binding specificity to human serum albumin comprises one amino acid sequence of any one of SEQ ID NOs: 28 to 30. In a preferred embodiment, the ankyrin repeat domain having binding specificity to human serum albumin is located at the N-terminus of the ankyrin repeat domain having binding specificity to CD3 within the binding protein.

[0092] In a preferred embodiment, the designed ankyrin-binding domain having binding specificity to human serum albumin includes an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity with SEQ ID NO: 29. Thus, in one embodiment, the recombinant binding protein of the present invention includes an ankyrin repeat domain having binding specificity to CD3 as more specifically described in any aspect or embodiment of this specification, and further includes an ankyrin-binding domain having binding specificity to human serum albumin having an amino acid sequence having at least 80% amino acid sequence identity with SEQ ID NO: 29. In one embodiment in humans, the ankyrin repeat domain having binding specificity to human serum albumin includes an amino acid sequence having at least 90% amino acid sequence identity with SEQ ID NO: 29. In another embodiment, the ankyrin repeat domain having binding specificity to human serum albumin includes an amino acid sequence having at least 93% amino acid sequence identity with SEQ ID NO: 29, and in a further embodiment, the ankyrin repeat domain having binding specificity to human serum albumin includes an amino acid sequence having at least 95% amino acid sequence identity with SEQ ID NO: 29. In one embodiment, in humans, the ankyrin repeat domain having binding specificity to human serum albumin includes an amino acid sequence having at least 98% amino acid sequence identity with SEQ ID NO: 29, and in a further embodiment, the ankyrin repeat domain having binding specificity to human serum albumin includes the amino acid sequence of SEQ ID NO: 29. Therefore, in one embodiment, the recombinant binding protein of the present invention includes an ankyrin repeat domain having binding specificity to CD3, further including an ankyrin repeat domain having binding specificity to human serum albumin, the ankyrin repeat domain having binding specificity to CD3 includes one of the amino acid sequences of SEQ ID NOs: 1 to 4, and the ankyrin repeat domain having binding specificity to human serum albumin includes the amino acid sequence of SEQ ID NO: 29.In a preferred embodiment, the ankyrin repeat domain having binding specificity to human serum albumin is located at the N-terminus of the ankyrin repeat domain having binding specificity to CD3.

[0093] In one embodiment, the recombinant binding protein of the present invention, which includes an ankyrin repeat domain having binding specificity to CD3 and further includes an ankyrin repeat domain having binding specificity to human serum albumin, exhibits an increased terminal phase half-life, preferably at least 5%, preferably 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or 250% increased terminal phase half-life compared to a corresponding recombinant binding protein that includes the ankyrin repeat domain having binding specificity to CD3 but does not include the ankyrin repeat domain having binding specificity to human serum albumin.

[0094] In one embodiment, the recombinant binding protein of the present invention comprises an ankyrin repeat domain having binding specificity to CD3 as more specifically described in any aspect or embodiment of this specification, and further comprises two ankyrin repeat domains having binding specificity to human serum albumin as more specifically described in any aspect or embodiment of this specification.

[0095] In one embodiment, the recombinant binding protein of the present invention further comprises a polypeptide tag. The polypeptide tag is an amino acid sequence bound to a polypeptide / protein, where the amino acid sequence is useful for the purification, detection, or targeting of the polypeptide / protein, or the amino acid sequence improves the physicochemical behavior of the polypeptide / protein, or the amino acid sequence has an effector function. Individual polypeptide tags of the binding protein may be attached to other sites of the binding protein directly or via a peptide linker. Polypeptide tags are well known in the art and are readily available to those skilled in the art. Examples of polypeptide tags include small polypeptide sequences, such as His tags, HA tags, myc tags, FLAG tags, or Strep tags, or polypeptides such as enzymes (e.g., alkaline phosphatase), which enable the detection of the polypeptide / protein, or polypeptides that can be used for targeting (such as immunoglobulins or their fragments) and / or polypeptides that can be used as effector molecules.

[0096] In one embodiment, the recombinant binding protein of the present invention further comprises a peptide linker. The peptide linker is an amino acid sequence that can link, for example, two protein domains, a polypeptide tag and a protein domain, a protein domain and a non-protein compound or a polymer such as polyethylene glycol, or a protein domain and a biologically active molecule, a protein domain and a localizer, or two sequence tags. Peptide linkers are known to those skilled in the art. A list of examples is provided in the specification of Japanese Patent Application Publication No. WO2002 / 020565. Specific examples of such linkers are glycine-serine linkers and variable-length proline-threonine linkers. Examples of glycine-serine linkers are the amino acid sequences GS and SEQ ID NO: 41, and examples of proline-threonine linkers are the amino acid sequences SEQ ID NOs: 31 and 42-46.

[0097] In another embodiment, the present invention relates to nucleic acids encoding amino acid sequences of the ankyrin repeat domain or recombinant binding protein of the present invention. In one embodiment, the present invention relates to nucleic acids encoding amino acid sequences of the recombinant binding protein of the present invention. In one embodiment, the present invention relates to nucleic acids encoding amino acid sequences selected from the group consisting of SEQ ID NOs: 1 to 4. In one embodiment, the present invention relates to nucleic acids encoding the amino acid sequence of SEQ ID NOs: 1. In one embodiment, the present invention relates to nucleic acids encoding the amino acid sequence of SEQ ID NOs: 2. In one embodiment, the present invention relates to nucleic acids encoding the amino acid sequence of SEQ ID NOs: 3. In one embodiment, the present invention relates to nucleic acids encoding the amino acid sequence of SEQ ID NOs: 4. Furthermore, the present invention relates to vectors comprising any of the nucleic acids of the present invention. Nucleic acids are well known to those skilled in the art. In the examples, nucleic acids were used to produce the designed ankyrin repeat domain or recombinant binding protein of the present invention in E. coli. Examples of nucleic acids of the present invention are provided by SEQ ID NOs: 37 to 40, which each encodes the amino acid sequences of SEQ ID NOs: 1 to 4.

[0098] In one embodiment, the present invention relates to a pharmaceutical composition comprising the recombinant binding protein and / or designed ankyrin repeat domain of the present invention, and / or a nucleic acid encoding the recombinant binding protein and / or designed ankyrin repeat domain of the present invention, and optionally a pharmaceutically acceptable carrier and / or diluent.

[0099] In one embodiment, the present invention relates to a pharmaceutical composition comprising a recombinant binding protein, or a nucleic acid encoding the recombinant binding protein of the present invention, and optionally a pharmaceutically acceptable carrier and / or diluent.

[0100] Pharmacopoeia-acceptable carriers and / or diluents are known to those skilled in the art and are described in more detail below.

[0101] The pharmaceutical composition includes recombinant binding proteins and / or designed ankyrin repeat domains and / or nucleic acids as described herein, preferably recombinant binding proteins and / or nucleic acids, and for example, Remington's Pharmaceutical Sciences 16 th Includes pharmaceutically acceptable carriers, excipients, or stabilizers, as described in edition, Osol, A.Ed., 1980.

[0102] Suitable carriers, excipients, or stabilizers known to those skilled in the art include, for example, physiological saline, Ringer's solution, dextrose solution, Hanks' solution, fixative oil, ethyl oleate, 5% dextrose physiological saline, substances that enhance isotonicity and chemical stability, buffers, and preservatives. Other suitable carriers include any carrier that does not induce the production of antibodies harmful to the individual administered the composition, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, and amino acid copolymers. The pharmaceutical composition may also be a combination containing additional active ingredients such as anticancer agents or anti-angiogenic agents, or further bioactive compounds.

[0103] Preparations used for in vivo administration must be sterile or sterilized. This can be easily achieved by filtering through a sterile filtration membrane.

[0104] One embodiment of the present invention relates to the use of a recombinant binding protein of the present invention comprising an ankyrin repeat domain having binding specificity to CD3, and further comprising an ankyrin repeat domain having binding specificity to serum albumin for the production of a pharmaceutical composition, wherein the recombinant binding protein comprises the ankyrin repeat domain having binding specificity to CD3, but does not comprise the ankyrin repeat domain having binding specificity to human serum albumin, and exhibits an increased terminal phase half-life, preferably at least 5%, preferably 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%, or 250% increased terminal phase half-life compared to a corresponding recombinant binding protein comprising the ankyrin repeat domain having binding specificity to CD3, and further comprising two ankyrin repeat domains having binding specificity to serum albumin.

[0105] In one embodiment, the pharmaceutical composition comprises at least one recombinant binding protein as described herein, as well as a detergent such as a nonionic detergent, a buffer such as a phosphate buffer, and a sugar such as sucrose. In one embodiment, such a composition comprises the recombinant binding protein and PBS as described above.

[0106] In another embodiment, the present invention provides a method for CD3-mediated T cell activation in tumor cells or tissues in a mammal, comprising the step of administering to the mammal a recombinant binding protein of the present invention comprising an ankyrin repeat domain having binding specificity to CD3, and further comprising a localizer molecule. In one embodiment, the localizer molecule is a binding protein having binding specificity to a target different from CD3, preferably TAA or VAA. In one embodiment, the mammal is human, and the TAA-expressing cells or tissues are located within a tumor, including a primary tumor, metastasis, and / or tumor stroma.

[0107] In another aspect, the present invention provides a method for tumor-local activation of T cells in a mammal, including a human, comprising the step of administering to the mammal a binding protein of the present invention, a nucleic acid of the present invention, or a pharmaceutical composition of the present invention.

[0108] In another embodiment, the present invention provides a method for local activation of T cells in a mammal, including a human, comprising the step of administering to the mammal a binding protein of the present invention, a nucleic acid of the present invention, or a pharmaceutical composition of the present invention.

[0109] In another embodiment, the present invention provides a method for treating a medical condition, comprising the step of administering to a patient in need of treatment for the medical condition a therapeutically effective amount of the recombinant binding protein of the present invention, a nucleic acid encoding the binding protein, or a pharmaceutical composition comprising the binding protein, further comprising a binding agent having binding specificity to a disease-related antigen. In another embodiment, the present invention provides a method for treating a medical condition, comprising the step of administering to a patient in need of treatment for the medical condition a therapeutically effective amount of the recombinant binding protein of the present invention, further comprising a binding agent having binding specificity to a disease-related antigen, wherein the binding agent is effective in localizing the binding protein to a target tumor tissue or infected tissue, and such localization of the binding protein results in T cell activation in the target tumor or infected tissue. In one embodiment, the binding agent having binding specificity to a disease-related antigen is a binding protein, preferably an ankyrin repeat-binding protein having binding specificity to TAA or VAA.

[0110] In one embodiment, the present invention relates to a pharmaceutical composition, recombinant binding protein, or nucleic acid according to the present invention for use in the treatment of a disease. For this purpose, the pharmaceutical composition, nucleic acid, or recombinant binding protein according to the present invention is administered in a therapeutically effective dose to a patient in need thereof. Dosage methods include topical administration, oral administration, and parenteral administration. The typical route of administration is parenteral administration. In parental administration, the pharmaceutical composition of the present invention is formulated in a unit dose injectable form such as a solution, suspension, or emulsion, together with pharmaceutically acceptable excipients as defined above. The dose and method of administration will vary depending on the individual being treated and the specific disease.

[0111] Furthermore, any of the aforementioned pharmaceutical compositions, nucleic acids, or recombinant binding proteins are considered to be for use in the treatment of disorders.

[0112] In one embodiment, the recombinant binding protein or other such pharmaceutical compositions described herein are administered intravenously. For parenteral use, the recombinant binding protein or the pharmaceutical composition may be administered in a therapeutically effective dose as a bolus injection or by slow infusion.

[0113] In one embodiment, the present invention relates to a method for treating a medical condition, comprising the step of administering a therapeutically effective amount of the recombinant binding protein of the present invention, the nucleic acid of the present invention, or the pharmaceutical composition of the present invention to a patient in need of such treatment. In one embodiment, the present invention relates to the use of the recombinant binding protein of the present invention, the nucleic acid of the present invention, or the pharmaceutical composition of the present invention for the treatment of a disease. In one embodiment, the present invention relates to the recombinant binding protein of the present invention, the nucleic acid of the present invention, or the pharmaceutical composition of the present invention for use in the treatment of a disease. In one embodiment, the present invention relates to the recombinant binding protein of the present invention, the nucleic acid of the present invention, or the pharmaceutical composition of the present invention for use in the treatment of a medical condition. In one embodiment, the present invention relates to the use of the recombinant binding protein of the present invention, the nucleic acid of the present invention, or the pharmaceutical composition of the present invention as a pharmaceutical for the treatment of a disease. In one embodiment, the present invention relates to the use of the recombinant binding protein of the present invention, the nucleic acid of the present invention, or the pharmaceutical composition of the present invention for the manufacture of a pharmaceutical for the treatment of a disease. In one embodiment, the present invention relates to a process for the manufacture of a pharmaceutical for the treatment of a disease, wherein the recombinant binding protein of the present invention, the nucleic acid molecule of the present invention, or the pharmaceutical composition of the present invention is the active ingredient of the pharmaceutical. In one embodiment, the present invention relates to a method for treating a disease using the recombinant binding protein of the present invention, the nucleic acid of the present invention, or the pharmaceutical composition of the present invention.

[0114] The use of the recombinant binding proteins, nucleic acids, or pharmaceutical compositions of the present invention for the treatment of cancer or infectious diseases may also be in combination with one or more other therapies known in the art. The term “in combination with ~” as used herein means co-administration carried out under a given dosing schedule. This includes simultaneous administration of different compounds and administration of different compounds at different times (for example, administering compound A once and compound B several times thereafter, or vice versa, or administering both compounds simultaneously and then administering one of them again at a later stage).

[0115] In one embodiment, the medical condition is cancer. In another embodiment, the medical condition is an infectious disease, preferably a viral infection.

[0116] In one particular embodiment, the medical condition is cancer, and the cancer or tumor tissue comprises cells expressing or presenting a tumor-associated antigen, and the binder having binding specificity to the disease-associated antigen binds to the tumor-associated antigen expressed or presented in the cells. In one particular embodiment, the disease-associated antigen is the extracellular domain of a cell surface protein expressed or overexpressed in the cancer or tumor tissue. In another particular embodiment, the binder having binding specificity to the tumor-associated antigen binds to a peptide-MHC complex, the peptide being derived from a protein expressed in tumor cells (e.g., NY-0ESO-1 or MAGE-A3). In one particular embodiment, the MHC is MHC class I. In one embodiment, the cancer is selected from adenocarcinoma and squamous cell carcinoma. In one embodiment, the cancer is selected from osteosarcoma or osteogenic sarcoma (bone), chondrosarcoma (cartilage), leiomyosarcoma (smooth muscle), rhabdomyosarcoma (skeletal muscle), mesosarcoma or mesothelioma (inner lining of body cavities), fibrosarcoma (fibrous tissue), angiosarcoma or hemangioendothelioma (blood vessels), liposarcoma (adipose tissue), glioma or astrocytoma (neurogenic connective tissue found in the brain), myxosarcoma (primitive embryonic connective tissue), and mesenchymal or mixed mesodermal tumor (mixed connective tissue type). In one embodiment, the cancer is selected from myeloid or granulocytic leukemia (malignant tumors of myeloid and granulocytic leukocyte lineages), lymphoid, lymphocytic, or lymphoblastic leukemia (malignant tumors of lymphoid and lymphocytic hematological cell lineages), and polycythemia vera or anemia (malignant tumors of various hematological cell products, but with a predominance of red blood cells). In one embodiment, the cancer is selected from Hodgkin lymphoma and non-Hodgkin lymphoma. In one embodiment, the cancer is selected from adenosquamous carcinoma, mixed mesodermal tumor, carcinosarcoma and teratoma. In one embodiment, the cancer is selected from colorectal cancer, gastric cancer, non-small cell lung cancer, breast cancer, head and neck cancer, ovarian cancer, lung cancer, invasive bladder cancer, pancreatic cancer, metastatic carcinoma of the brain and neck, esophageal squamous cell carcinoma, lung squamous cell carcinoma, cutaneous squamous cell carcinoma, melanoma, breast cancer, lung adenocarcinoma, cervical squamous cell carcinoma, pancreatic squamous cell carcinoma, colon squamous cell carcinoma, or gastric squamous cell carcinoma, prostate cancer, osteosarcoma, or soft tissue sarcoma and benign tumors.In one embodiment, such cancers are selected from epithelial malignancies (primary and metastatic), including lung cancer, colorectal cancer, gastric cancer, bladder cancer, ovarian cancer, and breast cancer, as well as bone and soft tissue sarcomas.

[0117] In another specific embodiment, the medical condition is an infectious disease, preferably a viral infection, and the infected tissue comprises cells expressing or presenting an infection-related antigen, preferably a virus-related antigen, and the binder having binding specificity to the disease-related antigen binds to the infection-related antigen expressed or presented in the cells. In another specific embodiment, the disease-related antigen is the extracellular domain of a viral protein expressed or overexpressed in the infected tissue. In yet another specific embodiment, the binder having binding specificity to the virus-related antigen binds to a peptide-MHC complex, the peptide being derived from a protein of an infectious agent, such as a bacterial or viral infectious agent, preferably a viral infectious agent (e.g., HBcAg or EBNA-1). In another specific embodiment, the MHC is MHC class I. In one embodiment, the infection is selected from viral infections caused by hepatitis B virus (HBV), viral infections caused by Epstein-Barr virus (EBV), HIV infection, West Nile virus infection, hepatitis A, B, and C, smallpox, tuberculosis, varicella stomatitis virus (VSV) infection, respiratory syncytial virus (RSV) infection, human papillomavirus (HPV) infection, SARS, influenza, Ebola hemorrhagic fever, viral meningitis, herpes, anthrax, Lyme disease, and Escherichia coli infection. In one embodiment, the present invention relates to a recombinant binding protein comprising any of the above-described ankyrin repeat domains.

[0118] In one embodiment, the present invention relates to a kit comprising the recombinant binding protein of the present invention. In one embodiment, the present invention relates to a kit comprising a nucleic acid encoding the recombinant binding protein of the present invention. In one embodiment, the present invention relates to a kit comprising the pharmaceutical composition of the present invention. In one embodiment, the present invention relates to a kit comprising the recombinant binding protein of the present invention and / or the nucleic acid of the present invention and / or the pharmaceutical composition of the present invention. In one embodiment, the present invention relates to a kit comprising a recombinant binding protein comprising an ankyrin repeat domain having binding specificity to CD3, for example, SEQ ID NOs: 1 to 4, and / or a nucleic acid encoding a recombinant binding protein comprising an ankyrin repeat domain having binding specificity to CD3, for example, SEQ ID NOs: 1 to 4, and / or a pharmaceutical composition comprising a recombinant binding protein comprising an ankyrin repeat domain having binding specificity to CD3, for example, SEQ ID NOs: 1 to 4.

[0119] In one embodiment, the present invention relates to a method for producing recombinant binding proteins of the present invention. In one embodiment, the present invention relates to a method for producing recombinant binding proteins, for example, recombinant binding proteins comprising the amino acid sequences of SEQ ID NOs: 1 to 4, comprising the steps of (i) expressing the recombinant binding protein in a suitable host cell (e.g., in bacteria) and (ii) purifying the recombinant binding protein (e.g., using chromatography). The method may include additional steps. Such a method for producing recombinant binding proteins of the present invention is described in Example 1.

[0120] The present invention is not limited to the specific embodiments described in the examples.

[0121] This specification refers to many amino acid sequences, nucleic acid sequences, and sequence numbers disclosed in the attached sequence listings, which are incorporated herein by reference in their entirety.

[0122] definition Unless otherwise defined herein, all technical and scientific terms used herein shall have the meanings generally understood by those skilled in the art to which the present invention belongs.

[0123] In the context of the present invention, the term "protein" means a molecule comprising polypeptides, where at least a portion of the polypeptides 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. If a protein comprises two or more polypeptide chains, the individual polypeptide chains may be linked by non-covalent or covalent bonds, for example, by disulfide bonds between the 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 called "protein domains." Such protein domains are well known to those skilled in the art.

[0124] The term "recombinant" as used in relation to recombinant proteins and recombinant 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 encoding a polypeptide (e.g., produced by gene synthesis) can be cloned into a bacterial expression plasmid (e.g., pQE30, QIAgen), a yeast expression plasmid, a mammalian expression plasmid, or a plant expression plasmid, or into DNA that enables in vitro expression. For example, if such a recombinant bacterial expression plasmid is inserted into a suitable bacterium (e.g., E. coli), these bacteria can produce the polypeptide encoded by this recombinant DNA. The polypeptide or protein produced in this manner is called a recombinant polypeptide or recombinant protein.

[0125] In the context of the present invention, the term "binding protein" refers to a protein containing a binding domain. The binding protein may also contain two, three, four, five or more binding domains. Preferably, the binding protein is a recombinant binding protein. The binding protein of the present invention contains an ankyrin repeat domain that has binding specificity to CD3.

[0126] Furthermore, any such binding protein may include additional polypeptides well known to those skilled in the art (e.g., polypeptide tags, peptide linkers, fusion to other protein domains with binding specificity, cytokines, hormones, or antagonists), or chemical modifications (e.g., coupling to polyethylene glycol, toxins (e.g., DM1 from immunogens), small molecules, antibiotics, etc.). The binding protein of the present invention may also include a localizer molecule.

[0127] The term "binding domain" refers to a protein domain that exhibits binding specificity to a target. Preferably, this binding domain is a recombinant binding domain.

[0128] The term "target" refers to individual molecules such as nucleic acid molecules, polypeptides or proteins, carbohydrates, or any other naturally occurring molecules, or to any part of such individual molecules, or to complexes of two or more such molecules, or to whole cells or tissue samples, or to any non-natural compounds. Preferably, the target is a naturally occurring or non-natural polypeptide or protein, or a polypeptide or protein that is chemically modified, for example, by naturally occurring or non-natural phosphorylation, acetylation, or methylation. In the context of the present invention, T cells are targets of the CD30-specific binding protein and the localizer target protein, and cells and tissues are targets of the localizer.

[0129] In the context of the present invention, the term "polypeptide" refers to a molecule consisting of multiple, i.e., chains of 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 ​​SS crosslinks. Polypeptides are well known to those skilled in the art.

[0130] Patent Application No. 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, techniques, and applications of repeat proteins and repeat domains. The term “repeat protein” refers to a protein containing one or more repeat domains. Preferably, a repeat protein contains one, two, three, four, five, or six repeat domains. Furthermore, the repeat protein may also contain additional non-repeat protein domains, polypeptide tags, and / or peptide linkers. The repeat domains may be binding domains.

[0131] The term “repeatable domain” refers to a protein domain comprising two or more consecutive repeating modules as structural units, wherein these repeating modules have structural homology and sequence homology. Preferably, the repeating domain further comprises N-terminal and / or C-terminal capping modules. For clarity, the capping modules may be repeating modules. Such repeating domains, repeating modules, and capping modules, sequence motifs, and their structural and sequence homology are well known to those skilled in the art from examples of ankyrin repeating domains (International Publication No. 2002 / 020565), leucine-rich repeating domains (International Publication No. 2002 / 020565), tetratricopeptide repeating domains (Main, ER, Xiong, Y., Cocco, MJ, D'Andrea, L., Regan, L., Structure 11(5), 497~508, 2003), and armadillo repeating domains (International Publication No. 2009 / 040338). It is more well known to those skilled in the art that such repeating domains are different from proteins containing repeating amino acid sequences, and all repeating amino acid sequences can form individual domains (for example, the FN3 domain of fibronectin).

[0132] The term "designed" as used in terms such as designed repeat proteins and designed repeat domains refers to the characteristic that such repeat proteins and repeat domains are artificial and do not occur in nature. The binding proteins of the present invention are designed repeat proteins, which include at least one designed ankyrin repeat domain.

[0133] The term "target interaction residue" refers to amino acid residues in a repeating module that contribute to direct interaction with a target.

[0134] The term "framework residue" refers to an amino acid residue in a repeating module that contributes to the folding topology, i.e., to the folding of the repeating module or to interactions with adjacent modules. Such contributions may include interactions with other residues within the repeating module, influences on the polypeptide scaffold structure observed in α-helices or β-sheets, or involvement in amino acid stretching to form linear polypeptides or loops.

[0135] Such frameworks and target interaction residues can be identified by analyzing structural data obtained by physicochemical methods such as X-ray crystallography, NMR and / or CD spectroscopy, or by comparing them with known relevant structural information well known to those skilled in the art in structural biology and / or bioinformatics.

[0136] The term “repeatable module” originally refers to the repeated amino acid sequence and structural unit of a designed repeatable domain, derived from the repeating units of naturally occurring repeat proteins. Each repeatable module contained within a repeatable domain is derived from one or more repeating units of a naturally occurring family or subfamily of repeatable proteins, such as the ankyrin repeatable protein family. Furthermore, each repeatable module contained within a repeatable domain may contain a “repeatable sequence motif” derived from a homologous repeatable module having the same target specificity, as described, for example, in Example 1, which is obtained from a repeatable domain selected on a target.

[0137] Therefore, the term "ankyrin repeat module" refers to a repeat module derived from the repeating unit of the naturally occurring ankyrin repeat protein. The ankyrin repeat protein is well known to those skilled in the art.

[0138] The repeating module may include positions with amino acid residues that are not randomized in the library ("unrandomized positions") and positions with amino acid residues that are randomized in the library ("randomized positions") for the purpose of selecting a target-specific repeating domain. Unrandomized positions include framework residues. Randomized positions include target interaction residues. "Randomized" means, for example, that two or more amino acids are permitted at an amino acid position in the repeating module, and any of the usual 20 naturally occurring amino acids are permitted, or most of the 20 naturally occurring amino acids are permitted, such as amino acids other than cysteine, or amino acids other than glycine, cysteine, and proline. These positions are generally not substituted in variants of the module sequences of SEQ ID NOs. 5-9 disclosed herein.

[0139] The term “repetitive sequence motif” refers to an amino acid sequence inferred from one or more repetitive modules. Preferably, the repetitive modules are derived from repetitive domains having binding specificity to the same target. Such a repetitive sequence motif includes framework residue locations and target interaction residue locations. The framework residue locations correspond to the positions of framework residues in the repetitive module. Similarly, the target interaction residue locations correspond to the positions of target interaction residues in the repetitive module. The repetitive sequence motif includes non-randomized and randomized locations.

[0140] The term “repeat unit” refers to an amino acid sequence containing one or more naturally occurring protein sequence motifs, which are found in multiple copies and exhibit a defined folding topology common to all such motifs that determine the protein's folding. Examples of such repeat units include leucine-rich repeat units, ankyrin repeat units, armadillo repeat units, tetratricopeptide repeat units, HEAT repeat units, and leucine-rich mutant repeat units.

[0141] The terms "having binding specificity to the target," "binding specifically to the target," "binding to the target with high specificity," "specific to the target," or "target specificity" all mean that the binding protein or binding domain binds to the target in PBS with a lower dissociation constant (i.e., with higher affinity) than it would to unrelated proteins such as E. coli maltose-binding protein (MBP). Preferably, the dissociation constant in PBS for the target is ("K"). D ) is at least 10 times greater than the corresponding dissociation constant for MBP. 2 times, more preferably at least 10 3 times, more preferably at least 10 4 double, or more preferably at least 10 5 It is twice as low. Methods for measuring the dissociation constant of protein-protein interactions, such as techniques based on surface plasmon resonance (SPR) (e.g., SPR equilibrium analysis) or isothermal titration calorimetry (ITC), are well known to those skilled in the art. The K of a particular protein-protein interaction D The measured values ​​may vary when measured under different conditions (e.g., salt concentration, pH). Therefore, K D The value is preferably measured using a standardized protein solution and a standardized buffer such as PBS. The dissociation constant (K) of the recombinant binding protein of the present invention having binding specificity to CD3. D A typical and preferred determination of ) by surface plasmon resonance (SPR) analysis is described in Example 4.

[0142] The term "binding agent" refers to any molecule that can specifically bind to a target molecule. The term "binding agent" includes, for example, antibodies, antibody fragments, aptamers, peptides (e.g., Williams et al., J Biol Chem 266:5182-5190 (1991)), antibody mimetic bodies, repeat proteins, such as designed aniclinic repeat proteins, receptor proteins, and other naturally occurring interaction partners of any of the target molecules, and may include both native proteins and proteins that have been modified or genetically engineered to, for example, include non-native residues and / or lack native residues.

[0143] The term "approximately" means ±20% of the stated value; for example, "approximately 50" means between 40 and 60.

[0144] The term "PBS" refers to a phosphate-buffered aqueous solution containing 137 mM NaCl, 10 mM phosphate, and 2.7 mM KCl, with a pH of 7.4.

[0145] The term "mouse serum albumin" refers to UniProt accession number P07724, the term "cynomolgus monkey serum albumin" (i.e., cynomolgus monkey (macaca fascicularis)) refers to UniProt accession number A2V9Z4, and the term "human serum albumin" refers to UniProt accession number P02768.

[0146] Preferably, clearance, and / or exposure, and / or terminal phase half-life are evaluated in mammals, more preferably mice and / or cynomolgus monkeys. Clearance, and / or exposure, and / or terminal phase half-life may be evaluated as described in Example 3. Preferably, when clearance, and / or exposure, and / or terminal phase half-life are measured in mice, the evaluation is carried out considering data up to a maximum of 48 hours after injection. More preferably, the evaluation of terminal phase half-life in mice is calculated from 24 to 48 hours. Preferably, when clearance, and / or exposure, and / or terminal phase half-life are measured in cynomolgus monkeys, the evaluation is carried out considering data up to a maximum of 7 days after injection. More preferably, the evaluation of terminal phase half-life in cynomolgus monkeys is calculated from day 1 to day 5. Those skilled in the art may further identify effects such as target-mediated clearance and take them into consideration when calculating terminal phase half-life. The term "terminal phase half-life" for drugs such as recombinant binding proteins of the present invention refers to the time required to reach half the plasma concentration of the drug after it has reached false equilibrium in a mammal (for example, calculated as 24 to 48 hours in mice, or as 1 to 5 days in cynomolgus monkeys). Terminal phase half-life is not defined as the time required to excrete half of the dose of the drug administered to a mammal. The term terminal phase half-life is well known to those skilled in the art. Preferably, pharmacokinetic comparisons are made at any dose, more preferably equivalent doses (i.e., the same mg / kg dose) or equimolar doses (i.e., the same mol / kg dose), more preferably equimolar dosages (i.e., the same mol / kg dose). It is understood to those skilled in the art that equivalent and / or equimolar dosages in animals involve experimental dose variability of at least 20%, more preferably 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%. Preferably, the dosage used for measuring pharmacokinetics is selected from 0.001 to 1000 mg / kg, more preferably 0.01 to 100 mg / kg, more preferably 0.1 to 50 mg / kg, and more preferably 0.5 to 10 mg / kg.

[0147] The term “CD3” or “differentiation cluster 3” refers to a multimeric protein complex composed of four distinct polypeptide chains, epsilon (ε), gamma (γ), and zeta (ζ), which assemble as three pairs (εγ, εδ, ζζ). The CD3 complex functions as a T cell coreceptor, non-covalently associating with the T cell receptor. It may refer to any form of CD3, as well as its variants, isoforms, and species homologs that retain at least some of the activity of CD3. Thus, the binding proteins defined and disclosed herein may also bind to CD3 from non-human species. In other cases, the binding proteins may be perfectly specific to human CD3 and may not exhibit species or other types of cross-reactivity. Unless otherwise indicated, such as by a specific reference to human CD3, CD3 includes the natural sequence CD3 of all mammalian species, e.g., humans, dogs, cats, horses, and cattle. The amino acid sequences of human CD3 gamma, delta, and zeta are shown in the NCBI (www.ncbi.nlm.nih.gov / ) reference sequences NP_000064.1, NP_000723.1, and NP_932170.1, respectively.

[0148] As used herein, the term “CD3-expressing cell” refers to any cell that expresses CD3 (differentiation cluster 3) on its cell surface, including but not limited to T cells such as cytotoxic T cells (CD8+ T cells) and T helper cells (CD4+ T cells).

[0149] The term "tumor-specific activation of T cells" refers to the preferential activation of T cells in tumor tissue compared to non-tumor tissue.

[0150] The term "localized activation of T cells in infection" means that T cells are preferentially activated in infected tissue compared to non-infected tissue.

[0151] Furthermore, the term "peptide" also includes peptides modified by glycosylation, for example, and proteins comprising two or more polypeptide chains, each with a length of 4 to 600 amino acids, such as insulin and immunoglobulins, which are crosslinked by disulfide bonds. The term "chemical or biochemical agent" is intended to include any naturally occurring or synthetic compound that may be administered to a recipient.

[0152] The term “medical condition” (or disorder or disease) includes autoimmune disorders, inflammatory disorders, retinopathy (particularly proliferative retinopathy), neurodegenerative disorders, infectious diseases, metabolic diseases, and neoplastic diseases. Any of the recombinant binding proteins described herein may be used in the preparation of pharmaceuticals for the treatment of such disorders, particularly those selected from the group including autoimmune disorders, inflammatory disorders, retinopathy, and neoplastic diseases. A “medical condition” may be characterized by inappropriate cell proliferation. A medical condition may be a state of hyperproliferation. The present invention relates, in particular, 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 the recombinant binding protein of the present invention or the pharmaceutical composition described above. In a preferred embodiment, the medical condition is a neoplastic disease. The term “neoplastic disease,” as used herein, refers to an abnormal state or condition of cells or tissue characterized by rapidly growing cell proliferation or tumors. In one embodiment, the medical condition is a malignant neoplastic disease. In one embodiment, the medical condition is cancer. The term "therapeutic dose" refers to the amount sufficient to produce the desired effect in a patient.

[0153] The term "antibody" encompasses not only intact antibody molecules but also any fragments and variants of antibody molecules that retain immunogenic binding ability. Such fragments and variants are also well known in the art and are commonly used both in vitro and in vivo. Therefore, the term "antibody" includes intact immunoglobulin molecules, e.g., Fab, Fab', F(ab')2, and single-chain V-region fragments (scFv), bispecific antibodies, chimeric antibodies, antibody-fusion polypeptides, and unconventional antibodies.

[0154] The terms “cancer” and “malignant” are used herein to refer to or describe a physiological condition in mammals typically characterized by unregulated cell proliferation. Cancer encompasses solid tumors and humoral tumors, as well as primary and metastatic tumors. A “tumor” includes one or more cancerous cells. A solid tumor typically also includes the tumor stroma. Examples of cancer include, but are not limited to, primary and metastatic cancers, lymphomas, blastomas, sarcomas, and leukemias, as well as any other epithelial and lymphoid malignancies. More specific examples of such cancers include brain cancer, bladder cancer, breast cancer, ovarian cancer, clear cell kidney cancer, head and neck squamous cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, malignant melanoma, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer (SCLC), triple-negative breast cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), and chronic myeloid leukemia (CLL). Examples include ML, diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), squamous cell carcinoma of the head and neck (SCCHN), chronic myeloid leukemia (CML), small lymphocytic lymphoma (SLL), malignant mesothelioma, colorectal cancer, or gastric cancer.

[0155] The terms “infection” and “infection” are used herein to refer to or describe the infiltration and proliferation of microorganisms in body tissues, particularly those causing pathological symptoms. Examples of infections include, but are not limited to, viral and bacterial diseases such as HIV infection, West Nile virus infection, hepatitis A, B, and C, smallpox, tuberculosis, varicella-stomatitis virus (VSV) infection, respiratory syncytial virus (RSV) infection, human papillomavirus (HPV) infection, SARS, influenza, Ebola virus disease, viral meningitis, herpes zoster, anthrax, Lyme disease, and Escherichia coli (E. coli) infection. [Examples]

[0156] The starting materials and reagents disclosed below are known to those skilled in the art, are commercially available, and / or can be prepared using well-known techniques.

[0157] material Chemicals were purchased from Sigma-Aldrich (USA). Oligonucleotides were purchased from Microsynth (Switzerland). Unless otherwise specified, DNA polymerase, restriction enzymes, and buffers were purchased from New England Biolabs (USA) or Fermentas / Thermo Fisher Scientific (USA). Inducible E. coli expression strains were used for cloning and protein production, such as E. coli XL1-blue (Stratagene, USA) or BL21 (Novagen, USA).

[0158] molecular biology Unless otherwise specified, the methods will be 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).

[0159] Designed Ankyrin Repeat Protein Library Methods for generating designed ankyrin repeat protein libraries are described, for example, in U.S. Patent No. 7,417,130; Binz et al. 2003, cited above; Binz et al. (2004), cited above. By such methods, designed ankyrin repeat protein libraries having randomized ankyrin repeat modules and / or randomized capping modules can be constructed. For example, such a library may be assembled based on an immobilized N-terminal capping module (e.g., the N-terminal capping module of SEQ ID NOs. 5, 6, or 7), the N-terminal capping module of SEQ ID NOs. 10, 11, 12, 13, 14, or 15), or a randomized N-terminal capping module of SEQ ID NO. 16, and an immobilized C-terminal capping module (e.g., the C-terminal capping module of SEQ ID NOs. 17, 18, 19, 20, 21, 22, 23, 24, or 25), or a randomized C-terminal capping module of SEQ ID NO. 26. Preferably, such a library is assembled such that none of the amino acids C, G, M, N (before the G residue) and P are present at randomized positions in the repeating or capping modules.

[0160] Furthermore, such randomized modules within such libraries may include additional polypeptide loop insertions having randomized amino acid positions. Examples of such polypeptide loop insertions are complementarity determining region (CDR) loop libraries of antibodies or de novo generated peptide libraries. For example, such loop insertions could be designed using the structure of the N-terminal ankyrin repeat domain of human ribonuclease L as guidance (Tanaka, N., Nakanishi, M, Kusakabe, Y., Goto, Y., Kitade, Y., Nakamura, K.T., EMBO J.. 23(30), 3929-3938, 2004). Similar to this ankyrin repeat domain where 10 amino acids are inserted into the β-turn near the boundary of two ankyrin repeats, an ankyrin repeat protein library may contain randomized loops of variable length (e.g., 1 to 20 amino acids) inserted into one or more β-turns of the ankyrin repeat domain (having fixed and randomized positions).

[0161] Any such N-terminal capping module of an ankyrin repeat protein library preferably has a RILLAA motif, a RILLKA motif, or a RELLKA motif (e.g., present at positions 21-26 of SEQ ID NO: 29), and any such C-terminal capping module of an ankyrin repeat protein library preferably has a KLN motif, a KLA motif, or a KAA motif (e.g., present in the last 3 amino acids of SEQ ID NO: 1). SEQ ID NOs: 10, 11, 12, 13, 14, or 15 provide examples of N-terminal capping modules containing the RILLAA, RILLKA, or RELLKA motif, and SEQ ID NOs: 17, 18, 19, 20, 21, 22, 23, 24, or 25 provide examples of C-terminal capping modules containing the KLN, KLA, or KAA motif.

[0162] The design of such ankyrin repeat protein libraries can be guided by the known structures of ankyrin repeat domains that interact with a target. Examples of such structures, identified by their unique accession codes or identification codes (PDB-ID) in the Protein Data Bank (PDB), are 1WDY, 3V31, 3V30, 3V2X, 3V2O, 3UXG, 3TWQ - 3TWX, 1N11, 1S70, and 2ZGD.

[0163] Examples of designed ankyrin repeat protein libraries, such as the designed ankyrin repeat protein library at N2C and N3C, are described (U.S. Patent No. 7,417,130, Binz et al., 2003, cited above; Binz et al., 2004, cited above). The numbers N2C and N3C describe the number of randomized repeat modules present between the N-terminal and C-terminal capping modules.

[0164] The nomenclature used to define positions within repeats and modules is based on Binz et al. (2004), cited above, with the modification that the boundary between an ankyrin repeat module and an ankyrin repeat unit shifts by 1 amino acid position. For example, position 1 of the ankyrin repeat module in Binz et al. (2004), cited above, corresponds to position 2 of the ankyrin repeat module of the present disclosure, and as a result, position 33 of the ankyrin repeat module in Binz et al. (2004), cited above, corresponds to position 1 of the following ankyrin repeat module of the present disclosure.

[0165] All DNA sequences were confirmed by sequencing, and the calculated molecular weights of the selected proteins were confirmed by mass spectrometry.

[0166] Example 1: Selection of a binding protein containing an ankyrin repeat domain with binding specificity for CD3 Using a ribosome display (Hanes, J. and Pluckthun, A., "PNAS" Vol. 94, pp. 4937-42 (1997)), many ankyrin repeat proteins with binding specificity to human scCD3 were selected from the DARPin® library, similar to the description cited above by Binz et al. (2004). Binding of the selected clones to recombinant human CD3 targets was evaluated by homogeneous time-resolved fluorescence (HTRF) of crude extracts, demonstrating the successful selection of hundreds of human scCD3-specific binding proteins. For example, the ankyrin repeat domain of SEQ ID NO: 1 constitutes the amino acid sequence of the selected binding protein, containing an ankyrin repeat domain with binding specificity to scCD3. Individual ankyrin repeat modules from such ankyrin repeat domains with binding specificity to scCD3, e.g., those in SEQ ID NOs: 5-9, are provided.

[0167] Human recombinant CD3 target preparation The selected target format is based on a single-chain format consisting of human CD3ε and CD3γ heterodimers linked by a 26-amino acid linker (scCD3εγ) and a C-terminal Avi tag for site-directed biotinylation. The target protein contains only the CD3 extracellular domain and lacks the C-terminal cysteine ​​"knob" as well as the entire transmembrane and cytoplasmic domains.

[0168] The extracellular domain of human scCD3εγ (SEQ ID NO: 32_scCD3εγ_Avi-Bio) was expressed in a single-stranded format similar to that previously described in *E. coli* (Kjer-Nielsen et al., PNAS, 2004, 101(20):7675-7680), followed by refolding from inclusion bodies and purification by preparative size exclusion chromatography (SEC). The material was concentrated to 3.4 mg / ml in 10 mM Tris-HCl, 50 mM NaCl, pH 8.0, and biotinylated in vitro using recombinant BirA. To isolate the functional target material, the material was re-purified using an OKT3 packed column (GE HiTrap NHS activated HP column). The final material was monomeric by size exclusion and stored at a final concentration of 0.39 mg / ml in 10 mM Tris, 100 mM NaCl, pH 8.0, and 10% glycerol.

[0169] An overview of the recombinant human scCD3 target preparation process is described in Dunstone et al., Acta Crystallographica 2004.

[0170] Selection of CD3-specific ankyrin repeat proteins by ribosome display Selection of CD3-specific ankyrin repeat proteins was performed by ribosome display (Hanes and Pluckthun (cited above)) using a portion of the extracellular domain of human CD3 (SEQ ID NO: 32) as the target protein, and the above-mentioned library of ankyrin repeat proteins, as well as an established protocol (see, e.g., Zahn, C., Amstutz, P., and Pluckthun, A., Nat. Methods 4, 69-79, 2007). The number of reverse transcription (RT)-PCR cycles after each selection round was consistently reduced from 45 to 28 to adjust for yield due to enrichment of the binder. The selection of the first four rounds employed standard ribosome display selection, decreasing the target concentration (400 nM, 133 nM, 45 nM, and 15 nM, respectively), increasing the wash stringency, and increasing the selection pressure from round 1 to round 4 (Binz et al. 2004, cited above). In rounds 2-4, mRNA was recovered by competitive elution using excess CD3-binding antibody OKT3 (the excess of the competitor increased consistently from 35-fold to 300-fold in each round).

[0171] The selected clone activates T cells in a bivalent format. Individual ankyrin repeat protein clones that bind to the CD3 target were selected by ribosome display, cloned into derivatives of the expression vector pQE30 (Qiagen), transformed into E. coli XL1-Blue (Stratagene), plated on LB agar (containing 1% glucose and 50 μg / ml ampicillin), and incubated overnight at 37°C. The expression vector, Jun leucine zipper construct with both His-tag and Myc-tag and CD3-specific ankyrin repeat domains, was used for screening in a bivalent format (with respect to the CD3-specific binding domain) to enable testing of functionality by crosslinking in T cells. Single colonies were picked up into 96-well plates (each clone in a single well) containing 160 μL of growth medium (TB containing 1% glucose and 50 μg / mL ampicillin), incubated overnight at 37°C, and shaken at 800 rpm. 8.5 μL of overnight cultures were seeded into 150 μL of fresh TB medium containing 50 μg / mL ampicillin in a new 96-well plate. After incubation at 37°C and 850 rpm for 120 minutes, expression was induced with IPTG (final concentration 0.5 mM) for 4 hours. Cells were collected, the pellet was frozen overnight at -20°C, and then resuspended in 8.5 μL of B-PERII (Thermo Scientific) and incubated at room temperature for 1 hour with shaking (600 rpm). Subsequently, 160 μL of PBS was added, and cell fragments were removed by centrifugation (at 3220 g for 15 minutes) and stored at -20°C for further use.

[0172] In the first step, T cell activation screening was performed using BK112 CD8+ monoclonal T cells. Extracts from each lysed clone were applied to a 96-well plate coated with anti-pentaHis antibody (Qiagen) as a 1:20 dilution (final concentration) in PBSB (PBS pH 7.4 supplemented with 12% (w / v) FBS) and incubated overnight at 4°C. After washing the plate five times with PBS, 100 μl of 100,000 BK112 T cells were added per well and cultured in T cell assay medium RPMI-1640 + 10% FBS + 1% L-glutamine + 1% Pen Strep + 200 IU IL2. 0.1 μg of 100 μL of Golgi Stop was added, the plate was centrifuged at 20 g for 3 minutes at room temperature, and then incubated in a CO2 incubator at 37°C for 4-5 hours. Cells were decanted by centrifuging at 350 g for 5 minutes at 4°C. After staining the cells for surface CD8 expression, the cells were preserved using BD Cytofix and incubated overnight at 4°C. Cells were stained for intracellular IFNγ by washing them with 1×PBS + 2% FBS, adding 50 μl of IFNγ-APC antibody in Cell Perm (BD), and incubating at 4°C for 30 minutes. Cells were washed again with PBS and analyzed using Cytometer FACS Canto II from BD.

[0173] The selected clones exhibit binding to CD3 (indicated by competition between HTRF and OKT3) and functionality in a bispecific format. To construct T cell engager (TCE) constructs, identified functionally designed ankyrin repeat domain hits were subcloned into derivatives of the pQE30 (Qiagen) expression vector containing an N-terminal His tag, a tumor-associated antigen 1 (TAA1)-specific ankyrin repeat domain, and a CD3-specific ankyrin repeat domain. The constructs were expressed in E. coli cells and purified using their His tags according to a standard protocol. 25 ml of static overnight culture (TB, 1% glucose, 50 μg / mL ampicillin; 37°C) was used to inoculate 500 mL of culture (TB, 50 μg / mL ampicillin, 37°C). The cultures were induced with 0.5 mM IPTG at 600 nm with absorbances of 1.0–1.5 and incubated at 37°C for 4–5 hours with shaking. The cultures were centrifuged, and the resulting pellet was added to 25 mL of TBS. 500 The sample was resuspended in (50 mM Tris-HCl, 500 mM NaCl, pH 8) and dissolved (sonicated). After dissolution, the sample was mixed with 50 KU DNase / mL and incubated for 15 minutes before a heat treatment step of 30 minutes at 62.5°C, then centrifuged, the supernatant was collected and filtered. Triton X100 (final concentration 1% (v / v)) and imidazole (final concentration 20 mM) were added to the homogenate. The protein was purified by Ni-nitrilotriacetic acid (Ni-NTA) column followed by size exclusion chromatography on an AKTAxpress® system according to standard protocols and resins known to those skilled in the art.

[0174] In the first step, binding to recombinant proteins was tested using an HTRF assay. Titer analysis of ankyrin repeat proteins (5–640 nM) in PBS-TC (PBS supplemented with 0.1% (w / v) casein and 0.1% Tween20, pH 7.4) was performed in wells of a 384-well plate against 48 nM (final concentration) human biotinylated scCD3εγ, 1:100 (final concentration) anti-6 His-D2 HTRF antibody-FRET acceptor conjugate (Cisbio), and 1:100 (final concentration) anti-strep-Tb antibody-FRET donor conjugate (Cisbio), incubated at room temperature for 120 minutes. HTRF was read using a Tecan M1000 with an excitation wavelength of 340 nm and an emission filter of 665 ± 10 nm. Several candidates showed dose-dependent binding and were used for further evaluation. For all of these constructs, the binding signal was at background levels when competing with a 20-fold excess of CD3-binding antibody (OKT3 containing the human Fc region, final concentration 2.4 mM) that binds to the conformational epitope of CD3ε (Kjer-Nielsen et al., PNAS, 2004, 101(20):7675-7680). Dose-dependent in vitro T cell activation was confirmed using a BK112 T cell activation assay (BK112 CD8 monoclonal T cells pre-activated with CD3 / CD28 Dynabeads) in the presence of TAA1-expressing tumor cells. Intracellular IFNγ levels were measured on CD8+ or CD4+ T cells after 5-hour incubation of BK112 and SKOV3 cells (E:T=1:10) in the presence of bispecific TCE constructs ranging from 1 to 100,000 pM. The most potent constructs showed EC50 values ​​of 0.5 nM and 0.4 nM for CD4+ and CD8+ cells, respectively.

[0175] Affinity maturation and rational design of selected CD3-specific ankyrin repeat proteins Several rounds of affinity maturation, combined with rational design, were applied to the parental low-affinity-binding CD3-specific ankyrin repeat protein (referred to as Precursor A) to obtain four higher-affinity CD3-specific ankyrin repeat proteins (referred to as Precursors B, C, and D). These precursor molecules were then engineered into the final CD3-specific ankyrin repeat proteins DARPin® Protein #1, DARPin® Protein #2, DARPin® Protein #3, and DARPin® Protein #4.

[0176] Affinity maturation was performed on one of the parental CD3-specific ankyrin repeat proteins (precursor A), selected for both sufficient binding ability to the CD3 target and the ability to efficiently activate T cells in vitro, by introducing diversity using error-prone PCR and DNA I shuffling as described in Zahnd et al., Nat Methods, 2007, 4:269-279. Briefly, three rounds of ribosome display were performed using different concentrations of dNTP analogs (mutagenesis kit from Jena Biosciences) to introduce approximately 1-2 mutations per round of CD3-specific ankyrin repeat protein, with increased selection pressure (washing step increased from round 1 (3 × 15 min) to 3 × 30 min (round 2) to 3 × 45 min (round 3)), but the target concentration was kept constant at 5 nM. In the second step, the DNA pool was DNA shuffled and backcrossed with parental clones in one or two rounds of ribosome display using DNAse I incubation time of 90 seconds and DNA polymerase HotStarTaq DNA polymerase (Qiagen) as previously described (Cadwell & Joyce, PCR Methods Appl, 1992, 2:28-33; Stemmer, Nature, 1994 370:389-391; Zaccolo et al., J Mol Biol 1996, 255:589-603). Affinity-matured CD3-specific ankyrin repeat protein pools were subcloned into derivatives of the pQE30(Qiagen) expression vector, which ultimately contained an N-terminal His-Flag tag, an HSA-binding ankyrin repeat domain for half-life extension, a TAA 1-binding ankyrin repeat domain, and a CD3-specific ankyrin repeat domain. These were expressed and screened for binding to recombinant scCD3εγ by HTRF as described above. Read clones were selected (precursor B, generated after 5 rounds of affinity maturation including 3 rounds of error-prone PCR using 10 μM dNTPs analog at all steps, and 2 rounds of DNA shuffling and backcross).

[0177] In the same process, several potentially beneficial mutations for increased T cell activation were identified by the percentage of IFNγ+ T cells in the BK112 assay, including N-cap mutations at positions 5 and 20, first internal repeat mutations at positions 2 and 4, second internal repeat mutations at positions 2, 5 and 20, and C-cap mutations at positions 1 and 18. While minimizing N and C-cap framework mutations to maintain thermal stability, combinations of these mutations resulted in a designed set of variants, which were tested again in the same format for improved T cell activation. This generated a new, more mature variant (precursor C) with higher T cell activation potency compared to the parent and the first matured variant.

[0178] To further increase CD3 affinity and T cell activation efficacy, a second affinity maturation was performed on the precursor C clone, similar to that described above. Briefly, four rounds of ribosome display were performed. In the first three rounds, mutations were introduced by error-prone PCR using a 7.5 μM dNTP analog. In rounds 1 and 3, off-rate selection was applied using either the more mature clone itself or a non-biotinylated CD3 target protein for competition. A 1 nM target was used in rounds 1 and 2, while a 5 nM target was applied in rounds 3 and 4, which were less stringent. CD3 variants were screened using an off-rate HTRF assay with 250x access to the more mature clone as a competitor in a triple-specific format containing an HSA-binding domain and a TAA1-binding ankyrin repeat domain. A total of 3 × 96 clones with the highest residual HTRF signal after competition were sequenced. Beneficial mutations identified for improved binding (including N-cap position 16, first internal repeats 1, 12, 18, 19, 26, 30, and 33, second internal repeats 2, 3, 7, 20, 21, 26, and 32, and C-cap positions 9, 11, and 18) or reduced domain interactions (including N-cap positions 11, 18, 19, and 26, and C-cap positions 3, 19, and 22) were first tested for BK112 T cell activation as individual or paired mutations in purified protein variants. The most beneficial variants were then recombined in a second step, and the variants were screened for the highest T cell activation, which led to the identification of precursor D.

[0179] In the final step, mutant DARPin® protein #1, DARPin® protein #2, DARPin® protein #3, and DARPin® protein #4 were constructed based on CD3-specific ankyrin repeat protein precursors A, B, C, and D, respectively, to improve serum half-life and biophysical properties. This introduced N-cap mutations at positions 23 and / or 26 and removed some of the framework mutations (positions 19 and 18).

[0180] An affinity matured ankyrin repeat domain having binding specificity for human CD3 was cloned into a pQE (QIAgen, Germany)-based expression vector, providing an N-terminal His tag (SEQ ID NO: 27) to facilitate simple protein purification as described below. For example, expression vectors encoding the following ankyrin repeat proteins were constructed. DARPin® protein #1 (having SEQ ID NO: 1 with a His tag (SEQ ID NO: 27) fused to its N-terminus), DARPin® protein #2 (having SEQ ID NO: 2 with a His tag (SEQ ID NO: 27) fused to its N-terminus), DARPin® protein #3 (having SEQ ID NO: 3 with a His tag (SEQ ID NO: 27) fused to its N-terminus), DARPin® protein #4 (having SEQ ID NO: 4 with a His tag (SEQ ID NO: 27) fused to its N-terminus),

[0181] High-level and soluble expression of CD3-specific ankyrin repeat proteins For detailed analysis, selected clones showing specific CD3 binding were expressed in E. coli cells either as monovalent or in combination with TAA and / or HSA-binding ankyrin repeat domains, purified using the His tag, and subsequently size-exclusion chromatography was performed on an AKTAxpress™ system according to standard protocols and resins known to those skilled in the art. For both monovalent and multivalent constructs, the proteins were monomeric and soluble when concentrated to 10 mg / ml in TBS pH 8.0 (50 mM Tris, 500 mM NaCl) or PBS pH 7.4. A representative example of such SDS-PAGE analysis is shown in FIG. 1.

[0182] Example 2: Evaluation of the stability of exemplary CD3-specific ankyrin repeat proteins Evaluation of exemplary CD3-specific ankyrin repeat proteins using UV spectroscopy, SDS-PAGE, and size-exclusion chromatography (SEC) Four selected CD3-specific ankyrin repeat proteins were analyzed in detail to evaluate their biophysical properties. Briefly, purified CD3-specific ankyrin repeat proteins DARPin®#1 (SEQ ID NO: 1), DARPin®#2 (SEQ ID NO: 2), DARPin®#3 (SEQ ID NO: 3), and DARPin®#4 (SEQ ID NO: 4) in TBS pH 8.0 (50 mM Tris, 500 mM NaCl) were aliquoted into sterile glass vials (Schmidlin: LPP 11 09 0620) and stressed by incubation at 50°C for 18 days. The applied stress conditions allowed for prediction of biophysical properties after storage at 4°C for 2 years. For each CD3-specific ankyrin repeat protein, an aliquot was stored at -70°C as a reference. Both the reference sample and the heat-stressed sample were subjected to the same number of freeze-thaw cycles before analysis. The samples were evaluated for aggregation and turbidity (size range >100 μm) by UV spectroscopy, and for polymerization, aggregation, and fragmentation by SDS-PAGE and analytical SEC. The stressed samples were then compared to their respective reference samples (Figures 2, 3, and 4).

[0183] Thermal stability evaluation of exemplary CD3-specific ankyrin repeat proteins using circular dichroism (CD) spectroscopy. The same CD3-specific ankyrin repeat proteins were also evaluated for their thermal stability and unfolding / refolding tendencies using a Jasco J-815 spectrophotometer (only unstressed proteins). The Tm (melting temperature) of the selected proteins was determined by CD as a parameter of thermal stability. Briefly, ellipticity was recorded at 222 nM, and after applying a temperature range of 20°C to 95°C, a reverse scan was performed to record the refolding behavior. The Tm of the selected CD3-specific ankyrin repeat proteins represents the midpoint of protein unfolding.

[0184] The samples were prepared at a concentration of 2 μM in TBS pH 8.0 (50 mM Tris, 500 mM NaCl) (Figure 5). For all four proteins tested, a Tm higher than 65°C was obtained.

[0185] [Table 1]

[0186] Aggregation initiation temperature T due to dynamic light scattering (DLS) agg decision The same CD3-specific ankyrin repeat proteins were analyzed for their temperature-dependent aggregation tendencies (T agg The following was evaluated: A temperature range of 25°C to 85°C was applied, and the results were plotted against the hydrodynamic radius of the protein. For evaluation, a Wyatt DynaPro Plate Reader II (serial number 140-WPR2) device was used, and the measurements were processed with Dynamics 7.8.1.30 software. Ankyrin replicate samples (Table 2) were prepared at a concentration of 1 mg / ml in TBS pH 8.0 (50 mM Tris, 500 mM NaCl), and measured in five replicates (A1, A2, A3, A4, and A5) (Figure 6).

[0187] As shown in Figure 6, the ankyrin repeat proteins of all four ankyrins tested tended to aggregate at temperatures much higher than 65°C.

[0188] [Table 2]

[0189] Example 3: Pharmacokinetic analysis of CD3-specific ankyrin repeat protein in female BALB / c mice To determine whether the CD3-specific ankyrin repeat domain of the present invention has an appropriate serum half-life in vivo for use in the development of therapeutic drugs, the pharmacokinetic profiles of DARPin® protein #1, DARPin® protein #2, DARPin® protein #3, and DARPin® protein #4 were analyzed in mice. For this purpose, the DARPin construct was subcloned and expressed as described above in a derivative of the pQE30 (Qiagen) expression vector containing an N-terminal His tag, an HSA-binding ankyrin repeat domain for half-life extension, and subsequently one of the CD3-specific binding domains. The two ankyrin repeat domains were linked to each other by a peptide linker such as the linker of SEQ ID NO: 31.

[0190] For example, we constructed an expression vector encoding the following ankyrin repeat protein. DARPin® protein #5 (SEQ ID NO: 33, with a His tag fused to its N-terminus (SEQ ID NO: 27)), DARPin® protein #6 (SEQ ID NO: 34, with a His tag fused to its N-terminus (SEQ ID NO: 27)), DARPin® protein #7 (SEQ ID NO: 35, with a His tag fused to its N-terminus (SEQ ID NO: 27)), DARPin® protein #8 (SEQ ID NO: 36, with a His tag fused to its N-terminus (SEQ ID NO: 27)),

[0191] In vivo administration and sample collection DARPin® protein #5, DARPin® protein #6, DARPin® protein #7, and DARPin® protein #8, formatted with human serum albumin-specific ankyrin repeat domains (SEQ ID NO: 29), were administered as a single intravenous bolus injection into the tail vein of six mice, for each ankyrin repeat fusion protein. The target dose level was 1 mg / kg at an applicable volume of 5 mL / kg. The ankyrin repeat fusion proteins were mixed in phosphate-buffered saline (PBS) solution.

[0192] The mice were divided into two groups, each containing an equal number of animals. Four serum samples were collected from each mouse. Blood samples for pharmacokinetic studies were collected at 5 minutes, 4 hours, 24 hours, 48 ​​hours, 76 hours, 96 hours, and 168 hours after compound administration. The blood was allowed to coagulate at room temperature, followed by centrifugation to collect the serum.

[0193] ELISA-based bioanalysis for measuring ankyrin repeat proteins in serum samples 100 μl of 10 nM polyclonal goat anti-rabbit IgG antibody (Ab18) in PBS was coated per well of a NUNC Maxisorb ELISA plate overnight at 4°C. After washing each well five times with 300 μl of PBST (PBS supplemented with 0.1% Tween20), the wells were blocked at room temperature (RT) for 1 hour with 300 μl of PBST supplemented with 0.25% casein (PBST-C) using a Heidolph Titramax 1000 shaker (450 rpm). The plate was washed as described above. 100 μl of 5 nmol / L rabbit anti-DARPin® 1-1-1 antibody in PBST-C was added, and the plate was incubated at room temperature (22°C) for 1 hour with orbital shaking (450 rpm). The plate was washed as described above.

[0194] 100 μl of diluted serum sample (1:20-1:30 with a 1:5 dilution step) or ankyrin repeat protein standard curve sample (0 and 50-0.0008 nmol / L with a 1:3 dilution step) was applied at room temperature for 2 hours with shaking at 450 rpm. The plate was washed as described above.

[0195] Next, the wells were incubated with 100 μl of mouse anti-RGS-His-HRP IgG (Ab06, 1:2000 in PBST-C) and incubated at room temperature at 450 rpm for 1 hour. The plates were washed as described above. The ELISA was developed by using 100 μl / well of TMB substrate solution for 5 minutes and stopping the process by adding 100 μl of 1 mol / L H2SO4. The difference between the absorbance at 450 nm and the absorbance at 620 nm was calculated. The samples were measured in a double-row system on two different plates. Figure 7A shows the serum concentrations of DARPin® protein #5, DARPin® protein #6, DARPin® protein #7, and DARPin® protein #8 as a function of time after a single intravenous administration to mice. Traces show the approximate single exponential removal of the compounds.

[0196] Pharmacokinetic analysis Pharmacokinetic data analysis was performed using Molecular Partners with version 7.0 of the WinNonlin program as part of Phoenix 64, Pharsight, and North Carolina. Pharmacokinetic parameters were calculated based on mean concentration-time data from animals administered via intravenous bolus injection using non-compartmental analysis (NCA model 200-202, IV bolus, linear trapezoidal interpolation). The following pharmacokinetic parameters were calculated: AUCinf, AUClast, AUC_%extrapol, Cmax, Tmax, Cl_pred, Vss_pred, t1 / 2.

[0197] The maximum serum concentrations (Cmax) and their time to occurrence (Tmax) were obtained directly from the serum concentration-time profile. The area under the serum concentration-time curve (AUCinf) was determined by the linear trapezoidal rule up to the last sampling point (Tlast) and extrapolation to infinity assuming a single exponential decrease in the terminal phase. Extrapolation to infinity was performed using Clast / λz, where λz represents the final rate constant estimated by log-linear regression, and Clast represents the concentration estimated at Tlast by the final log-linear regression. Total serum clearance (Cl_pred) and apparent terminal phase half-life were calculated as follows: Cl_pred = intravenous dose / AUCinf and t1 / 2 = ln2 / λz. The steady-state volume of distribution Vss was determined as follows: Vss = iv dose·AUMCinf / (AUCinf)². AUMCinf represents the total area under the first instant of the drug concentration-time curve extrapolated to infinity, using the same extrapolation procedure described for calculating AUCinf. To calculate the PK parameters based on concentrations given in nmol / L, the dose values ​​given in mg / kg were converted to nmol / kg using the molecular weight of the ankyrin repeat protein. Table 3 summarizes the pharmacokinetic properties of the four ankyrin repeat proteins tested, DARPin® protein #5, DARPin® protein #6, DARPin® protein #7, and DARPin® protein #8, after a single intravenous administration of 1 mg / kg.

[0198] [Table 3]

[0199] Example 4: Dissociation constant (K) of ankyrin repeat protein with binding specificity to human CD3 determined by surface plasmon resonance (SPR) analysis. D ) decision The binding affinity of four purified ankyrin repeat proteins to recombinant human CD3 targets was analyzed using a ProteOn instrument (BioRad) and measured according to standard procedures known to those skilled in the art. For this purpose, DARPin® protein #1, DARPin® protein #2, DARPin® protein #3, and DARPin® protein #4 of the present invention were subcloned and expressed in derivatives of the pQE30 (Qiagen) expression vector containing an N-terminal His tag, two different TAAs (TAA2 and TAA3, respectively) that bind to the ankyrin repeat domain, and subsequently one of the four CD3-specific ankyrin repeat protein constructs, as described above.

[0200] In short, biotinylated human scCD3εγ was diluted in PBST (PBS, pH 7.4, containing 0.005% Tween20®) and coated onto an NLC tip (BioRad) to a level of approximately 700–1400 resonance units (RU). The interaction between ankyrin repeat proteins and human CD3 was then measured for multi-trace SPR by injecting 300 μL of electrophoresis buffer (PBS, pH 7.4, containing 0.005% Tween20®) containing serially diluted ankyrin repeat proteins covering a concentration range of 64 nM–4 nM, followed by injecting the electrophoresis buffer stream at a constant flow rate of 60 μL / min for at least 10 minutes (off-rate measurement). Regeneration was performed using 30 μL of 10 mM glycine pH 2. The signal between spots (i.e., resonance unit (RU) values) and reference injection (i.e., injection of running buffer only) were subtracted from the RU trace obtained after injection of ankyrin repeat proteins (double reference). For affinity-matured constructs, the binding parameter (K) to CD3 was determined. D The on-rate and off-rate were determined. Binding equilibrium was not adequately reached even at the highest sample concentration, resulting in an unoptimal fit for the on-rate, therefore the on-rate (k on ) and dissociation constant (K D ) is given only as an approximation.

[0201] As a representative example, Figure 7B shows the SPR trace obtained for DARPin® molecule #3 formatted with TAA2-binding ankyrin repeat domains and TAA3-binding ankyrin repeat domains. Dissociation constant (K D The K of the binding interaction of selected ankyrin repeat proteins by human CD3 was calculated from the estimated on-rate and off-rate using standard procedures known to those skilled in the art. D The value was determined to be in the range of 6 to 35 nM (see Table 4).

[0202] [Table 4]

[0203] Example 5: Determination of CD3 T cell binding CD3 binding was determined in primary human T cells using Mirrorball laser scanning imaging cytometry. Therefore, primary T cells were isolated from human peripheral blood mononuclear cells (PBMCs) using a pan-T cell purification kit (Miltenyi Biotec). The titers of DARPin® protein #1, DARPin® protein #2, DARPin® protein #3, and DARPin® protein #4, hereafter referred to herein as DARPin® protein #A, DARPin® protein #B, DARPin® protein #C, and DARPin® protein #D, respectively, in a triple-specific format (containing TAA2 and TAA3-binding ankyrin repeat domains), or in a quadruple-specific format (containing an additional ankyrin repeat domain that specifically binds to human serum albumin), were incubated with 50,000 pan-T cells per well at 4°C for 30 minutes in the presence of 600 μM human serum albumin (to mimic physiological serum concentrations). To generate the triplicate TCE protein, DARPin® protein #1, DARPin® protein #2, DARPin® protein #3, and DARPin® protein #4 were each subcloned and expressed as described above in derivatives of the pQE30 (Qiagen) expression vector encoding an N-terminal His tag, followed by two different TAA-specific ankyrin repeat domains (in the order of TAA2-specific and TAA3-specific, respectively), and then one of the CD3-specific ankyrin repeat domains of the present invention. For the quadruplicate TCE protein, a human serum albumin-specific ankyrin repeat domain was further included between the His tag and the TAA2-specific domain. The ankyrin repeat domains were linked to each other by a peptide linker such as the linker of SEQ ID NO: 31. Two benchmark T cell engagers targeting either TAA2 or TAA3 were applied as controls. After washing, CD3 binding was detected using a 1:100 dilution of anti-penta-His Alexa Fluor488 antibody (Qiagen).After incubation at 4°C for 30 minutes, the cells were washed, resuspended in Cytofix fixation buffer (BD Biosciences), and counterstained with 5 μM DRAQ5 (Abcam) at room temperature for 15 minutes. The median mean fluorescence intensity of Alexa Fluor 488 bound cells on far-red counterstained cells was measured using Mirrorball with Cellista software (SPT Labtech), and the data were plotted using GraphPad Prism 8.

[0204] As shown in Figures 8A-B, the DARPin® protein exhibits a wide range of affinities, from no detectable binding to #A to comparable binding to benchmark molecules for #D (known benchmark T cell engagers: TCE1 with binding specificity to TAA2 and TCE2 with binding specificity to TAA3). CD3 binding to T cells is consistent with CD3 affinity measured by SPR. The presence of an additional HSA-binding domain (see Figure 8B) had only a slight effect on binding to T cells. Table 5 lists the ECs of four exemplary ankyrin repeat proteins. 50 This indicates the CD3 binding affinity represented by the value.

[0205] [Table 5]

[0206] Example 6: Evaluation of target-specific short-term tumor cell death induced by CD3-specific ankyrin repeat protein using an LDH cytotoxicity assay. The specificity and potency of four CD3-specific ankyrin repeat proteins (DARPin® proteins #1-#4) in a format described for cell binding were evaluated by an in vitro short-term cytotoxicity assay induced by LDH release.

[0207] Effector cells and target cells were co-incubated in two rows in a 96-well plate at a 5:1 E:T ratio in the presence of 600 μM human serum albumin (to mimic physiological concentrations). Untouched T cells were isolated from human PBMCs using a pan-T cell isolation kit (Miltenyi). 100,000 purified pan-T cells (effector cells) and 20,000 TAA2 / TAA3-expressing tumor cells (target cells) per well were incubated at 37°C for 48 hours with a series of control dilutions containing selected CD3-specific ankyrin repeat protein, a control benchmark molecule, or 1% Triton X-100. After 48 hours of incubation, the cells were spun down and incubated with 100 μl of supernatant per well and 100 μl of LDH reaction mixture (LDH detection kit; Roche Applied Science) per well for 30 minutes. Absorbance was measured from 492nm to 620nm using a TECAN infinite M1000Pro reader. After background correction, OD values ​​were plotted using a GraphPad Prism8.

[0208] As shown in Figure 9A, DARPin protein without half-life extension, DARPin® protein #2, and DARPin® protein #3 induced tumor cell death comparable to that of the benchmark molecule (a known benchmark T cell engager, TCE1 with binding specificity to TAA2), while DARPin® protein #1 and DARPin® protein #4 showed 10 to 100 times lower potency. In Figure 9B, DARPin® protein with extended half-life showed approximately 4 to 70 times lower potency compared to the corresponding molecule without extended half-life.

[0209] Example 7: Evaluation of target-specific short-term T cell activation and IFNγ secretion. The specificity and efficacy of the CD3-specific ankyrin repeat proteins described above (Examples 5 and 6) were evaluated in an in vitro short-term T cell activation assay using FACS, which measures the CD25 activation marker on CD8+ T cells, and ELISA, which measures IFNγ secretion.

[0210] Therefore, 100,000 purified pan-T cells (effector cells) and 100,000 TAA2 / TAA3-expressing tumor cells (target cells) per well were co-incubated in 2 series with serial dilutions of selected binding proteins or control benchmark molecules in the presence of 600 μM human serum albumin at 37°C for 24 hours (E:T ratio 1:1). After 24 hours, 100 μl of supernatant per well was transferred for measurement of IFNγ secretion using the Human IFNγ Standard ABTS ELISA Development Kit (PeproTech) according to the manufacturer's protocol. The cells were washed and stained at 4°C for 30 minutes with 1:1,000 Live / Dead Aqua (Thermo Fisher), 1:250 Mouse Anti-Human CD8 Pacific Blue (BD), and 1:100 Mouse Anti-Human CD25 PerCP-Cy5.5 (eBiosciences) antibody. After washing and fixation, cells were analyzed using a FACS Canto II (BD) instrument. T cell activation was evaluated by measuring CD25+ cells on live / dead-negative CD8+ gated T cells. FACS data was analyzed using FlowJo software, and the data was plotted using GraphPad Prism8.

[0211] As shown in Figure 10A, DARPin® protein #2 and DARPin® protein #3 induced specific short-term T cell activation comparable to the benchmark molecule (a known benchmark T cell engager, TCE1 with binding specificity to TAA2) without half-life extension, while DARPin® protein #1 and DARPin® protein #4 showed 10 to 100 times lower potency. In Figure 10B, DARPin® proteins with extended half-lives showed approximately 3 to 100 times lower potency compared to the corresponding molecules without extended half-lives.

[0212] Example 8: Evaluation of target-specific long-term tumor cell death by IncuCyte The specificity and efficacy of CD3-specific ankyrin repeat proteins (described in Examples 5, 6, and 7) were evaluated by an in vitro long-term death assay using the IncuCyte S3 platform.

[0213] TAA2 / TAA3-expressing tumor cells were first transduced with NucLight Red (NLR) lentiviral particles (Sartorius), and red fluorescent cells were selected by 0.7 μg / ml puromycin and / or FACS sorting. Long-term tumor cell death was then evaluated using the IncuCyte S3 system (Sartorius). Effector and target cells were co-incubated in pairs on 96-well plates coated with 0.01% poly-L-ornithine in a 5:1 E:T ratio in the presence of 1:200 Annexin V green (Sartorius) and 600 μM human serum albumin (to mimic physiological concentrations). 50,000 purified (Miltenyi)pan-T cells (isolated from healthy donor PBMCs) + 10,000 TAA2 / TAA3-expressing tumor cells (transduced via NLR) were incubated per well with serial dilutions of selected ankyrin repeat proteins or control benchmark molecules at 37°C for up to 6 days. Cell proliferation (red fluorescence from NLR) and cell death (green fluorescence from annexin V) were evaluated. Total cell proliferation and tumor cell death were analyzed by calculating the area under the curve after 6 days of co-culture using GraphPad Prism8.

[0214] As shown in Figure 12, the tested DARPin® proteins #2, #3, and #4 exhibit potent and specific tumor cell death comparable to benchmark molecules (known benchmark T cell engagers, TCE1 with binding specificity to TAA2 and TCE2 with binding specificity to TAA3), regardless of half-life extension. Only DARPin® #1, with its lower affinity, showed reduced killing efficacy.

[0215] Example 9: Evaluation of target-specific long-term T cell activation and proliferation by FACS The specificity and efficacy of CD3-specific ankyrin repeat proteins (described in Examples 5, 6, 7, and 8) were evaluated by a FACS-based in vitro long-term T cell activation assay.

[0216] To evaluate drug and target-specific T cell activation and proliferation, effector and target cells were co-incubated in two series at a 1:1 E:T ratio in the presence of serial dilutions of selected molecules. Purified (Miltenyi)pan-T cells (isolated from healthy donor PBMCs) were first labeled with 5 μM CellTrace Violet (CTV) (Thermo Fischer) at 37°C for 20 minutes. 50,000 CTV-labeled pan-T cells + 50,000 TAA2 / TAA3-expressing tumor cells per well were then incubated at 37°C with serial dilutions of selected CD3-specific ankyrin repeat proteins or control benchmark molecules (TCE1 and TCE2) in the presence of 600 μM human serum albumin (to mimic physiological concentrations). After 5 days, the cells were washed with PBS and stained with 1:5,000 Live / Dead Green (Thermo Fisher), 1:100 Mouse anti-human CD8 PE (BD), and 1:100 Mouse anti-human-CD25 PerCP-Cy5.5 (eBiosciences) antibody at 4°C for 30 minutes. After two washes with PBS, the cells were fixed using CellFIX (BD) at 4°C for 20 minutes, and finally the buffer was replaced with PBS. The stained cells were analyzed using a FACS Canto II (BD) instrument. T cell activation was assessed by measuring CD25+ cells on Live / Dead-negative and CD8+ gated T cells. T cell proliferation was assessed by gating Live / Dead-negative and CTV-positive cells. FACS data were analyzed using FlowJo software, and the data were plotted using GraphPad Prism8.

[0217] As shown in Figures 13 and 14, DARPin® protein #2 and DARPin® protein #3 induced long-term T cell activation and proliferation comparable to benchmark molecules (known benchmark T cell engagers TCE1 with binding specificity to TAA2 and TCE2 with binding specificity to TAA3) without extension of half-life, while DARPin® protein #1 and DARPin® protein #4 showed 10 to 100 times lower potency. CD3-specific ankyrin repeat proteins with extended half-lives showed approximately 5 to 30 times lower potency compared to the corresponding molecules without extended half-lives.

[0218] Example 10: In vivo efficacy evaluation of exemplary multispecific TCE-binding proteins in PBMC humanized mice and TAA-expressing tumor models. Experiment A. DARPin® protein #2, DARPin® protein #3, and DARPin® protein #4 were formatted into a quadruple-specific TCE molecule, which, in addition to DARPin® protein #2, DARPin® protein #3, or DARPin® protein #4, contains ankylin repeat domains that have binding specificity to TAA2, TAA3, and human serum albumin. To this end, DARPin® protein #2, DARPin® protein #3, and DARPin® protein #4 were subcloned and expressed in derivatives of the pQE30(Qiagen) expression vector encoding an N-terminal His tag, followed by a human serum albumin-specific ankylin repeat domain, followed by two different TAA-specific (TAA2-specific and TAA3-specific, respectively) ankylin repeat domains, followed by one of the CD3-specific ankylin repeat domains of the present invention, as described above. Ankyrin repeat domains were linked together by peptide linkers such as the linker of Sequence ID No. 31. The corresponding TCE proteins are named herein as DARPin® Protein #F (containing DARPin® Protein #2), DARPin® Protein #G (containing DARPin® Protein #3), and DARPin® Protein #H (containing DARPin® Protein #4), respectively. DARPin® Protein #2 was similarly formatted into a triple-specific TCE molecule, which, in addition to DARPin® Protein #2, contains ankyrin repeat domains that have binding specificity to TAA2 and TAA3, but lacks an HSA-specific ankyrin repeat domain. This TCE protein is named herein as DARPin® Protein #E.DARPin® protein #E, DARPin® protein #F, DARPin® protein #G, and DARPin® protein #H were tested in a humanized mouse model of peripheral blood mononuclear cells (PBMCs) carrying TAA2 / TAA3-expressing tumor cell lines as solid subcutaneous tumors, and compared with known TAA3-targeted T cell engager molecules currently being tested in clinical trials.

[0219] Materials and methods Animals: 60 female NOG mice, 65 days old at the start of the study (provided by Taconic Biosciences)

[0220] Test molecules and control molecules: DARPin® protein #E, DARPin® protein #F, DARPin® protein #G, and DARPin® protein #H were produced at a concentration of 5 mg / ml as previously described; the control was benchmark TAA3-targeted TCE 0.59 mg / ml, provided by Evitria AG.

[0221] Treatment group: 60 mice were enrolled in the study. All animals were randomly assigned to one of eight different study groups. The date of tumor cell inoculation is indicated as day 0.

[0222] Methods: Body weight was recorded two days before the start of the experiment, and mice were randomized to have equal mean body weight and similar standard deviation in each group. The average weight was 19.6 g.

[0223] -On the second day, the mice were given 5 x 10 6 Individual PBMCs were injected intraperitoneally.

[0224] On day 0, 10 6 Individual TAA2 / TAA3-expressing tumor cells were subcutaneously injected.

[0225] On the fifth day, treatment by intraperitoneal injection was initiated according to Table 8. The final treatment was on the 16th day.

[0226] Tumor measurements and weighing were performed on days 7, 10, 12, 14, 17, and 19. Tumor volume was calculated using the following formula: [length × (width)² × π] / 6.

[0227] Tumor volume data were analyzed by comparing growth curves using Anova, followed by a nonparametric Kruskal-Wallis test corrected for multiple comparisons (Dunn test).

[0228] The tumor volume in the treatment group was compared to that of the control group. Data from two different PBMC donors were analyzed together and separately.

[0229] [Table 6]

[0230] result Tumor growth inhibition The tumor growth curves upon exposure to the tested multispecific binding proteins and known benchmark T cell enforcers are summarized in Figures 15 A–C, including both donors together and separately.

[0231] [Table 7] +=p<0.1; ** =p<0.05;+++=p<0-01;++++=p<0.001;ns=not significant

[0232] PBMCs derived from Donor B reduced the amount of CD8-positive lymphocytes in mouse blood. Generally, antitumor effects were observed for all molecules tested. DARPin® protein #F and the known TAA3 T-cell engager showed effects starting from day 5 after the initiation of treatment, while the effects of DARPin® protein #G and DARPin® protein #H were significant and relevant starting from day 7 after the initiation of treatment. When comparing subgroup A only, statistically significant effects were observed for all tested molecules starting from day 5 after the initiation of treatment. The effects of DARPin® protein #F, DARPin® protein #G, and DARPin® protein #H were stronger than those of the benchmark TAA3 T-cell engager and DARPin® protein #E. When comparing subgroup B, significant antitumor effects were observed for DARPin® protein #G and the benchmark TAA3 T-cell engager.

[0233] The results obtained from the benchmark TCE confirmed the model used. At the same time, the less pronounced effect of the benchmark observed in mice humanized with donor B-derived PBMCs indicates that mice belonging to this subgroup are less sensitive. This is also reflected by the significantly lower levels of human CD8-positive lymphocytes found at the end of the experiment in mice humanized with donor B-derived PBMCs.

[0234] Therefore, data obtained from humanized mice using PBMCs derived from donor A will be more relevant.

[0235] The high variability in tumor size is due to the fact that randomization and range selection were not performed in the mice because treatment was initiated before the tumors became recognizable in all mice. Despite this high variability, statistical analysis could be performed without the need for normalization to the initial tumor volume.

[0236] The inventors confirmed that all multispecific binding proteins tested exhibited antitumor activity, and that the half-life extension molecules DARPin® protein #F, DARPin® protein #G, and DARPin® protein #H had stronger antitumor activity than DARPin® protein #E, which does not have an extended half-life, at the doses and regimens used. While not theoretically bound, exposure to DARPin® protein #E may be lower due to the shorter half-life of this molecule.

[0237] Overall, all four multispecific binding proteins tested exhibited in vivo antitumor activity comparable to or stronger than that of benchmark T cell engagers. These results demonstrate that the CD3-specific ankyrin repeat domain of the present invention can be combined with the TAA-specific binding domain (and half-life extension portion) to form a T cell engager molecule with in vivo antitumor activity. Thus, when formatted in such a TCE molecule, the CD3-specific ankyrin repeat domain of the present invention maintains its ability to bind to CD3 in vivo and activate T cells.

[0238] Experiment B. In a similar experimental setup, DARPin® protein #3 was formatted into a hexaspecific TCE molecule further comprising three engineered ankyrin repeat domains with binding specificity to TAA2, TAA3, and TAA4, respectively, and two engineered ankyrin repeat domains with binding specificity to human serum albumin. To this end, DARPin® protein #3 was subcloned and expressed as described above in a derivative of the pQE30 (Qiagen) expression vector encoding an N-terminal His tag, followed by two human serum albumin-specific ankyrin repeat domains, then three different TAA-specific ankyrin repeat domains (in the order of TAA3-specific, TAA2-specific, and TAA4-specific, respectively), and then one of the CD3-specific ankyrin repeat domains of the present invention (DARPin® protein #3). The ankyrin repeat domains were linked to each other by a peptide linker such as the linker of Sequence ID No. 31. This hexaspecific TCE molecule is described in detail in U.S. Provisional Patent Application No. 63 / 265,184, filed on 9 December 2021, the disclosure of which is incorporated herein by reference in its entirety. DARPin® protein #3 in such a multispecific TCE format was tested in a humanized mouse model of peripheral blood mononuclear cells (PBMCs) having a TAA2 / TAA3 / TAA4-expressing tumor cell line (Molm-13) as a solid subcutaneous tumor, and compared to known TAA3-targeted T cell engagers tested in clinical trials.

[0239] In short, in vivo experiments were conducted using 6-9 week old female immunodeficient NXG mice (provided by Anvier Labs). The mice were maintained under standardized environmental conditions in a standard rodent micro-isolator cage (room temperature 20±1°C, relative humidity 50±10%, and a 12-hour light-dark cycle). The mice were given irradiated food and bedding, as well as 0.22 μm filtered drinking water. All experiments were conducted with authorization from the cantonal and federal veterinary authorities in accordance with Swiss animal protection laws.

[0240] NXG mice were injected with hPBMCs intraperitoneally, followed by xenografting of cancer cells. TAA2 / TAA3 / TAA4-expressing tumor cells were (sc) xenografted subcutaneously into the right flank of NXG mice. Two hPBMC donors were used. The procedure was administered intravenously (iv) four days after cancer cell transplantation. Most tumors had not yet been established at this point. The procedure was performed as follows: DARPin® protein #3 (multispecific TCE format) was administered intravenously at a dose of 0.5 mg / kg three times a week for two weeks. The benchmark TAA3-targeted T-cell engager was administered intravenously at a dose of 0.5 mg / kg daily for two weeks.

[0241] Tumor size was evaluated by caliper measurement. Tumor volume was calculated using the following formula: Tumor volume (mm²) 3 ) = 0.5 × length × width 2 .

[0242] As shown in Figure 16(A-B), DARPin® protein #3 in a multispecific TCE format demonstrated good efficacy in inhibiting tumor growth and tumor volume throughout the entire duration of the experiment (Figure 16A) and 17 days after the initial injection (Figure 16B). This result further demonstrates that the CD3-specific ankyrin repeat domain of the present invention can be combined with a TAA-specific binding domain and a half-life extension portion to form a T cell engager molecule with in vivo antitumor activity. Thus, when formatted in such a TCE molecule, the CD3-specific ankyrin repeat domain of the present invention maintains its ability to bind to CD3 in vivo and activate T cells.

[0243] This specification is best understood in light of the teachings of the references cited herein. The embodiments herein are provided as 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 within the invention. All publications, patents, and GenBank sequences cited herein are incorporated by reference in their entirety. Unless the material incorporated by reference is inconsistent with or contradicts this specification, this specification takes precedence over any such material. The references herein do not constitute an admission that such references are prior art to the invention.

[0244] Those skilled in the art will recognize, or can confirm by routine experimentation, many equivalents to the particular aspects of the present invention described herein. Such equivalents are intended to be covered by the following claims.

Claims

1. A recombinant binding protein comprising an ankyrin repeat domain with binding specificity for CD3, wherein the ankyrin repeat domain comprises an ankyrin repeat module having an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 5 to 9 and (2) sequences in which up to 9 amino acids in any of SEQ ID NOs: 5 to 9 are replaced by other amino acids.

2. 2. The binding protein according to claim 1, wherein the ankyrin repeat module is a first ankyrin repeat module and the ankyrin repeat domain further comprises a second ankyrin repeat module having an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 5 to 9 and (2) sequences in which up to 9 amino acids in any of SEQ ID NOs: 5 to 9 are replaced by other amino acids.

3. Binding protein according to claim 2, wherein the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 5 and (2) sequences in which up to 9 amino acids in SEQ ID NO: 5 are replaced by other amino acids, and the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 6 and (2) sequences in which up to 9 amino acids in SEQ ID NO: 6 are replaced by other amino acids.

4. Binding protein according to claim 2, wherein the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 7 and (2) sequences in which up to 9 amino acids in SEQ ID NO: 7 are replaced by other amino acids, and the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 6 and (2) sequences in which up to 9 amino acids in SEQ ID NO: 6 are replaced by other amino acids.

5. Binding protein according to claim 2, wherein the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 7 and (2) sequences in which up to 9 amino acids in SEQ ID NO: 7 are replaced by other amino acids, and the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 8 and (2) sequences in which up to 9 amino acids in SEQ ID NO: 8 are replaced by other amino acids.

6. Binding protein according to claim 2, wherein the first ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 7 and (2) sequences in which up to 9 amino acids in SEQ ID NO: 7 are replaced by other amino acids, and the second ankyrin repeat module comprises an amino acid sequence selected from the group consisting of (1) SEQ ID NO: 9 and (2) sequences in which up to 9 amino acids in SEQ ID NO: 9 are replaced by other amino acids.

7. 7. The binding protein according to any one of claims 2 to 6, wherein the first ankyrin repeat module is located N-terminal to the second ankyrin repeat module within the ankyrin repeat domain.

8. 1. A recombinant binding protein comprising an ankyrin repeat domain with binding specificity for CD3, said ankyrin repeat domain comprising an amino acid sequence having at least 80% amino acid sequence identity with any one of SEQ ID NOs: 1 to 4, wherein A at the penultimate position of SEQ ID NOs: 1 to 4 is optionally replaced by L and / or A at the last position of SEQ ID NOs: 1 to 4 is optionally replaced by N.

9. The binding protein is -7 Dissociation constant (K D 9. The binding protein of claim 1, which binds to human CD3 in PBS at 100 .mu.m.

10. The binding protein has an EC 50 10. The binding protein of claim 1, which binds to human CD3 on T cells at

11. The binding protein of any one of claims 1 to 10, wherein the binding protein further comprises a binding agent that has binding specificity for a disease associated antigen.

12. The binding protein of any one of claims 1 to 11, wherein the binding protein further comprises a half-life extending moiety.

13. A nucleic acid encoding a binding protein according to any one of claims 1 to 12.

14. A pharmaceutical composition comprising a binding protein according to any one of claims 1 to 12 or a nucleic acid according to claim 13, and optionally a pharma- ceutically acceptable carrier and / or diluent.

15. A pharmaceutical composition for treating a medical condition comprising a binding protein according to any one of claims 1 to 12 or a nucleic acid according to claim 13, and optionally a pharma- ceutically acceptable carrier and / or diluent.

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