Novel DARPin-based CD70 engagers

Abstract

The present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD70. Additionally, the present invention relates to a nucleic acid encoding such a recombinant binding protein, a pharmaceutical composition comprising such a protein or nucleic acid, and the use of such a binding protein, nucleic acid or pharmaceutical composition in a mammal, including a human, in a method for treating or diagnosing a disease, such as cancer, e.g. acute myeloid leukemia (AML).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Application No. 63 / 172,973, filed April 9, 2021, U.S. Provisional Application No. 63 / 173,186, filed April 9, 2021, and U.S. Provisional Application No. 63 / 265,181, filed December 9, 2021. The disclosures of these patent applications are incorporated herein by reference in their entirety for all purposes.

[0002] The present invention relates to recombinant binding proteins comprising an ankyrin repeat domain, which has binding specificity for human CD70. In addition, the present invention relates to nucleic acids encoding such recombinant binding proteins, pharmaceutical compositions comprising such proteins or nucleic acids, and the use of such binding proteins, nucleic acids, or pharmaceutical compositions in mammals, including humans, in methods for treating or diagnosing diseases such as cancer, e.g., acute myeloid leukemia (AML). [Background technology]

[0003] Acute myeloid leukemia (AML) is a heterogeneous and complex malignant disease characterized by rapid cell proliferation, an aggressive clinical course, and generally high mortality. Resistance to treatment remains the leading cause of AML-related death (Winer & Stone, Ther Adv Hematol; 2019; 10). While standard chemotherapy-based protocols remain the primary therapeutic approach worldwide, recent advances in immunotherapy have provided effective treatment options for chemotherapy-resistant AML. Such immunotherapeutic approaches include monoclonal antibodies, bispecific antibodies, and chimeric antigen receptor-expressing T cells (CAR-T cells).

[0004] CD70 is expressed on approximately 86–100% of AML blast cells, making it an attractive target for the treatment of cancer, particularly AML (Perna et al., Cancer Cell, 32(4), 2017, 506–519). CD70 is also highly specific for leukemia stem cells. A monoclonal antibody targeting CD70 (cusatuzumab) is currently in clinical trials. Phase I trials showed promising results, but Phase II trials showed disappointing efficacy (see https: / / www.evaluate.com / vantage / articles / news / snippets / argenxs-cusatuzumab-culminates-disappointment). As with many existing antibody therapies, concerns exist regarding its potential safety profile (see https: / / www.clinicaltrialsarena.com / comment / argenxs-cusatuzumab-in-previously-untreated-aml-draws-varied-expert-forecasts / ). Furthermore, downregulation of tumor surface markers targeted by antibodies can lead to tumor resistance to antibody therapy.

[0005] CAR-T cell therapy is an approach that has had a profound impact on the management of lymphoid malignancies. There has been great interest in applying this technology to AML, but in practice, this has proven challenging. With regard to monoclonal antibodies, CD70 is considered one of the most promising targets for CAR-T cell therapy in AML.

[0006] AML is a type of cancer that in many ways exemplifies the challenges of cancer therapy and the shortcomings of currently available cancer therapies, such as low efficacy and a problematic side effect profile. AML remains a significant medical need due to its high mortality rate, and treatment of relapsed or refractory AML remains difficult to treat.

[0007] Thus, there remains a need for new CD70-specific binding proteins with beneficial properties. Such binding proteins may be useful in therapeutic and diagnostic approaches for treating and characterizing diseases such as cancer, e.g., AML. In particular, there is a need for new CD70-specific binding proteins that can serve to specifically target CD70 on cancer cells and that can also be easily combined with other functional moieties, e.g., one or more binding moieties. Summary of the Invention

[0008] The present invention relates to recombinant binding proteins comprising an ankyrin repeat domain, which has binding specificity for human CD70. In addition, the present invention relates to nucleic acids encoding such recombinant binding proteins, pharmaceutical compositions comprising such proteins or nucleic acids, and the use of such binding proteins, nucleic acids, or pharmaceutical compositions in mammals, including humans, in methods for treating or diagnosing diseases such as cancer, e.g., acute myeloid leukemia (AML).

[0009] The recombinant binding proteins of the present invention specifically bind to or target the tumor-associated antigen (TAA) CD70. Such binding proteins of the present invention can serve as tools or building blocks for generating new therapeutic or diagnostic agents. Also disclosed herein are recombinant binding proteins in which a CD70-specific ankyrin repeat domain is combined with one or more other functional moieties in a single molecule. Such other functional moieties include a binding moiety with binding specificity for a target expressed on immune cells, a half-life extending moiety, a binding moiety with binding specificity for another tumor-associated antigen, and / or a cytotoxic agent. Thus, the recombinant binding proteins of the present invention with binding specificity for CD70 are useful for generating novel therapeutic molecules that may offer improved toxicity profiles and / or therapeutic windows compared to current therapies.

[0010] Based on the disclosure provided herein, those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by embodiment (E) below.

[0011] 1. In a first embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity for human CD70, and the ankyrin repeat domain comprises an ankyrin repeat module having an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 24 to 45 and 73 to 77, and (2) sequences in which up to 9 amino acids in any of SEQ ID NOs: 24 to 45 and 73 to 77 are substituted by other amino acids.

[0012] 2. In a second embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity for human CD70, and wherein the ankyrin repeat domain comprises an amino acid sequence having at least 85% amino acid sequence identity to any one of SEQ ID NOs: 1 to 12 and 71 to 72.

[0013] 3. In a third embodiment, the present invention provides an ankyrin repeat domain having a dissociation constant (K D 3. The recombinant binding protein of embodiment 1 or 2, which binds to human CD70 at the C3 domain.

[0014] 4. In a fourth embodiment, the present invention provides a method for treating rheumatoid arthritis, wherein the ankyrin repeat domain has an EC value in the range of about 0.2 nM to about 500 nM. 50 The present invention relates to a recombinant binding protein according to any of the preceding embodiments, which binds to human CD70 at

[0015] 5. In a fifth embodiment, the invention relates to a recombinant binding protein according to any of the previous embodiments, further comprising a binding moiety having binding specificity for a target expressed on an immune cell.

[0016] 6. In a sixth embodiment, the invention relates to a recombinant binding protein according to embodiment 5, wherein said immune cells are T cells and said target expressed on immune cells is CD3.

[0017] 7. In a seventh embodiment, the present invention relates to a recombinant binding protein according to embodiment 5 or 6, wherein the binding moiety having binding specificity for a target expressed on an immune cell is an ankyrin repeat domain.

[0018] 8. In an eighth embodiment, the present invention relates to a recombinant binding protein according to any of embodiments 5 to 7, wherein the binding moiety having binding specificity for a target expressed on an immune cell is an ankyrin repeat domain having binding specificity for human CD3.

[0019] 9. In a ninth embodiment, the present invention relates to a recombinant binding protein according to any of embodiments 5 to 7, wherein the binding moiety with binding specificity for a target expressed on an immune cell is an ankyrin repeat domain with binding specificity for human CD3, and wherein the ankyrin repeat domain with binding specificity for human CD3 comprises an amino acid sequence with at least 85% amino acid sequence identity to any one of SEQ ID NOs: 55 to 59.

[0020] 10. In a tenth embodiment, the present invention relates to a recombinant binding protein according to embodiment 9, wherein said ankyrin repeat domain with binding specificity for human CD3 comprises the amino acid sequence of any one of SEQ ID NOs: 55 to 59.

[0021] 11. In an eleventh embodiment, the present invention relates to a recombinant binding protein according to any of embodiments 5 to 10, wherein the ankyrin repeat domain having binding specificity for human CD70 and the binding moiety having binding specificity for a target expressed on an immune cell are covalently linked via a peptide linker.

[0022] 12. In the twelfth embodiment, the present invention relates to a recombinant binding protein according to embodiment 11, wherein said peptide linker is a proline-threonine rich peptide linker.

[0023] 13. In a thirteenth embodiment, the present invention relates to a recombinant binding protein according to embodiment 11 or 12, wherein the amino acid sequence of the peptide linker has a length of 1 to 50 amino acids.

[0024] 14. In the fourteenth embodiment, the present invention relates to a recombinant binding protein according to any of the previous embodiments, wherein said binding protein further comprises a half-life extending moiety.

[0025] 15. In a fifteenth embodiment, the invention relates to a recombinant binding protein according to embodiment 14, wherein said half-life extending moiety is an ankyrin repeat domain having binding specificity for human serum albumin.

[0026] 16. In a sixteenth embodiment, the present invention relates to a recombinant binding protein according to embodiment 15, wherein the ankyrin repeat domain having binding specificity for human serum albumin comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 52 to 54.

[0027] 17. In the seventeenth embodiment, the present invention relates to a recombinant binding protein according to embodiment 15 or 16, wherein the ankyrin repeat domain having binding specificity for human serum albumin comprises the amino acid sequence of any one of SEQ ID NOs: 52 to 54.

[0028] 18. In the eighteenth embodiment, the invention relates to a recombinant binding protein according to any of the previous embodiments, wherein the binding protein further comprises at least one binding moiety having binding specificity for a target expressed on tumor cells, wherein the target expressed on tumor cells is different from human CD70.

[0029] 19. In a nineteenth embodiment, the present invention relates to a nucleic acid encoding a recombinant binding protein according to any of the previous embodiments.

[0030] 20. In a twentieth embodiment, the present invention relates to a pharmaceutical composition comprising a recombinant binding protein according to any one of embodiments 1 to 18 or a nucleic acid according to embodiment 19, and a pharmaceutically acceptable carrier and / or diluent.

[0031] 21. In a twenty-first embodiment, the present invention relates to a method for immune cell activation in tumor tissue of a human patient, comprising administering to said patient a recombinant binding protein according to any one of embodiments 1 to 18, a nucleic acid according to embodiment 19, or a pharmaceutical composition according to embodiment 20.

[0032] 22. In the 22nd embodiment, the invention relates to the method according to embodiment 21, wherein the immune cells are T cells.

[0033] 22a. In embodiment 22a, the present invention relates to the method of embodiment 21, wherein the immune cells are Natural Killer (NK) cells.

[0034] 23. In a twenty-third embodiment, the present invention relates to a method for treating a medical condition, comprising administering to a patient in need of treatment for the medical condition a therapeutically effective amount of a recombinant binding protein according to any one of embodiments 1 to 18, a nucleic acid according to embodiment 19, or a pharmaceutical composition according to embodiment 20.

[0035] 24. In the 24th embodiment, the invention relates to the method of embodiment 23, wherein the medical condition is cancer.

[0036] 25. In the 25th embodiment, the invention relates to the method according to embodiment 23, wherein the medical condition is a cancer characterized by a liquid tumor.

[0037] 26. In the 26th embodiment, the invention relates to the method of embodiment 23, wherein the medical condition is leukemia.

[0038] 27. In the 27th embodiment, the invention relates to the method of embodiment 23, wherein the medical condition is acute myeloid leukemia.

[0039] 28. In a 28th embodiment, the present invention relates to a recombinant binding protein according to any one of embodiments 1 to 18, a nucleic acid according to embodiment 19, or a pharmaceutical composition according to embodiment 20, for use in therapy.

[0040] 29. In a twenty-ninth embodiment, the present invention relates to a recombinant binding protein according to any one of embodiments 1 to 18, to a nucleic acid according to embodiment 19, or to a pharmaceutical composition according to embodiment 20, for use in the treatment of cancer, optionally for use in the treatment of cancer characterized by liquid tumors.

[0041] 30. In a thirtieth embodiment, the present invention relates to a recombinant binding protein, or a nucleic acid, or a pharmaceutical composition for use according to embodiment 29, wherein the cancer is leukemia, optionally wherein the cancer is acute myeloid leukemia. [Brief explanation of the drawings]

[0042] [Figure 1]Surface plasmon resonance (SPR) analysis of DARPin® protein #24 binding to human CD70. Various concentrations of purified ankyrin repeat protein were applied to a GLC chip with immobilized human CD70 for on-rate and off-rate measurements. The resulting SPR traces were used to analyze and determine the binding of ankyrin repeat protein to CD70. RU, resonance units; s, time (seconds). [Figure 2] Binding of exemplary binding proteins of the invention to CD70-expressing tumor cells. Concentration-dependent binding curves for DARPin® Protein #2, DARPin® Protein #9, DARPin® Protein #24, and DARPin® Protein 25 are shown. [Figure 3] Short-term T cell activation was determined by measuring the activation marker CD25. Pan-effector T cells and Molm-13 target cells were incubated at an E:T ratio of 5:1, and T cell activation was assessed 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. Figure 1 shows T cell activation induced by selected ankyrin repeat proteins: DARPin® Protein #28, DARPin® Protein #29, DARPin® Protein #26, and DARPin® Protein #27. [Figure 4] Tumor cell killing assessed by a cytotoxicity assay measuring LDH release. Pan-effector T cells and Molm-13 target cells were incubated at an E:T ratio of 5:1, and tumor cell killing was assessed by FACS after 24 hours of co-culture in the presence of serial dilutions of the indicated molecules. Figure 1 shows tumor cell killing by T cells induced by selected ankyrin repeat proteins: DARPin® Protein #28, DARPin® Protein #29, DARPin® Protein #26, and DARPin® Protein #27. [Figure 5A]Binding of different CD70-binding molecules immobilized on a plate to the human CD70 target in the presence or absence of DARPin® Protein #2 or mAb ARGX-110 analog as competitors by ELISA. After preincubation of biotinylated human CD70 target with or without competitor (DARPin® Protein #2 or ARGX-110 analog), binding to immobilized DARPin® Protein #2 (FIG. 5A), CD27 (FIG. 5B), or ARGX-110 analog (FIG. 5C) was determined using an anti-streptavidin-POD detection agent. Competitive binding to CD70 was observed for all molecules tested, i.e., DARPin® Protein #2, CD27, and ARGX-110 analog. [Figure 5B] Binding of different CD70-binding molecules immobilized on a plate to the human CD70 target in the presence or absence of DARPin® Protein #2 or mAb ARGX-110 analog as competitors by ELISA. After preincubation of biotinylated human CD70 target with or without competitor (DARPin® Protein #2 or ARGX-110 analog), binding to immobilized DARPin® Protein #2 (FIG. 5A), CD27 (FIG. 5B), or ARGX-110 analog (FIG. 5C) was determined using an anti-streptavidin-POD detection agent. Competitive binding to CD70 was observed for all molecules tested, i.e., DARPin® Protein #2, CD27, and ARGX-110 analog. [Figure 5C]Binding of different CD70-binding molecules immobilized on a plate to the human CD70 target in the presence or absence of DARPin® Protein #2 or mAb ARGX-110 analog as competitors by ELISA. After preincubation of biotinylated human CD70 target with or without competitor (DARPin® Protein #2 or ARGX-110 analog), binding to immobilized DARPin® Protein #2 (FIG. 5A), CD27 (FIG. 5B), or ARGX-110 analog (FIG. 5C) was determined using an anti-streptavidin-POD detection agent. Competitive binding to CD70 was observed for all molecules tested, i.e., DARPin® Protein #2, CD27, and ARGX-110 analog. [Figure 6A] Figure 6A: Tumor growth over time in mice (n=5 mice per donor / 2 hPBMC donors) injected intraperitoneally with hPBMCs, xenografted subcutaneously with MOLM-13 tumor cells 2 days after hPBMC injection, and treated with PBS 1x (filled circles) or 0.5 mg / kg of DARPin® Protein #24 in multidomain format (filled squares). Treatment began 4 days after tumor cell xenografting. Data are presented as mean + SEM. Figure 6B: Assessment of tumor volume 17 days after tumor cell xenografting in the mice described in Figure 6A. [Figure 6B] Figure 6A: Tumor growth over time in mice (n=5 mice per donor / 2 hPBMC donors) injected intraperitoneally with hPBMCs, xenografted subcutaneously with MOLM-13 tumor cells 2 days after hPBMC injection, and treated with PBS 1x (filled circles) or 0.5 mg / kg of DARPin® Protein #24 in multidomain format (filled squares). Treatment began 4 days after tumor cell xenografting. Data are presented as mean + SEM. Figure 6B: Assessment of tumor volume 17 days after tumor cell xenografting in the mice described in Figure 6A. [Figure 7] Potency titration curve (T cell activation) of DARPin® Protein #24 in multidomain format using wild-type or CD70 knockout Molm-13 tumor cells. EC50 values ​​are shown in pM. DETAILED DESCRIPTION OF THE INVENTION

[0043] The present invention relates to recombinant binding proteins comprising an ankyrin repeat domain, which has binding specificity for human CD70. In addition, the present invention relates to nucleic acids encoding such recombinant binding proteins, pharmaceutical compositions comprising such proteins or nucleic acids, and the use of such binding proteins, nucleic acids, or pharmaceutical compositions in mammals, including humans, in methods for treating or diagnosing diseases such as cancer, e.g., acute myeloid leukemia (AML).

[0044] Ankyrin repeat domain The described recombinant binding proteins or binding domains thereof comprising a designed ankyrin repeat motif or module are also referred to herein as DARPin® proteins (see Stumpp et al., Curr Opin Drug Discov Devel. 10(2):153-9 (2007) and Binz et al., Nature Biotech. 22(5):575-582 (2004)). DARPin® proteins can be considered as antibody mimics with high specificity and high binding affinity for target proteins. Generally, DARPin® proteins comprise at least one ankyrin repeat domain, for example, at least one, two, three, four, five or more ankyrin repeat domains.

[0045] The ankyrin repeat domains described herein generally comprise a core scaffold that provides structure and target binding residues that bind to the target: the structural core comprises conserved amino acid residues, and the target binding surface comprises amino acid residues that vary depending on the target.

[0046] Designed ankyrin repeat protein libraries (WO 2002 / 020565; Binz et al., Nat. Biotechnol. 22, 575-582, 2004; Stumpp et al., Drug Discov. Today 13, 695-701, 2008) can be used to select target-specific designed 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 are a type of binding molecule that has the potential to overcome the limitations of monoclonal antibodies, thus enabling novel therapeutic approaches. Such ankyrin repeat proteins may contain a single designed ankyrin repeat domain or a combination of two or more designed ankyrin repeat domains with the same or different target specificities (Stumpp et al., Drug Discov. Today 13, 695-701, 2008; U.S. Pat. No. 9,458,211). Ankyrin repeat proteins containing only a single designed ankyrin repeat domain are small proteins (14 kDa) that can be selected to bind to a given target protein with high affinity and specificity. These features, and the ability to combine two or more designed ankyrin repeat domains into one protein, make designed ankyrin repeat proteins ideal agonist, antagonist, and / or inhibitor candidates. Furthermore, such ankyrin repeat proteins can be engineered to carry various effector functions, such as cytotoxic agents or half-life extenders, enabling entirely new drug formats.

[0047] Designed ankyrin repeat proteins can also target epitopes that are not easily accessible by monoclonal antibodies. A further advantage of the described designed ankyrin repeat proteins is that they are generally less immunogenic and have no or minimal off-target effects. DARPin® candidates also exhibit favorable development properties, such as being produced quickly, at low cost, with high yields, and being storable at 4°C for up to several years. In summary, designed ankyrin repeat proteins are an example of a next-generation protein therapeutic that has the potential to surpass existing antibody drugs.

[0048] DARPin® is a trademark owned by Molecular Partners AG, Switzerland.

[0049] As mentioned above, CD70 is an attractive therapeutic target for the treatment of certain cancers, particularly AML. The recombinant binding proteins described herein contain ankyrin repeat domains and ankyrin repeat modules that specifically bind to human CD70.

[0050] 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 for human CD70, and the ankyrin repeat domain comprises an ankyrin repeat module having an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 24 to 45 and 73 to 77, and (2) sequences in which up to 9 amino acids in any of SEQ ID NOs: 24 to 45 and 73 to 77 are substituted by other amino acids.

[0051] 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 for human CD70, and the ankyrin repeat domain comprises an ankyrin repeat module having an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 24 to 45 and 73 to 77, and (2) sequences in which at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 amino acid in any of SEQ ID NOs: 24 to 45 and 73 to 77 is substituted by another amino acid.

[0052] 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 for human CD70, and wherein the ankyrin repeat domain comprises an ankyrin repeat module having an amino acid sequence selected from the group consisting of SEQ ID NOs: 24 to 45 and 73 to 77.

[0053] 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 for human CD70, and wherein the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity to any one of SEQ ID NOs: 1-12 and 71-72.

[0054] In a further embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity for human CD70, and wherein the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% amino acid sequence identity, to any one of SEQ ID NOs: 1-12 and 71-72.

[0055] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity for human CD70, and wherein the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% amino acid sequence identity, to SEQ ID NO: 1.

[0056] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity for human CD70, and wherein the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% amino acid sequence identity, to SEQ ID NO:2.

[0057] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity for human CD70, and wherein the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% amino acid sequence identity, to SEQ ID NO:3.

[0058] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity for human CD70, and wherein the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% amino acid sequence identity, to SEQ ID NO:4.

[0059] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity for human CD70, and wherein the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% amino acid sequence identity, to SEQ ID NO:5.

[0060] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity for human CD70, and wherein the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% amino acid sequence identity, to SEQ ID NO: 6.

[0061] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity for human CD70, and wherein the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% amino acid sequence identity, to SEQ ID NO: 7.

[0062] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity for human CD70, and wherein the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% amino acid sequence identity, to SEQ ID NO:8.

[0063] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity for human CD70, and wherein the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% amino acid sequence identity, to SEQ ID NO:9.

[0064] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity for human CD70, and wherein the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% amino acid sequence identity, to SEQ ID NO: 10.

[0065] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity for human CD70, and wherein the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% amino acid sequence identity, to SEQ ID NO: 11.

[0066] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity for human CD70, and wherein the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% amino acid sequence identity, to SEQ ID NO: 12.

[0067] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity for human CD70, and wherein the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% amino acid sequence identity, to SEQ ID NO: 71.

[0068] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity for human CD70, and wherein the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% amino acid sequence identity, to SEQ ID NO: 72.

[0069] In a further embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity for human CD70, and wherein the ankyrin repeat domain comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-12 and 71-72.

[0070] In a further aspect, the invention relates to a recombinant binding protein as described above, further comprising at least one binding moiety that has binding specificity for a target expressed on an immune cell. In one embodiment, the immune cell is a T cell. In another embodiment, the immune cell is a natural killer (NK) cell. Examples of binding moieties that have binding specificity for a target expressed on an immune cell for use in the invention include antibodies, surrogate scaffolds, and polypeptides.

[0071] Antibodies include any polypeptide or protein comprising an antigen-binding domain derived from an antibody or immunoglobulin molecule. The antigen-binding domain can be derived from, for example, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and single-domain antibodies, such as heavy chain variable domains (VH), light chain variable domains (VL), and variable domains (VHH), for example, of human or camelid origin. In some instances, it is beneficial for the antigen-binding domain to be derived from the same species in which the binding moiety will ultimately be used. For example, for use in humans, it may be beneficial for the antigen-binding domain of the binding moieties described herein to comprise a human or humanized antigen-binding domain. Antibodies can be obtained using techniques well known in the art.

[0072] In one embodiment, the binding moiety that has binding specificity for a target expressed on an immune cell is an antibody.

[0073] In one embodiment, the binding moiety having binding specificity for a target expressed on immune cells is a camelid nanobody. Camelid nanobodies (also known as camelid single domain antibodies or VHHs) are derived from the camelid family of mammals, including llamas, camels, and alpacas. Unlike other antibodies, camelid antibodies lack light chains and are composed of two identical heavy chains. Camelid antibodies typically have a relatively low molecular weight, roughly in the range of 15 kDa.

[0074] In one embodiment, the binding moiety that has binding specificity for a target expressed on an immune cell is a shark antibody domain. Like camelid nanobodies, shark antibody domains also lack light chains.

[0075] Alternative scaffolds include any polypeptide or protein that can bind to an antigen (such as a drug molecule) and that contains a binding domain that is not derived from an antibody or immunoglobulin molecule. The binding domain of alternative scaffolds may comprise or be derived from a variety of different polypeptide or protein structures. Alternative scaffolds include, but are not limited to, adnectins (monobodies), affibodies, affilins, affimers and aptamers, affitins, alphabodies, anticalins, armadillo repeat protein-based scaffolds, atrimers, avimers, ankyrin repeat protein-based scaffolds (e.g., DARPin® proteins), fynomers, knottins, and Kunitz domain peptides. Alternative scaffolds are described, for example, in Yu et al., Annu Rev Anal Chem (Palo Alto Calif). 2017 June 12;10(1):293-320. doi:10.1146 / annurevanchem-061516-045205.

[0076] In one embodiment, the binding moiety that has binding specificity for a target expressed on an immune cell is a surrogate scaffold. In one embodiment, the binding moiety that has binding specificity for a target expressed on an immune cell comprises an adnectin, a monobody, an affibody, an affilin, an affimer, an aptamer, an affitin, an alphabody, an anticalin, a repeat protein domain, an armadillo repeat domain, an atrimer, an avimer, an ankyrin repeat domain, a fynomer, a knottin, a Kunitz domain, or an antigen binding domain derived from or related to a T cell receptor (TCR).

[0077] Adnectins originally originated from the tenth extracellular domain of the human fibronectin type III protein (10Fn3). The fibronectin type III domain has seven or eight beta strands distributed between two beta sheets that pack together to form the core of the protein, and further contains solvent-exposed loops (similar to CDRs) that connect the beta strands to each other. At least three such loops are present on each edge of the beta sheet sandwich, and the edges are the boundaries of the protein perpendicular to the direction of the beta strands (see U.S. Pat. No. 6,818,418). Because of this structure, these non-antibody scaffolds mimic the nature and affinity of antigen-binding properties of antibodies. These scaffolds can be used in in vitro loop randomization and shuffling techniques, which are similar to the in vivo process of antibody affinity maturation.

[0078] Affibody affinity ligands are composed of a three-helix bundle based on a scaffold from one of the IgG-binding domains of Protein A, a surface protein from the bacterium Staphylococcus aureus. This scaffold domain consists of 58 amino acids, 13 of which are randomized to generate affibody libraries with a large number of ligand variants (see, e.g., U.S. Pat. No. 5,831,012). Affibody molecules mimic antibodies, but have a much smaller molecular weight of approximately 6 kDa compared to the roughly 150 kDa of antibodies. Despite the difference in size, the binding site of affibody molecules shares similarities with that of antibodies.

[0079] Affilins are synthetic antibody mimics structurally derived from human ubiquitin (and historically from gamma-B crystallin). Affilins consist of two identical domains with a primarily beta-sheet structure and a total molecular mass of approximately 20 kDa. They contain several surface-exposed amino acids suitable for modification. Affilins resemble antibodies in their affinity and specificity for antigens, but not in structure.

[0080] Affimers are a type of peptide aptamer with a structure known as SQT (Stefin A quadruple mutant-Tracy). Aptamers and affimers are short peptides responsible for affinity binding, with an inert, rigid protein scaffold for structural constraint, where both the N- and C-termini of the binding peptide are embedded in the inert scaffold.

[0081] Affitins are variants of the DNA-binding protein Sac7d that have been engineered to have specific binding affinities. Sac7d, originally derived from the hyperthermophilic archaeon Sulfolobus acidocaldarius, binds to DNA and prevents its thermal denaturation. Affitins are commercially known as nanofitins.

[0082] Alphabodies are small (approximately 10 kDa) proteins engineered to bind to various antigens and are therefore antibody mimics. Alphabody scaffolds are computationally designed based on coiled-coil structures. A standard alphabody scaffold contains three α-helices, each composed of four heptad repeats (stretches of seven residues), connected via a glycine / serine-rich linker. The standard heptad sequence is "IAAIQKQ." The ability of alphabodies to target extracellular and intracellular proteins, combined with their high binding affinity, may enable them to bind to targets that cannot be reached by antibodies.

[0083] Anticalins are a group of binding proteins with a rigid and conserved β-barrel structure found in lipocalins, a type of extracellular protein containing a single peptide chain (150–190 amino acids) that is responsible for the recognition, storage, and transport of various biological molecules, including signaling molecules.

[0084] Armadillo repeat protein-based scaffolds are abundant in eukaryotes and are involved in a wide range of biological processes, particularly those related to nuclear transport. Armadillo repeat protein-based scaffolds typically consist of three to five internal repeats and two capping elements. They also possess a tandem extended superhelical structure, which can bind corresponding peptide ligands in an extended conformation.

[0085] Atrimers are scaffolds derived from a trimeric plasma protein known as tetranectin, which belongs to the family of C-type lectins, consisting of three identical units. The structure of the C-type lectin domain (CTLD) in tetranectin contains five flexible loops that mediate interactions with targeting molecules.

[0086] Avimers are derived from naturally occurring A-domain-containing proteins, such as HER3, and consist of several different "A-domain" monomers (2-10) joined via amino acid linkers. For example, methods described in U.S. Patent Application Publication Nos. 2004 / 0175756, 2005 / 0053973, 2005 / 0048512, and 2006 / 0008844 can be used to generate avimers capable of binding to target antigens.

[0087] Fynomer is a small globular protein (approximately 7 kDa) evolved from amino acids 83-145 of the Src homology domain 3 (SH3) of the human Fyn tyrosine kinase. Fynomer is an attractive binding molecule due to its high thermal stability, cysteine-free scaffold, and human origin, reducing potential immunogenicity.

[0088] Knottins, also known as cysteine ​​knot miniproteins, are typically 30 amino acid long proteins containing three antiparallel β-sheets and a constraining loop bound by disulfide bonds that create a cysteine ​​knot, which confers high thermal stability and makes knottins attractive antibody mimics.

[0089] Kunitz domain peptides or Kunitz domain inhibitors are a class of protease inhibitors with an irregular secondary structure containing approximately 60 amino acids with three disulfide bonds and three loops that can be mutated without destabilizing the structural framework.

[0090] In one embodiment, the binding moiety that has binding specificity for a target expressed on an immune cell is a polypeptide or protein that comprises an antigen-binding domain derived from a T cell receptor (TCR).

[0091] In a preferred embodiment, the binding moiety with binding specificity for a target expressed on an immune cell is an ankyrin repeat domain.

[0092] The nature of the target expressed on the immune cell is not particularly limited. In one embodiment, the target is expressed on an immune cell that is a T cell, and the target expressed on the immune cell is CD3.

[0093] Thus, in a preferred embodiment, the present invention relates to a recombinant binding protein comprising: (i) a first ankyrin repeat domain having binding specificity for human CD70 and comprising an ankyrin repeat module having an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 24 to 45 and 73 to 77, and (2) sequences in which at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 amino acid in any of SEQ ID NOs: 24 to 45 and 73 to 77 is substituted by another amino acid; and (ii) a second ankyrin repeat domain having binding specificity for CD3, more preferably human CD3.

[0094] In another embodiment, the present invention provides a method for the production of a medicament for the treatment of dengue fever, comprising: (i) a first ankyrin repeat domain, which has binding specificity for human CD70 and comprises an ankyrin repeat module having an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 24 to 45 and 73 to 77; and (2) sequences in which at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 amino acid in any of SEQ ID NOs: 24 to 45 and 73 to 77 is substituted by another amino acid; and (ii) a second ankyrin repeat domain, which has binding specificity for human CD70 and comprises an ankyrin repeat module having an amino acid sequence selected from the group consisting of: and a second ankyrin repeat domain having binding specificity for SEQ ID NO:3 and comprising an amino acid sequence having at least about 85% amino acid sequence identity, e.g., at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% amino acid sequence identity, to any one of SEQ ID NOs:55 to 59.

[0095] In a further embodiment, the invention relates to a recombinant binding protein comprising: (i) a first ankyrin repeat domain, which has binding specificity for human CD70 and comprises an ankyrin repeat module having an amino acid sequence selected from the group consisting of SEQ ID NOs: 24 to 45 and 73 to 77; and (ii) a second ankyrin repeat domain, which has binding specificity for human CD3 and comprises an amino acid sequence having at least about 85% amino acid sequence identity, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% amino acid sequence identity, to any one of SEQ ID NOs: 55 to 59.

[0096] In another embodiment, the present invention relates to a recombinant binding protein comprising: (i) a first ankyrin repeat domain, which has binding specificity for human CD70 and comprises an ankyrin repeat module having an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 24 to 45 and 73 to 77, and (2) sequences in which at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 amino acid in any of SEQ ID NOs: 24 to 45 and 73 to 77 is substituted by another amino acid; and (ii) a second ankyrin repeat domain, which has binding specificity for human CD3 and comprises the amino acid sequence of any one of SEQ ID NOs: 55 to 59.

[0097] In another embodiment, the present invention relates to a recombinant binding protein comprising: (i) a first ankyrin repeat domain, which has binding specificity for human CD70 and comprises an ankyrin repeat module having an amino acid sequence selected from the group consisting of SEQ ID NOs: 24 to 45 and 73 to 77; and (ii) a second ankyrin repeat domain, which has binding specificity for human CD3 and comprises the amino acid sequence of any one of SEQ ID NOs: 55 to 59.

[0098] In another preferred embodiment, the present invention relates to a recombinant binding protein comprising: (i) a first ankyrin repeat domain, which has binding specificity for human CD70 and comprises an amino acid sequence having at least about 85% amino acid sequence identity, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% amino acid sequence identity, to any one of SEQ ID NOs: 1-12 and 71-72; and (ii) a second ankyrin repeat domain, which has binding specificity for CD3, more preferably human CD3.

[0099] In another embodiment, the present invention provides (i) a first ankyrin repeat domain, which has binding specificity for human CD70 and comprises an amino acid sequence having at least about 85% amino acid sequence identity, e.g., at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% amino acid sequence identity, to any one of SEQ ID NOs: 1-12 and 71-72. and (ii) a second ankyrin repeat domain, which has binding specificity for human CD3 and comprises an amino acid sequence having at least about 85% sequence identity, e.g., at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% amino acid sequence identity, to any one of SEQ ID NOs: 55 to 59.

[0100] In one embodiment, the invention relates to a recombinant binding protein comprising: (i) a first ankyrin repeat domain, which has binding specificity for human CD70 and comprises the amino acid sequence of any one of SEQ ID NOs: 1-12 and 71-72; and (ii) a second ankyrin repeat domain, which has binding specificity for human CD3 and comprises an amino acid sequence having at least about 85% sequence identity, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% amino acid sequence identity, to any one of SEQ ID NOs: 55-59.

[0101] In one embodiment, the invention relates to a recombinant binding protein comprising: (i) a first ankyrin repeat domain, which has binding specificity for human CD70 and comprises an amino acid sequence having at least about 85% amino acid sequence identity, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% amino acid sequence identity, to any one of SEQ ID NOs: 1-12 and 71-72; and (ii) a second ankyrin repeat domain, which has binding specificity for human CD3 and comprises the amino acid sequence of any one of SEQ ID NOs: 55-59.

[0102] In a further embodiment, the present invention relates to a recombinant binding protein comprising (i) a first ankyrin repeat domain, which has binding specificity for human CD70 and comprises the amino acid sequence of any one of SEQ ID NOs: 1 to 12 and 71 to 72, and (ii) a second ankyrin repeat domain, which has binding specificity for human CD3 and comprises the amino acid sequence of any one of SEQ ID NOs: 55 to 59.

[0103] The present invention further relates to a recombinant binding protein comprising (i) a first ankyrin repeat domain having binding specificity for human CD70 and (ii) a second ankyrin repeat domain having binding specificity for human CD3, wherein the recombinant binding protein comprises an amino acid sequence having at least about 85% amino acid sequence identity, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% amino acid sequence identity, to any one of SEQ ID NOs: 13-23 and 78-81.

[0104] In one embodiment, the present invention relates to a recombinant binding protein comprising (i) a first ankyrin repeat domain having binding specificity for human CD70 and (ii) a second ankyrin repeat domain having binding specificity for human CD3, wherein the recombinant binding protein comprises an amino acid sequence having at least about 85% amino acid sequence identity to any one of SEQ ID NOs: 13-23 and 78-81.

[0105] Half-life extension moiety A "half-life extending moiety" extends the in vivo serum half-life of a recombinant binding protein described herein compared to the same protein without the half-life extending moiety. Examples of half-life extending moieties include, but are not limited to, polyhistidine, Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A, protein G, an immunoglobulin domain, maltose binding protein (MBP), a human serum albumin (HSA) binding domain, or polyethylene glycol (PEG).

[0106] In some embodiments, the recombinant binding proteins described herein comprise an ankyrin repeat domain that specifically binds to serum albumin (e.g., preferably human serum albumin), also referred to herein as a "serum albumin binding domain." The recombinant binding proteins described herein may also comprise more than one serum albumin binding domain, e.g., two or three serum albumin binding domains. Thus, the recombinant binding proteins described herein may comprise a first and a second serum albumin binding domain, or a first, second, and third serum albumin binding domain. The embodiments provided below describe such a first serum albumin binding domain, a second serum albumin binding domain, and / or a third serum albumin binding domain.

[0107] In some embodiments, the half-life extending moieties described herein comprise a serum albumin-specific ankyrin repeat domain comprising an amino acid sequence that is at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical to any one of SEQ ID NOs: 52-54. In exemplary embodiments, the half-life extending moieties described herein comprise an amino acid sequence that is at least about 90% identical to any one of SEQ ID NOs: 52-54. In one embodiment, the half-life extending moieties described herein comprise an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 53. In an exemplary embodiment, the half-life extending moieties described herein comprise an amino acid sequence that is at least 90% identical to SEQ ID NO: 53.

[0108] In some embodiments, the serum albumin binding domain is located at the N-terminus of the recombinant binding protein of the invention. In some embodiments, two or more serum albumin binding domains are preferred. In some embodiments, two serum albumin binding domains are located at the N-terminus of the recombinant binding protein of the invention.

[0109] In some embodiments, the half-life extending moiety comprises an immunoglobulin domain. In some embodiments, the immunoglobulin domain comprises an Fc domain. In some embodiments, the Fc domain is derived from any one of the known heavy chain isotypes: IgG(γ), IgM(μ), IgD(δ), IgE(ε), or IgA(α). In some embodiments, the Fc domain is derived from any one of the known heavy chain isotypes or subtypes: IgG1(γ1), IgG2(γ2), IgG3(γ3), IgG4(γ4), IgA1(α1), or IgA2(α2). In some embodiments, the Fc domain is a human IgG1 Fc domain.

[0110] In some embodiments, the Fc domain comprises the uninterrupted native sequence of the Fc domain (i.e., the wild-type sequence). In some embodiments, the immunoglobulin Fc domain comprises a variant Fc domain that results in altered biological activity. For example, at least one point mutation or deletion may be introduced into the Fc domain to reduce or eliminate effector activity (e.g., WO 2005 / 063815) and / or to increase homogeneity during production of the recombinant binding protein. In some embodiments, the Fc domain is the Fc domain of human IgG1 and comprises one or more of the following effector-null substitutions: L234A, L235A, and G237A (Eu numbering). In some embodiments, the Fc domain does not comprise the lysine located at the C-terminal position of human IgG1 (i.e., K447 according to Eu numbering). The absence of a lysine may increase homogeneity during production of the recombinant binding protein. In some embodiments, the Fc domain comprises a lysine located at the C-terminal position (K447, Eu numbering).

[0111] Further binding moieties with binding specificity for tumor-associated antigens In a further aspect, the present invention relates to the above-described recombinant binding protein, further comprising at least one binding moiety having binding specificity for a tumor-associated antigen (TAA) other than CD70. In one embodiment, the one or more TAAs other than CD70 are TAA co-expressed with CD70 in cells derived from the same cancer. In a further embodiment, the one or more TAAs other than CD70 are TAA co-expressed with CD70 in cancers characterized by liquid tumors. In a preferred embodiment, the one or more TAAs other than CD70 are TAA co-expressed with CD70 in leukemia, e.g., TAA co-expressed with CD70 by AML cancer cells. Examples of binding moieties having binding specificity for a tumor-associated antigen (TAA) other than CD70 for use in the present invention include antibodies, surrogate scaffolds, and polypeptides. Many TAAs, including TAA expressed in AML cancer cells, are known in the art.

[0112] replacement In some embodiments, in any ankyrin repeat module of a recombinant binding protein of the invention, the sequences of SEQ ID NOs: 24-45 and 73-77 have 9 or less, 8 or less, 7 or less, 6 or less, 5 or less, 4 or less, 3 or less, 2 or less, or 1 or less substitutions. In some embodiments, the sequences of SEQ ID NOs: 24-45 and 73-77 have 5 or less substitutions. In some embodiments, the sequences of SEQ ID NOs: 24-45 and 73-77 have 4 or less substitutions. In some embodiments, the sequences of SEQ ID NOs: 24-45 and 73-77 have 3 or less substitutions. In some embodiments, the sequences of SEQ ID NOs: 24-45 and 73-77 have 2 or less substitutions. In some embodiments, the sequences of SEQ ID NOs: 24-45 and 73-77 have 1 or less substitutions.

[0113] In some embodiments, 15% or less, 14% or less, 13% or less, 12% or less, 11% or less, 10% or less, 9% or less, 8% or less, or 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, or 1% or less of the amino acid sequence of any ankyrin repeat domain of a recombinant binding protein of the invention is altered by substitutions relative to the sequence of SEQ ID NOs: 1-12 and 71-72. In some embodiments, 10% or less of the amino acid sequence is altered by substitutions relative to the sequence of SEQ ID NOs: 1-12 and 71-72. In some embodiments, 8% or less of the amino acid sequence is altered by substitutions relative to the sequence of SEQ ID NOs: 1-12 and 71-72. In some embodiments, 6% or less of the amino acid sequence is altered by substitutions relative to the sequence of SEQ ID NOs: 1-12 and 71-72. In some embodiments, 4% or less of the amino acid sequence is altered by substitutions relative to the sequence of SEQ ID NOs: 1-12 and 71-72. In some embodiments, no more than 2% of the amino acid sequence is changed by substitution relative to the sequences of SEQ ID NOs: 1-12 and 71-72.

[0114] In some embodiments, the amino acid substitutions made to the binding agent are D The value is the K DFor example, in some embodiments, the amino acid substitutions do not alter the K D The value is the K of a binding agent comprising any of the sequences of SEQ ID NOs: 1 to 12, 24 to 45, and 71 to 77 against CD70. D The value does not change by more than about 1000-fold, more than about 300-fold, more than about 100-fold, more than about 50-fold, more than about 25-fold, more than about 10-fold, or more than about 5-fold compared to the value.

[0115] In certain embodiments, the substitutions are conservative substitutions as set out in Table 1. In certain embodiments, the substitutions are made outside the structural core residues of the ankyrin repeat domain, for example within the beta loops connecting the alpha helices. [Table 1]

[0116] In certain embodiments, substitutions are made within the structural core residues of the ankyrin repeat domain. In other embodiments, substitutions are made outside the structural core residues of the ankyrin repeat domain. For example, the ankyrin domain may comprise the consensus sequence: xDxxGxTPLHLAxxxGxxxlVxVLLxxGADVNA (SEQ ID NO: 62), where "x" represents any amino acid (preferably not cysteine, glycine, or proline), or xDxxGxTPLHLAAxxGHLEIVEVLLKzGADVNA (SEQ ID NO: 63), where "x" represents any amino acid (preferably not cysteine, glycine, or proline) and "z" is selected from the group consisting of asparagine, histidine, or tyrosine. In one embodiment, substitutions are made in residues designated as "x." In another embodiment, substitutions are made in residues not designated as "x."

[0117] In addition, the penultimate position of any ankyrin repeat domain of the recombinant binding protein of the invention may be "A" or "L" and / or the last position may be "A" or "N". Thus, in some embodiments, each ankyrin repeat domain comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 1-12, 52-59, and 71-72, optionally wherein A in the penultimate position is substituted with L and / or A in the last position is substituted with N, or optionally wherein L in the penultimate position is substituted with A and / or N in the last position is substituted with A. In exemplary embodiments, each ankyrin repeat domain comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 1-12, 52-58, and 71-72, optionally in which A at the penultimate position is substituted with L and / or A at the last position is substituted with N. Furthermore, the sequence of any ankyrin repeat domain comprised in a binding protein of the invention may optionally comprise G, S, or GS at its N-terminus (see below).

[0118] In addition, each ankyrin repeat domain comprised in a recombinant binding protein of the invention may optionally comprise a "G", "S", or "GS" sequence at its N-terminus. Thus, in some embodiments, each ankyrin repeat domain comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to any one of SEQ ID NOs: 1-12, 52-59, and 71-72, and further comprises GS at its N-terminus (e.g., as in SEQ ID NOs: 61 and 64), or contains only G or S instead of GS.

[0119] binding affinity In certain embodiments, the affinity between the recombinant binding protein and its target (i.e., human CD70) is D In an exemplary embodiment, K D is about 10 -1 M or less, about 10 -2 M or less, about 10 -3 M or less, about 10 -4 M or less, about 10 -5 M or less, about 10 -6 M or less, about 10 -7 M or less, about 10 -8 M or less, about 10 -9 M or less, about 10 -10 M or less, about 10 -11 M or less, about 10 -12 M or less, about 10 -13 M or less, about 10 -14 M or less, about 10 -5 M ~ about 10 -15 M, about 10 -6 M ~ about 10 -15 M, about 10 -7 M ~ about 10 -15 M, about 10 -8 M ~ about 10 -15 M, about 10 -9 M ~ about 10-15 M, about 10 -10 M~about 10 -15 M, about 10 -5 M~about 10 -14 M, about 10 -6 M~about 10 -14 M, about 10 -7 M~about 10 -14 M, about 10 -8 M~about 10 -14 M, about 10 -9 M~about 10 -14 M, about 10 -10 M~about 10 -14 M, about 10 -5 M~about 10 -13 M, about 10 -6 M~about 10 -13 M, about 10 -7 M~about 10 -13 M, about 10 -8 M~about 10 -13 M, about 10 -9 M~about 10 -13 M, about 10 -10 M~about 10 -13 M, about 10 -5 M~about 10 -12 M, about 10 -6 M~about 10 -12 M, about 10 -7 M~about 10 -12 M, about 10 -8 M~about 10 -12 M, about 10 -9 M~about 10 -12 M, about 10 -10 M~about 10 -12 M, about 10 -5 M~about 10 -11 M, about 10 -6 M~about 10 -11 M, about 10 -7 M~about 10 -11 M, about 10 -8 M~about 10 -11 M, about 10 -9 M~about 10 -11 M, about 10 -10 M~about 10 -11 M, about 10 -5 M~about 10 -10 M, about 10 -6 M~about 10 -10 M, about 10 -7M ~ about 10 -10 M, about 10 -8 M ~ about 10 -10 M, about 10 -9 M ~ about 10 -10 M, about 10 -5 M ~ about 10 -9 M, about 10 -6 M ~ about 10 -9 M, about 10 -7 M ~ about 10 -9 M, or about 10 -8 M ~ about 10 -9 I am M.

[0120] In exemplary embodiments, the recombinant binding protein has a K of about 150 nM, about 100 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM, about 10 nM, about 5 nM, about 2 nM, about 1 nM, about 900 pM, about 800 pM, about 700 pM, about 600 pM, about 500 pM, about 400 pM, about 300 pM, about 200 pM, about 100 pM, about 10 pM, or about 1 pM or less. D In an exemplary embodiment, the recombinant binding protein binds to human CD70 with a K value of 100 nM or less. D In another exemplary embodiment, the recombinant binding protein binds to CD70 with a K value of 10 nM or less. D It binds to CD70 at high levels.

[0121] In one aspect, the recombinant binding protein has an EC50 of less than about 500, about 400, about 300, about 200, about 150, about 100, about 70, about 60, about 50, about 40, about 30, about 20, about 15, about 10, about 7, about 5, about 3, about 1, about 0.5, or about 0.1 nM. 50 Thus, in one embodiment, the binding protein binds to human CD70 with an EC 50 in another embodiment, the binding protein binds to human CD70 on T cells with an EC 50 in another embodiment, the binding protein binds to human CD70 on T cells with an EC 50 in another embodiment, the binding protein binds to human CD70 on T cells with an EC 50in another embodiment, the binding protein binds to human CD70 on T cells with an EC 50 in another embodiment, the binding protein binds to human CD70 on T cells with an EC 50 in another embodiment, the binding protein binds to human CD70 on T cells with an EC 50 in another embodiment, the binding protein binds to human CD70 on T cells with an EC 50 in another embodiment, the binding protein binds to human CD70 on T cells with an EC 50 in another embodiment, the binding protein binds to human CD70 on T cells with an EC 50 in another embodiment, the binding protein binds to CD70 with an EC 50 in another embodiment, the binding protein binds to CD70 with an EC 50 in another embodiment, the binding protein binds to CD70 with an EC 50 in another embodiment, the binding protein binds to CD70 with an EC 50 in another embodiment, the binding protein binds to CD70 with an EC 50 in another embodiment, the binding protein binds to CD70 with an EC 50 in another embodiment, the binding protein binds to CD70 with an EC 50 in another embodiment, the binding protein binds to CD70 with an EC 50 In a further embodiment, the binding protein binds to CD70 with an EC 50 It binds to CD70.

[0122] Additional Polypeptides In one embodiment, the recombinant binding protein of the present invention further comprises a polypeptide tag. A polypeptide tag is an amino acid sequence attached to a polypeptide / protein, where the amino acid sequence is useful for purifying, detecting, or targeting the polypeptide / protein, or where the amino acid sequence improves the physicochemical behavior of the polypeptide / protein, or where the amino acid sequence has an effector function. Individual polypeptide tags of a recombinant binding protein may be connected to other portions of the recombinant binding protein directly or via a peptide linker. Polypeptide tags are all well known in the art and are readily available to those skilled in the art. Examples of polypeptide tags are small polypeptide sequences that enable detection of the polypeptide / protein, such as His tags, HA tags, myc tags, FLAG tags, or Strep tags, or polypeptides such as enzymes (e.g., alkaline phosphatase), or polypeptides that can be used for targeting (e.g., immunoglobulins or fragments thereof) and / or as effector molecules.

[0123] In one embodiment, the recombinant binding protein of the present invention further comprises a peptide linker. A 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, a protein domain and a biologically active molecule, a protein domain and a localizer, or two sequence tags. Peptide linkers are known to those of skill in the art. A list of examples is provided in the specification of WO 2002 / 020565. In one embodiment, a peptide linker for use in the present invention has a length of 1 to 50 amino acids. In another embodiment, a peptide linker for use in the present invention has a length of about 5 to about 40 amino acids. In another embodiment, a peptide linker for use in the present invention has a length of about 10 to about 30 amino acids.

[0124] Specific examples of peptide linkers are glycine-serine linkers and proline-threonine-rich linkers of variable length. In the context of the present invention, a proline-threonine-rich linker contains at least about 20% proline residues and at least about 20% threonine residues in its amino acid sequence. Examples of glycine-serine linkers are the amino acid sequence GS and the amino acid sequence of SEQ ID NO: 67, and examples of proline-threonine-rich linkers are the amino acid sequences of SEQ ID NOs: 65 and 66.

[0125] N- and C-terminal capping sequences The ankyrin repeat domain of the recombinant binding proteins disclosed herein may comprise an N- or C-terminal capping sequence, which refers to an additional polypeptide sequence fused to the N- or C-terminus of the ankyrin repeat motif, which forms precise tertiary interactions (i.e., tertiary structure interactions) with the ankyrin repeat motif, thereby providing a cap that shields the hydrophobic core of the flanking ankyrin repeat domain from exposure to solvent.

[0126] The N- and / or C-terminal capping sequences may be derived from capping units or other structural units found in naturally occurring repeat proteins adjacent to the repeat units. Examples of capping sequences are described in WO 2002 / 020565 and WO 2012 / 069655, US Patent Application Publication No. 2013 / 0296221, and Interlandi et al., J Mol Biol. 2008 Jan 18;375(3):837-54. Examples of N-terminal ankyrin capping modules (i.e., N-terminal capping repeats) include SEQ ID NOs: 46-47 and 69-70, and examples of ankyrin C-terminal capping modules (i.e., C-terminal capping repeats) include SEQ ID NOs: 48-51.

[0127] Nucleic acids and methods In another aspect, the present invention relates to a nucleic acid encoding the amino acid sequence of the recombinant binding protein of the present invention. In one aspect, the present invention relates to a nucleic acid encoding the amino acid sequence of the recombinant protein of the present invention. Furthermore, the present invention relates to a vector comprising any of the nucleic acids of the present invention. Nucleic acids are well known to those skilled in the art. In the examples, the nucleic acids were used to produce the designed ankyrin repeat domain or recombinant binding protein of the present invention in E. coli.

[0128] Compositions, Uses, and Methods of Treatment In one aspect, the present invention relates to a pharmaceutical composition comprising a recombinant binding protein and / or designed ankyrin repeat domain of the invention and / or a nucleic acid encoding a recombinant binding protein and / or designed ankyrin repeat domain of the invention, and optionally a pharmaceutically acceptable carrier and / or diluent.

[0129] In one aspect, the present invention relates to a pharmaceutical composition comprising a recombinant binding protein or a nucleic acid encoding a recombinant binding protein of the present invention, and optionally a pharmaceutically acceptable carrier and / or diluent.

[0130] Pharmaceutically acceptable carriers and / or diluents are known to those skilled in the art and are described in more detail below.

[0131] Pharmaceutical compositions may be prepared by combining recombinant binding proteins and / or designed ankyrin repeat domains and / or nucleic acids, preferably recombinant binding proteins and / or nucleic acids as described herein, with the recombinant binding proteins and / or nucleic acids described, for example, in Remington's Pharmaceutical Sciences 16 th and a pharmaceutically acceptable carrier, excipient, or stabilizer as described in Osol, A. Ed., 1980.

[0132] Suitable carriers, diluents, excipients, or stabilizers known to those skilled in the art include, for example, saline, Ringer's solution, dextrose solution, Hank's solution, fixed oils, ethyl oleate, 5% dextrose in saline, substances that enhance isotonicity and chemical stability, buffers, and preservatives. Other suitable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, and amino acid copolymers. Pharmaceutical compositions may also be combinations containing additional active agents, such as anti-cancer or anti-angiogenic agents, or additional biologically active compounds. Compositions used for in vivo administration must be sterile or sterilized. This is readily accomplished by filtration through sterile filtration membranes.

[0133] In one aspect, a pharmaceutical composition comprises at least one recombinant binding protein described herein, a surfactant, such as a non-ionic surfactant, a buffer, such as a phosphate buffer, and a sugar, such as sucrose. In one aspect, such a composition comprises the recombinant binding protein and PBS.

[0134] 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 recombinant protein of the present invention, a nucleic acid of the present invention, or a pharmaceutical composition of the present invention.

[0135] In another aspect, the invention provides a method of treating a medical condition, the method comprising administering to a patient in need of treatment for the medical condition a therapeutically effective amount of a recombinant binding protein of the invention, a nucleic acid of the invention, or a pharmaceutical composition of the invention.

[0136] In another aspect, the invention provides a method of treating a medical condition, the method comprising administering to a patient in need of treatment for the medical condition a therapeutically effective amount of a recombinant binding protein of the invention, a nucleic acid encoding the recombinant binding protein, or a pharmaceutical composition comprising the binding protein, wherein the recombinant binding protein further comprises a binding agent having binding specificity for a disease-associated antigen.

[0137] In one aspect, 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 to a patient in need thereof in a therapeutically effective amount. Administration can include topical administration, oral administration, and parenteral administration. A typical administration route is parenteral administration. For parenteral administration, the pharmaceutical composition of the present invention can be formulated in a unit dose injection form such as a solution, suspension, or emulsion together with a pharmaceutically acceptable excipient as defined above. The dose and administration method can depend on the individual and the disease to be treated.

[0138] Furthermore, any of the pharmaceutical compositions, nucleic acids or recombinant proteins described above are contemplated for use in the treatment of disorders.

[0139] In one aspect, the recombinant binding proteins or other such pharmaceutical compositions described herein are administered intravenously. For parenteral use, the recombinant binding proteins or pharmaceutical compositions can be injected in a therapeutically effective amount as a bolus injection or by slow infusion.

[0140] In one aspect, the present invention relates to the use of a recombinant binding protein of the invention, a nucleic acid of the invention, or a pharmaceutical composition of the invention as a medicament for the treatment of a disease. In one aspect, the present invention relates to the use of a recombinant binding protein of the invention, a nucleic acid of the invention, or a pharmaceutical composition of the invention for the manufacture of a medicament. In one aspect, the present invention relates to the use of a recombinant binding protein of the invention, a nucleic acid of the invention, or a pharmaceutical composition of the invention for the manufacture of a medicament for the treatment of a disease. In one aspect, the present invention relates to a method for the manufacture of a medicament for the treatment of a disease, wherein a recombinant binding protein of the invention, a nucleic acid of the invention, or a pharmaceutical composition of the invention is an active ingredient of the medicament. In one aspect, the present invention relates to a method for the treatment of a disease using a recombinant binding protein of the invention, a nucleic acid of the invention, or a pharmaceutical composition of the invention.

[0141] In one aspect, the present invention further provides the use of such a recombinant binding protein for treating a medical condition in a subject in need thereof.

[0142] As used herein, the medical condition or disease is cancer, preferably a liquid tumor, more preferably a leukemia, even more preferably acute myeloid leukemia (AML).

[0143] The recombinant binding proteins of the invention, the nucleic acids of the invention, or the pharmaceutical compositions of the invention can also be used in combination with one or more other therapies known in the art. The term "in combination with," as used herein, refers to co-administration carried out under a given dosing regimen. This includes simultaneous administration of different compounds and administration of different compounds at different times (e.g., compound A is administered once and compound B is administered several times thereafter, or vice versa, or both compounds are administered simultaneously and one of the two is also administered at a later stage).

[0144] In one aspect, the present invention relates to a kit comprising a recombinant binding protein of the present invention. In one aspect, the present invention relates to a kit comprising a nucleic acid encoding the recombinant binding protein of the present invention. In one aspect, the present invention relates to a kit comprising a pharmaceutical composition of the present invention. In one aspect, the present invention relates to a kit comprising a recombinant protein of the present invention, and / or a nucleic acid of the present invention, and / or a pharmaceutical composition of the present invention. In one aspect, the present invention relates to a kit comprising a recombinant protein of the present invention comprising an ankyrin repeat domain having binding specificity for CD70, e.g., SEQ ID NOs: 1-12 and 71-72, and / or a nucleic acid encoding a recombinant protein having binding specificity for CD70, e.g., SEQ ID NOs: 1-12 and 71-72, and / or a pharmaceutical composition comprising a recombinant protein having binding specificity for CD70, e.g., SEQ ID NOs: 1-12 and 71-72. In one aspect, the present invention relates to a kit comprising a recombinant protein comprising any one of the amino acid sequences of SEQ ID NOs: 1-45 and 71-81, and / or a nucleic acid encoding the recombinant protein, and / or a pharmaceutical composition comprising the recombinant protein.

[0145] In one aspect, the present invention relates to a method for producing a recombinant protein of the present invention. In one aspect, the present invention relates to a method for producing a recombinant binding protein, e.g., a recombinant protein comprising the amino acid sequence of any one of SEQ ID NOs: 1-45 and 71-81, comprising: (i) expressing the recombinant binding protein in a suitable host cell (e.g., a bacterium); and (ii) purifying the recombinant binding protein (e.g., using chromatography). The method may include additional steps. Such a method for producing a recombinant binding protein of the present invention is described in Example 1.

[0146] The present invention is not limited to the specific embodiments described in the Examples. This specification makes reference to several amino acid sequences, nucleic acid sequences, and SEQ ID NOs disclosed in the accompanying Sequence Listing, which is incorporated herein by reference in its entirety.

[0147] definition Unless otherwise defined herein, all technical and scientific terms used herein shall have the meaning commonly understood by one of ordinary skill in the art to which this invention belongs. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclature used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry described herein are those well known and commonly used in the art.

[0148] The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms unless otherwise indicated. When an embodiment of the invention is described as "comprising" a certain feature, it is also contemplated that the embodiment "consists of" or "consists essentially of" that feature. Any and all examples provided herein, or the use of illustrative language (e.g., "such as"), are intended merely to better explain the disclosure and do not impose limitations on the scope of the disclosure unless otherwise claimed. No language herein should be construed as indicating any non-claimed element as essential to the practice of the disclosure. Except in the working examples or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about," as that term would be interpreted by one of ordinary skill in the art. As used herein, the term "about" corresponds to ±10% of the given numerical value, unless otherwise specified.

[0149] The recitation of ranges of values ​​herein, unless otherwise indicated herein, is merely intended to serve as a shorthand method of referring individually to each separate value and each endpoint falling within the range, and each separate value and endpoint is incorporated herein as if it were individually listed herein.

[0150] In the context of the present invention, the term "protein" refers to a molecule comprising a polypeptide, at least a portion of which has or can acquire a defined three-dimensional sequence by forming secondary, tertiary, and / or quaternary structures within a single polypeptide chain and / or between multiple polypeptide chains. When a protein comprises two or more polypeptide chains, the individual polypeptide chains can be linked by non-covalent or covalent bonds, for example, by disulfide bonds between two polypeptides. The portions of a protein that individually have or can acquire a defined three-dimensional sequence by forming secondary and / or tertiary structures are referred to as "protein domains." Such protein domains are well known to those skilled in the art.

[0151] The term "recombinant," as used in reference to recombinant proteins and polypeptides, means that the protein or polypeptide is produced by use of recombinant DNA techniques well known to those skilled in the art. For example, a recombinant DNA molecule (e.g., produced by gene synthesis) encoding a polypeptide can be cloned into a bacterial expression plasmid (e.g., pQE30, QIAgen), yeast expression plasmid, mammalian expression plasmid, or plant expression plasmid, or into DNA enabling in vitro expression. For example, when such a recombinant bacterial expression plasmid is inserted into suitable bacteria (e.g., E. coli), these bacteria can produce the polypeptide encoded by the recombinant DNA. The polypeptide or protein produced accordingly is referred to as a recombinant polypeptide or recombinant protein.

[0152] In the context of the present invention, the term "binding protein" refers to a protein comprising a binding domain. A binding protein may also comprise two, three, four, five or more binding domains. Preferably, the binding protein is a recombinant binding protein. The binding protein of the present invention comprises an ankyrin repeat domain that has binding specificity for CD70.

[0153] Additionally, any such binding proteins may comprise additional polypeptides known to those skilled in the art (e.g., polypeptide tags, peptide linkers, fusions to other protein domains with binding specificity, cytokines, hormones, or antagonists, etc.), or chemical modifications (e.g., coupling to polyethylene glycol, toxins (e.g., DM1 from immunogens), small molecules, antibiotics, etc.). The binding proteins of the present invention may also comprise a localizer molecule.

[0154] The term "binding domain" refers to a protein domain that exhibits binding specificity for a target. Preferably, the binding domain is a recombinant binding domain.

[0155] As used herein, the term "target" refers to an individual molecule (including any portion of such an individual molecule), such as a nucleic acid molecule, a polypeptide or protein, a carbohydrate, or any other naturally occurring molecule, or a complex of two or more such molecules, or a whole cell or tissue sample, or any non-natural compound. Preferably, the target is CD70. More preferably, the target is human CD70.

[0156] In the context of the present invention, the term "polypeptide" relates to a molecule consisting of a chain of multiple, i.e., two or more, amino acids linked by peptide bonds. Preferably, a polypeptide consists of more than eight amino acids linked by peptide bonds. The term "polypeptide" also includes multiple chains of amino acids linked by cysteine ​​S-S bridges. Polypeptides are well known to those skilled in the art.

[0157] WO 2002 / 020565 and Forrer et al., 2003 (Forrer, P., Stumpp, MT, Binz, HK, Plueckthun, A., 2003. FEBS Letters 539, 2-6) contain a general description of the characteristics of repeat proteins and of the characteristics, techniques and uses of repeat domains. The term "repeat protein" refers to a protein comprising one or more repeat domains. Preferably, a repeat protein comprises one, two, three, four, five or six repeat domains. Furthermore, the repeat protein may comprise additional non-repeat protein domains, polypeptide tags and / or peptide linkers. The repeat domain may be a binding domain.

[0158] The term "repeat domain" refers to a protein domain comprising two or more consecutive repeat modules as a structural unit, the repeat modules having structural and sequence homology. Preferably, the repeat domain further comprises an N-terminal and / or C-terminal capping module. For clarity, the capping module may be a repeat module. Such repeat domains, repeat modules, and capping modules, sequence motifs, and structural and sequence homology are well known to those skilled in the art from the examples of ankyrin repeat domains (WO 2002 / 020565), leucine-rich repeat domains (WO 2002 / 020565), tetratricopeptide repeat domains (Main, E.R., Xiong, Y., Cocco, M.J., D'Andrea, L., Regan, L., Structure 11(5), 497-508, 2003), and armadillo repeat domains (WO 2009 / 040338). It is further known to those skilled in the art that such repeat domains are distinct from proteins containing repeated amino acid sequences, all of which can form individual domains (e.g., the FN3 domain of fibronectin).

[0159] The term "ankyrin repeat domain" refers to a repeat domain that contains two or more consecutive ankyrin repeat modules as a structural unit. Ankyrin repeat domains can be modularly assembled into larger ankyrin repeat proteins, optionally with half-life extending domains, using standard recombinant DNA techniques (see, for example, Forrer, P., et al., FEBS letters 539, 2-6, 2003; WO 2002 / 020565; WO 2016 / 156596; WO 2018 / 054971).

[0160] The term "designed", as used in designed repeat proteins and designed repeat domains, refers to the property that such repeat proteins and repeat domains, respectively, are artificial and do not occur in nature. The binding proteins of the present invention are designed repeat proteins, which comprise at least one designed ankyrin repeat domain. Preferably, the designed repeat domain is a designed ankyrin repeat domain.

[0161] The term "target interacting residues" refers to amino acid residues of a repeat module that contribute to direct interaction with the target.

[0162] The term "framework residues" refers to amino acid residues of a repeat module that contribute to the folding topology, i.e., contribute to the folding of said repeat module or contribute to interactions with neighboring modules. Such contributions may be interactions with other residues within the repeat module, or influence on the polypeptide backbone structure found in alpha helices or beta sheets, or participation in amino acid stretches forming linear polypeptides or loops. Such framework and target interaction residues may be identified by analysis of structural data obtained by physicochemical methods such as X-ray crystallography, NMR and / or CD spectroscopy, or by comparison with known related structural information well known to those skilled in the art in structural biology and / or bioinformatics.

[0163] The term "repeat module" refers to the repeated amino acid sequence and structural unit of a designed repeat domain, which is originally derived from a repeat unit of a naturally occurring repeat protein. Each repeat module comprised in a repeat domain is derived from one or more repeat units of a family or subfamily of naturally occurring repeat proteins, e.g., the ankyrin repeat protein family. Furthermore, each repeat module comprised in a repeat domain may comprise a "repeat sequence motif" derived from a repeat domain selected on a target, e.g., as described in Example 1, and deduced from homologous repeat modules with the same target specificity.

[0164] Thus, the term "ankyrin repeat module" refers to a repeat module originally derived from the repeat unit of a naturally occurring ankyrin repeat protein. Ankyrin repeat proteins are well known to those skilled in the art. Designed ankyrin repeat proteins have been previously described, see, for example, WO 2002 / 020565, WO 2010 / 060748, WO 2011 / 135067, WO 2012 / 069654, WO 2012 / 069655, WO 2014 / 001442, WO 2014 / 191574, WO 2014 / 083208, WO 2016 / 156596, and WO 2018 / 054971, all of which are incorporated by reference in their entirety. Typically, ankyrin repeat modules contain approximately 31-33 amino acid residues that form two alpha helices separated by a loop.

[0165] A repeat module may comprise positions with amino acid residues that are not randomized in the library for the purpose of selecting target-specific repeat domains ("non-randomized positions") and positions with amino acid residues that are randomized in the library for the purpose of selecting target-specific repeat domains ("randomized positions"). Non-randomized positions include framework residues. Randomized positions include target-interaction residues. "Randomized" means that more than one amino acid is allowed at a certain amino acid position of a repeat module, for example, any of the 20 common naturally occurring amino acids is allowed, or most of the 20 naturally occurring amino acids are allowed, such as amino acids other than cysteine, or amino acids other than glycine, cysteine, and proline.

[0166] The term "repeat sequence motif" refers to an amino acid sequence deduced from one or more repeat modules. Preferably, the repeat modules are derived from repeat domains with binding specificity for the same target. Such a repeat sequence motif comprises framework residue positions and target interaction residue positions. The framework residue positions correspond to the framework residue positions of a repeat module. Similarly, the target interaction residue positions correspond to the target interaction residue positions of a repeat module. A repeat sequence motif comprises non-randomized positions and randomized positions.

[0167] The term "repeat unit" refers to an amino acid sequence comprising 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 determines protein folding. Examples of such repeat units include leucine-rich repeat units, ankyrin repeat units, armadillo repeat units, tetratricopeptide repeat units, HEAT repeat units, and leucine-rich variant repeat units.

[0168] The terms "having binding specificity for a target," "specifically binds to a target," "binds to a target with high specificity," "specific for a target," "target specific," or "specifically binds," etc., mean that a binding protein or binding domain binds to a target in PBS with a lower dissociation constant (i.e., binds with higher affinity) than it binds to an unrelated protein, such as E. coli maltose binding protein (MBP). Preferably, the dissociation constant for the target in PBS ("K D ") is at least 10 times smaller than the corresponding dissociation constant for MBP. -2 times, more preferably at least 10 -3 times, more preferably at least 10 -4 times, or more preferably at least 10 -5 Methods for determining the dissociation constant of a protein-protein interaction, such as surface plasmon resonance (SPR)-based techniques (e.g., SPR equilibrium analysis) or isothermal titration calorimetry (ITC), are well known to those skilled in the art. The K of a particular protein-protein interaction D Measurements of K may vary when measured under different conditions (e.g., salt concentration, pH). D Measurement of the dissociation constant (K) of the recombinant binding protein of the present invention with binding specificity for CD70 is preferably performed using a standardized solution of the protein and a standardized buffer, such as PBS. DA typical and preferred determination of the equilibrium dissociation constant (K) is performed using surface plasmon resonance (SPR) analysis. A variety of assay formats can be used to select or characterize binding moieties that specifically bind to a drug molecule of interest. For example, solid-phase ELISA immunoassays, immunoprecipitation, BIAcore™ (GE Healthcare, Piscataway, NJ), fluorescence-activated cell sorting (FACS), Octet™ (ForteBio, Inc., Menlo Park, CA), and Western blot analysis are some of the many assays that can be used to identify binding moieties that specifically bind to a target drug molecule. Typically, specific or selective binding will be at least two times the background signal or noise, more typically more than 10 times the background signal. More specifically, a binding agent will have an equilibrium dissociation constant (K D A target is said to "specifically bind" if its .DELTA..times ...

[0169] The term "binding agent" or "binding moiety" refers to any molecule capable of specifically binding to a target molecule. Binding agents include, for example, antibodies, antibody fragments, aptamers, peptides (e.g., Williams et al., J Biol Chem 266:5182-5190 (1991)), surrogate scaffolds, antibody mimetics, repeat proteins, such as designed ankyrin repeat proteins, receptor proteins, and any other naturally occurring interaction partners of a target molecule, including naturally occurring proteins and proteins that have been modified or engineered to include, for example, non-naturally occurring residues and / or lack natural residues.

[0170] The term "PBS" refers to an aqueous phosphate buffer solution containing 137 mM NaCl, 10 mM phosphate, and 2.7 mM KCl, with a pH of 7.4.

[0171] Preferably, clearance, and / or exposure, and / or terminal half-life are evaluated in mammals, more preferably mice and / or cynomolgus monkeys, more preferably cynomolgus monkeys. Preferably, when clearance, and / or exposure, and / or terminal half-life are measured in mice, the evaluation is performed taking into account data up to 48 hours after injection. More preferably, the evaluation of terminal half-life in mice is calculated from 24 hours to 48 hours. Preferably, when clearance, and / or exposure, and / or terminal half-life are measured in cynomolgus monkeys, the evaluation is performed taking into account data up to 7 days after injection. More preferably, the evaluation of terminal half-life in cynomolgus monkeys is calculated from days 1 to 5. Those skilled in the art can further identify effects such as target-mediated clearance and take them into account when calculating terminal half-life. The term "terminal half-life" of a drug, such as a recombinant binding protein of the present invention, refers to the time required to reach half of the plasma concentration of the drug administered to a mammal after pseudo-equilibrium has been reached (e.g., calculated from 24 to 48 hours in mice or from days 1 to 5 in cynomolgus monkeys). Terminal half-life is not defined as the time required to eliminate half of the dose of drug administered to a mammal. The term terminal half-life is well known to those skilled in the art. Preferably, pharmacokinetic comparisons are performed at any dose, more preferably at equivalent doses (i.e., the same mg / kg dose) or equimolar doses (i.e., the same mol / kg dose), more preferably equimolar doses (i.e., the same mol / kg dose). Those skilled in the art will understand that equivalent and / or equimolar administration in animals involves experimental dose variations of at least about 20%, more preferably about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100%. Preferably, the dose used for measuring pharmacokinetics is selected from about 0.001 to about 1000 mg / kg, more preferably about 0.01 to about 100 mg / kg, more preferably about 0.1 to about 50 mg / kg, and more preferably about 0.5 to about 10 mg / kg.

[0172] The term "CD3" or "cluster of differentiation 3" refers to a multimeric protein complex composed of four distinct polypeptide chains: epsilon (ε), gamma (γ), and zeta (ζ), assembled in three pairs (εγ, εδ, ζζ). The CD3 complex functions as a T cell coreceptor, noncovalently binding to the T cell receptor. It can 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 species other than humans. In other cases, the binding proteins may be completely specific for human CD3 and may not exhibit species or other types of cross-reactivity. Unless otherwise indicated, such as by specific reference to human CD3, CD3 includes native sequence CD3 from all mammalian species, e.g., human, canine, feline, equine, and bovine. The amino acid sequences of the human CD3 gamma, delta, and zeta chains are shown in the NCBI (www.ncbi.nlm.nih.gov / ) reference sequences NP_000064.1, NP_000723.1, and NP_932170.1, respectively.

[0173] As used herein, the term "CD3-expressing cell" refers to any cell that expresses CD3 (cluster of differentiation 3) on its cell surface, including, but not limited to, T cells such as cytotoxic T cells (CD8+ T cells) and helper T cells (CD4+ T cells).

[0174] The term "CD70" refers to the CD70 antigen, a cytokine that functions as a ligand for CD27. The CD70-CD27 pathway plays an important role in the generation and maintenance of T cell immunity, particularly during antiviral responses. The amino acid sequence of human CD70 (hCD70) is shown in UniProt (www.uniprot.org) reference number P32970.

[0175] The term "tumor-local activation of T cells" means that T cells are preferentially activated in tumor tissue compared to non-tumor tissue.

[0176] Furthermore, the term "peptide" also encompasses peptides modified, e.g., by glycosylation, and proteins comprising two or more polypeptide chains, e.g., cross-linked by disulfide bonds, each 4 to 600 amino acids in length, such as insulin and immunoglobulins. The term "chemical or biochemical agent" is intended to include any naturally occurring or synthetic compound that can be administered to a recipient. In a preferred embodiment, the localizer is a target-specific ankyrin repeat domain.

[0177] The term "medical condition" (or disorder or disease) includes autoimmune disorders, inflammatory disorders, retinopathies (particularly proliferative retinopathy), neurodegenerative disorders, infectious diseases, metabolic diseases, and neoplastic diseases. Any of the recombinant binding proteins described herein can be used in the preparation of a medicament for the treatment of such disorders, particularly disorders such as neoplastic diseases. A "medical condition" may be one characterized by inappropriate cell proliferation. A medical condition may be a hyperproliferative state. The present invention particularly relates to methods of treating a medical condition, comprising administering to a patient in need of such treatment a therapeutically effective amount of a recombinant binding protein of the present invention or a pharmaceutical composition thereof. In a preferred aspect, the medical condition is a neoplastic disease. The term "neoplastic disease," as used herein, refers to an abnormal state or condition of cells or tissues characterized by rapidly proliferating cell proliferation or neoplasia. In one aspect, the medical condition is a malignant neoplastic disease. In one aspect, the medical condition is cancer, preferably leukemia, more preferably acute myeloid leukemia. The term "therapeutically effective amount" means an amount sufficient to produce a desired effect in a patient.

[0178] The term "antibody" refers not only to intact antibody molecules, but also to any fragments and variants of antibody molecules that retain immunogen-binding ability. Such fragments and variants are also well known in the art and are routinely used both in vitro and in vivo. Thus, the term "antibody" encompasses intact immunoglobulin molecules, antibody fragments such as Fab, Fab', F(ab')2, and single-chain V-region fragments (scFv), bispecific antibodies, chimeric antibodies, antibody fusion polypeptides, and non-conventional antibodies.

[0179] The terms "cancer" and "cancerous" are used herein to refer to or describe the physiological condition in mammals that is typically characterized by uncontrolled cell growth. Cancer includes solid and liquid tumors, as well as primary tumors and metastases. A "tumor" contains one or more cancerous cells. Solid tumors typically also contain tumor stroma. Examples of cancer include, but are not limited to, primary and metastatic carcinomas, 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 renal 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), chronic myeloid leukemia (CML), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, Hodgkin's lymphoma (HL), mantle cell lymphoma (MCL), and leukemia. cell lymphoma (MCL), multiple myeloma (MM), myelodysplastic syndrome (MDS), non-Hodgkin's lymphoma (NHL), squamous cell carcinoma of the Head and Neck (SCCHN), chronic myelogenous leukemia (CML), small lymphocytic lymphoma (small lymphocytic lymphoma (SLL), malignant mesothelioma, colorectal cancer, or gastric cancer. [Example]

[0180] The starting materials and reagents disclosed below are known to those of ordinary skill in the art, are commercially available, and / or can be prepared using well-known techniques.

[0181] material Chemicals were purchased from Sigma-Aldrich (USA). Oligonucleotides were purchased from Microsynth (Switzerland). Unless otherwise stated, DNA polymerases, restriction enzymes, and buffers were purchased from New England Biolabs (USA) or Fermentas / Thermo Fisher Scientific (USA). Inducible E. coli expression strains, such as E. coli XL1-blue (Stratagene, USA) or BL21 (Novagen, USA), were used for cloning and protein production.

[0182] molecular biology Unless otherwise stated, the methods are carried out according to known protocols (see, for example, Sambrook J., Fritsch EF and Maniatis T., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory (1989), New York).

[0183] 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., J. Mol. Biol. 332, 489-503, 2003, and 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. Thus, for example, such libraries can be assembled based on a fixed N-terminal capping module (e.g., an N-terminal capping module of SEQ ID NO: 46, 69, or 70) or a randomized N-terminal capping module (e.g., according to SEQ ID NO: 47) and a fixed C-terminal capping module (e.g., a C-terminal capping module of SEQ ID NO: 48, 49, or 50) or a randomized C-terminal capping module (e.g., according to SEQ ID NO: 51). Preferably, such libraries are assembled so as to have none of the amino acids C, G, M, N (before a G residue) and P in the randomized positions of the repeat or capping module.

[0184] Furthermore, such randomized modules in such libraries may contain additional polypeptide loop insertions with randomized amino acid positions. Examples of such polypeptide loop insertions are complement determining region (CDR) loop libraries for antibodies or de novo generated peptide libraries. For example, such loop insertions can be designed using the structure of the N-terminal ankyrin repeat domain of human ribonuclease L as guidance (Tanaka, N., Nakanishi, M., Kusakabe, Y., Goto, Y., Kitade, Y., Nakamura, K.T., EMBO J., 23(30), 3929-3938, 2004). Similar to this ankyrin repeat domain in which 10 amino acids are inserted into a beta turn located near the boundary between two ankyrin repeats, ankyrin repeat protein libraries can contain randomized loops (with fixed and randomized positions) of variable length (e.g., 1 to 20 amino acids) inserted into one or more beta turns of the ankyrin repeat domain. Any such N-terminal capping module of an ankyrin repeat protein library preferably has a RILLAA, RILLKA, or RELLKA motif (e.g., present at positions 19-24 of SEQ ID NOs: 1, 2, and 9), and any such C-terminal capping module of an ankyrin repeat protein library preferably has a KLN, KLA, or KAA motif (e.g., present at the last three amino acids of SEQ ID NO: 1). SEQ ID NOs: 46, 69, and 70 provide examples of N-terminal capping modules comprising a RILLAA, RILLKA, or RELLKA motif, and SEQ ID NOs: 48-50 provide examples of C-terminal capping modules comprising a KLN, KLA, or KAA motif.

[0185] The design of such ankyrin repeat protein libraries can be guided by known structures of ankyrin repeat domains that interact with targets. Examples of such structures, identified by unique Protein Data Bank (PDB) accession or identification codes (PDB-IDs), are 1WDY, 3V31, 3V30, 3V2X, 3V2O, 3UXG, 3TWQ-3TWX, 1N11, 1S70, and 2ZGD.

[0186] Examples of designed ankyrin repeat protein libraries have been described, such as the N2C and N3C designed ankyrin repeat protein libraries (U.S. Pat. No. 7,417,130; Binz et al. 2003, supra; Binz et al. 2004, supra). The numbers in N2C and N3C describe the number of randomized repeat modules present between the N-terminal capping module and the C-terminal capping module.

[0187] The nomenclature used to define the repeat units and their positions within modules is based on Binz et al. 2004 (cited above), with the modification that the boundaries of ankyrin repeat modules and ankyrin repeat units are shifted by one amino acid position. For example, position 1 of the ankyrin repeat module of Binz et al. 2004 (cited above) corresponds to position 2 of the ankyrin repeat module of the present disclosure, and consequently, position 33 of the ankyrin repeat module of Binz et al. 2004 (cited above) corresponds to position 1 of the following ankyrin repeat module of the present disclosure:

[0188] Example 1: Selection of binding proteins containing ankyrin repeat domains with binding specificity for CD70 Using ribosome display (Hanes, J. and Plueckthun, A., PNAS 94, 4937-42, 1997), many ankyrin repeat proteins with binding specificity for human CD70 (hCD70) were selected from a DARPin® library similar to that described by Binz et al. 2004 (cited above). The binding of selected clones to recombinant human CD70 targets was assessed by homogeneous time-resolved fluorescence (HTRF) of crude extracts, demonstrating that several hundred hCD70-specific binding proteins were successfully selected. For example, the ankyrin repeat domains of SEQ ID NOS: 1-12 constitute the amino acid sequences of selected binding proteins containing ankyrin repeat domains with binding specificity for hCD70. SEQ ID NOS: 24-45 constitute the ankyrin repeat modules of selected binding proteins with binding specificity for hCD70.

[0189] Selection of CD70-specific ankyrin repeat proteins by ribosome display Selection of hCD70-specific ankyrin repeat proteins was performed by ribosome display (Hanes and Plückthun, supra) using the biotinylated extracellular domain of human CD70 (SEQ ID NO: 60) as the target protein, the library of ankyrin repeat proteins described above, and established protocols (see, e.g., Zahnd, C., Amstutz, P., and Plückthun, A., Nat. Methods 4, 69-79, 2007). The CD70 target (ACROBiosystems) contained a C-terminal Fc tag and was chemically biotinylated with a 5-fold excess of biotin. A total of four rounds of standard ribosome selection were employed, with increasing selection pressure from round 1 to round 4 using decreasing target concentrations and increasing washing stringency (Binz et al., 2004, supra). A deselection strategy was applied in each round by using streptavidin and neutravidin beads. The number of reverse transcription (RT)-PCR cycles after each selection round was consistently reduced from 45 to 28, adjusting for yield, to enrich for binders.

[0190] To enrich for high-affinity CD70-specific ankyrin repeat proteins, the output from the fourth round of standard ribosome display selection (described above) was subjected to an off-rate selection round with increased selection stringency (Zahnd, 2007, cited above). A final standard selection round was performed after the off-rate selection round to amplify and recover the off-rate-selected binding proteins. In rounds 5 and 6, the number of RT-PCR cycles was 30 and 35, respectively.

[0191] Selected clones specifically bind human CD70 as demonstrated by crude extract HTRF Individual selected ankyrin repeat proteins that specifically bind to hCD70 in solution were identified by a homogeneous time-resolved fluorometry (HTRF) assay using crude extracts of ankyrin repeat protein-expressing E. coli cells using standard protocols. Ankyrin repeat protein clones selected by ribosome display were cloned into a derivative of the pQE30 (Qiagen) expression vector (pMPDV25), transformed into E. coli XL1-Blue (Stratagene), plated on LB-agar (containing 1% glucose and 50 μg / ml ampicillin), and then incubated overnight at 37°C. Single colonies were picked and placed (each clone in a single well) into 96-well plates containing 160 μl of growth medium (TB containing 1% glucose and 50 μg / ml ampicillin) and incubated overnight at 37°C with shaking at 800 rpm. 150 μl of fresh TB medium containing 50 μg / ml ampicillin was inoculated with 8.5 μl of overnight culture in a new 96-well plate. After 120 minutes of incubation at 37°C and 700 rpm, expression was induced with IPTG (final concentration 0.5 mM) and continued for 4 hours. Cells were harvested, and the pellet was frozen overnight at -20°C before being resuspended in 8 μl of B-PERII (Thermo Scientific) and incubated for 1 hour at room temperature with shaking (900 rpm). 160 μl of PBS was then added, and cell debris was removed by centrifugation (3220 g for 15 minutes).

[0192] The lysed extracts of each clone were applied to wells of a 384-well plate at a final dilution of 1:2000 in PBSTB (PBS supplemented with 0.1% Tween 20® and 0.2% (w / v) BSA, pH 7.4) together with 2 nM (final concentration) biotinylated hCD70, 1:400 (final concentration) anti-strep-Tb HTRF antibody-FRET donor conjugate (Cisbio), and 1:400 (final concentration) anti-6His-D2 antibody FRET receptor conjugate (Cisbio) and incubated for 60 min at RT. HTRF readings were performed on a Tecan M1000 using an excitation wavelength of 340 nm and an emission filter of 665 ± 10 nm. Screening of hundreds of clones by such crude cell extract HTRF revealed an ankyrin repeat domain with specificity for hCD70. The amino acid sequences of the selected ankyrin repeat domains that specifically bind to hCD70 are SEQ ID NOs: 1-12: DARPin® Protein #1 (SEQ ID NO: 1), DARPin® Protein #2 (SEQ ID NO: 2), DARPin® Protein #3 (SEQ ID NO: 3), DARPin® Protein #4 (SEQ ID NO: 4), DARPin® Protein #5 (SEQ ID NO: 5), DARPin® Protein #6 (SEQ ID NO: 6), DARPin® Protein #7 (SEQ ID NO: 7), DARPin® Protein #8 (SEQ ID NO: 8), DARPin® Protein #9 (SEQ ID NO: 9), DARPin® protein #10 (SEQ ID NO: 10), DARPin® protein #11 (SEQ ID NO: 11), and The DARPin® protein is provided in DARPin® Protein #12 (SEQ ID NO: 12).

[0193] These DARPin® proteins optionally further comprise a G, S, or GS sequence at their N-terminus.

[0194] Engineering additional ankyrin repeat proteins with binding specificity for hCD70 SEQ ID NOs: 2 and 5 were engineered based on the sequences of SEQ ID NOs: 1 and 4, respectively. Both sequences were modified to alter the surface charge. In both N-terminal capping modules, the RILLAA motif was replaced with RILLKA, and the aspartic acid (position 15) was replaced with leucine. Additionally, the C-terminal capping module of SEQ ID NO: 1 was modified by replacing serine (position 112) with glycine and glutamic acid (position 114) with glutamine.

[0195] Expression of CD70-specific ankyrin repeat proteins For further analysis, selected clones that showed specific human CD70 binding in crude cell extract HTRF as described above were expressed in E. coli cells with a His tag (SEQ ID NO: 82) fused to their N-terminus for easy purification. The expressed proteins were purified using their His tag according to standard protocols. 0.11 ml of the static overnight culture (TB, 1% glucose, 50 mg / L ampicillin, 37°C) was used to inoculate 0.99 ml of culture (TB, 50 mg / L ampicillin, 37°C) in a 96-deep-well plate. After 2 hours of incubation at 37°C (700 rpm), the culture was induced with 0.5 mM IPTG and incubated with shaking (900 rpm) at 37°C for 6 hours. Cells were harvested and pellets were frozen overnight at -20°C. They were then resuspended in 50 μl of B-PERII (Thermo Scientific) supplemented with DNAase I (200 units / ml) and lysozyme (0.4 mg / ml) and incubated at room temperature for 1 hour with shaking (900 rpm). Sixty μl of low-salt sodium phosphate buffer was then added, and cell debris was removed by centrifugation (3,220 g for 15 minutes). Eight individual expression pools were pooled, and cell debris was removed by centrifugation (3,200 g for 60 minutes at 4°C). The supernatant was filtered using a MultiScreen filter plate (Millipore) and purified using a 96-well Thermo HisPur cobalt spin plate. The protein solution was rebuffered in PBS using a 96-well Thermo Zeba spin desalting plate. The purified protein was soluble and monomeric in PBS using a standard Sephadex 150 / 5 column on an Agilent 1200 HPLC system.

[0196] Generation of affinity-matured ankyrin repeat proteins with binding specificity for hCD70 In further development of the initially identified CD70-specific binding proteins, binding domains with very high affinity for and / or very low off-rates from the target protein were generated using affinity maturation. Two initially identified binding proteins (the "parent" binding proteins DARPin® Protein #2 and DARPin® Protein #9) were selected as suitable starting points for affinity maturation. The affinity maturation procedure involved saturation mutagenesis of each randomized position in the ankyrin repeat domain used as the starting point. The sequences generated by the affinity maturation procedure were screened for lower off-rates by competitive HTRF. Briefly, crude extracts of ankyrin repeat proteins containing an N-terminal His tag (SEQ ID NO: 82) were incubated with biotinylated targets, after which excess untagged parent CD70-specific binding protein was added, and the HTRF signal was measured over time. Beneficial mutations identified based on higher HTRF signals compared to the parent clones were combined in binding proteins by protein engineering. In this way, affinity-matured DARPin® protein #24 (SEQ ID NO: 71) and DARPin® protein #25 (SEQ ID NO: 72) were generated, which originated from the parent proteins DARPin® protein #2 (SEQ ID NO: 2) and DARPin® protein #9 (SEQ ID NO: 9), respectively.

[0197] Such affinity-matured CD70-specific binding domains were then subcloned into a derivative of the pQE30 (Qiagen) expression vector to obtain expression constructs encoding an N-terminal His tag (SEQ ID NO:82), followed by the CD70-specific binding domain (SEQ ID NO:71 or 72), a peptide linker (SEQ ID NO:65), and a C-terminal CD3-specific binding domain (SEQ ID NO:57). These constructs in T cell engager format were expressed in Escherichia coli cells and purified using their His tag according to standard protocols. The proteins were tested for dose-dependent in vitro T cell activation and tumor cell killing in assays using primary T cells isolated from healthy donor PBMCs as effector cells (E) and Molm-13-N1 tumor cells as target cells (T) (5:1 E:T ratio). Assay incubation of cocultures was performed for 48 hours and analyzed by flow cytometry and LDH release. DARPin® Protein #24 (SEQ ID NO: 71) and DARPin® Protein #25 (SEQ ID NO: 72) showed approximately 7-fold and 31-fold improvements in EC50 values, respectively, compared to their parent binding proteins, and showed greater than 95% monomericity as measured by analytical size exclusion chromatography. In a further step, the CD70-specific binding proteins DARPin® Protein #24 (SEQ ID NO: 71) and DARPin® Protein #25 (SEQ ID NO: 72) were subcloned into a derivative of the pQE30 (Qiagen) expression vector encoding an N-terminal His tag (SEQ ID NO: 82), expressed in E. coli as monovalent designed ankyrin repeat proteins, and purified and characterized according to standard protocols.

[0198] Example 2: Dissociation constants (K) of recombinant ankyrin repeat proteins with binding specificity for human CD70 by surface plasmon resonance (SPR) analysis D ) determination The binding affinity of purified ankyrin repeat proteins to the recombinant human CD70 target was analyzed using a ProteOn XPR36 instrument (BioRad) and measurements were performed according to standard procedures known to those skilled in the art.

[0199] Bio.CD70 was immobilized on a NLC chip and ankyrin repeat proteins were applied as titrations at a starting concentration of 100 nM, then titrated down to 1.2 nM. Signals were double-referenced against PBST-treated control lanes L1 and A6. Analyte injection was performed for 120 seconds, followed by dissociation for 1500 seconds. The K values ​​obtained in this study D The values ​​are summarized in Table 2a. [Table 2]

[0200] Furthermore, the binding affinity of two additional purified ankyrin repeat proteins to the recombinant human CD70 target was measured and analyzed using the procedure described above for DARPin proteins #2, #3, and #5-#12. Briefly, neutravidin was precoated onto a GLC chip (BioRad) to a level of 5300 RU. In a second step, bio.hCD70 was immobilized to a level of 510 RU. Binding of DARPin proteins #24 and #25 to bio.hCD70 was measured by injecting DARPin® molecules at serial dilutions of 100, 40, 16, 6.4, and 2.6 nM. For all measurements described, a constant flow of 100 μl / min was used, with 120 s of association and 1200 s of dissociation. The CD70 target was regenerated by applying 4 M MgCl2 for 30 s. Signals were double-referenced against interspot and running buffer (PBST: PBS pH 7.4 containing 0.005% Tween 20®) treated control lane A6. A 1:1 Langmuir model was used for fitting. The K values ​​obtained in this study D The values ​​are summarized in Table 2b. [Table 3]

[0201] FIG. 1 shows the SPR analysis of DARPin® protein #24 (SEQ ID NO: 71).

[0202] Example 3: Pharmacokinetic analysis of CD70-specific ankyrin repeat proteins in female BALB / c mice To determine whether the CD70-specific ankyrin repeat domains of the present invention have a suitable serum half-life in vivo that would be useful for the development of therapeutic agents, the pharmacokinetic profiles of DARPin® Protein #2, DARPin® Protein #9, DARPin® Protein #24, and DARPin® Protein #25 are analyzed in mice. To this end, the DARPin constructs are subcloned into a derivative of the pQE30 (Qiagen) expression vector as described above and expressed to obtain an expression construct encoding an N-terminal His tag (e.g., SEQ ID NO: 82), followed by a human serum albumin (HSA)-binding ankyrin repeat domain (e.g., SEQ ID NO: 53) for half-life extension, a peptide linker (e.g., SEQ ID NO: 65), and one of the CD70-specific binding domains at the C-terminus.

[0203] In vivo administration and sampling DARPin® Protein #2, DARPin® Protein #9, DARPin® Protein #24, and DARPin® Protein #25, formatted with the above-described human serum albumin-specific ankyrin repeat domain (SEQ ID NO: 53), are administered as a single intravenous bolus injection into the tail vein of six mice for each ankyrin repeat fusion protein. The target dose level is 1 mg / kg in an application volume of 5 mL / kg. The ankyrin repeat fusion proteins are formulated in phosphate-buffered saline (PBS) solution.

[0204] Mice are divided into two groups with equal numbers of animals. Four serum samples are collected from each mouse. Blood samples for pharmacokinetic studies are collected from saphenous vein at 5 minutes, 4 hours, 24 hours, 48 ​​hours, 76 hours, 96 hours and 168 hours after compound administration. Blood is kept at room temperature to clot, then centrifuged and serum is collected.

[0205] ELISA-based bioassay for measuring ankyrin repeat proteins in serum samples NUNC Maxisorb ELISA plates were coated overnight at 4°C with 10 nM polyclonal goat anti-rabbit IgG antibody (Ab18) in 100 μl of PBS per well. After washing five times with 300 μl of PBST (PBS supplemented with 0.1% Tween 20) per well, the wells were blocked with 300 μl of PBST supplemented with 0.25% casein (PBST-C) for 1 hour at room temperature (RT) in a Heidolph Titramax 1000 shaker (450 rpm). The plates were washed as above. 100 μl of 5 nmol / L rabbit anti-DARPin® 1-1-1 antibody in PBST-C was added, and the plates were incubated for 1 hour at RT (22°C) with orbital shaking (450 rpm). The plates were washed as above.

[0206] Apply 100 µl of diluted serum samples (1:20 to 1:3 in 1:5 dilution steps) or ankyrin repeat protein standard curve samples (0 and 50 to 0.0008 nmol / L in 1:3 dilution steps) for 2 h at RT with shaking at 450 rpm. Wash the plate as above.

[0207] The wells are then incubated with 100 μl of mouse anti-RGS-His-HRP IgG (Ab06, 1:2000 in PBST-C) and incubated for 1 hour at RT, 450 rpm. The plates are washed as above. The ELISA is developed using 100 μl / well of TMB substrate solution for 5 minutes and stopped by adding 100 μl of 1 mol / L H2SO4. The difference between the absorbance at 450 nm and the absorbance at 620 nm is calculated. Samples are measured in duplicate on two different plates.

[0208] Pharmacokinetic analysis Pharmacokinetic data analysis is performed at Molecular Partners using version 7.0 of the WinNonlin program as part of Phoenix 64, Pharsight, North Carolina. Calculation of pharmacokinetic parameters based on mean concentration-time data from animals dosed via intravenous bolus injection is performed using noncompartmental analysis (NCA model 200-202, IV bolus, linear trapezoidal linear interpolation). For example, the following pharmacokinetic parameters are calculated: AUCinf, AUClast, AUC_%extrapol, Cmax, Tmax, Cl_pred, Vss_pred, t1 / 2.

[0209] The maximum serum concentrations (Cmax) and their time of occurrence (Tmax) are obtained directly from the serum concentration-time profile. The area under the serum concentration-time curve (AUCinf) is determined by a linear trapezoidal rule up to the last sampling point (Tlast) and extrapolation to infinity assuming a monoexponential terminal decay. The extrapolation to infinity is performed using Clast / λz, where λz represents the terminal rate constant estimated by log-linear regression, and Clast represents the concentration estimated at Tlast by terminal log-linear regression. The total serum clearance (Cl_pred) and apparent terminal half-life are calculated as follows: Cl_pred = iv dose / AUCinf and t1 / 2 = ln2 / λz. The steady-state volume of distribution, Vss, is determined by Vss = iv dose·AUMCinf / (AUCinf)2. AUMCinf indicates the total area under the first moment of the drug concentration-time curve extrapolated to infinity using the same extrapolation procedure as described for calculating AUCinf. To calculate PK parameters based on concentrations given in nmol / L doses, values ​​given in mg / kg are converted to nmol / kg using the molecular weight of the ankyrin repeat protein. Table 3 shows the approximate predicted half-lives of the ankyrin repeat proteins of the invention, DARPin® Protein #2, DARPin® Protein #9, DARPin® Protein #24, and DARPin® Protein #25, formatted with the human serum albumin-specific ankyrin repeat domain described above. [Table 4]

[0210] In conclusion, the CD70-specific ankyrin repeat domains of the present invention can be combined with half-life extending moieties, such as serum albumin-specific binding domains, to achieve suitable serum half-lives in vivo that make them useful for the development of therapeutic agents.

[0211] Example 4: Determination of the binding of ankyrin repeat proteins of the invention to CD70-expressing tumor cells The binding of the binding proteins of the present invention to CD70 expressed on the cell surface was analyzed by fluorescence-activated cell sorting (FACS) flow cytometry. For this purpose, CD70-expressing tumor cells (Molm-13 N1) were seeded at 100,000 cells per well in a 96-well plate. DARPin Protein #2, DARPin Protein #9, DARPin Protein #24, and DARPin Protein #25 were titrated at a 1:5 dilution ratio starting at 2000 nM. Tumor cells were resuspended with the diluted DARPin proteins and incubated for 60 min at 4°C. The assay was performed in PBS containing 2% fetal bovine serum and without human serum albumin (HSA). After two washes with phosphate-buffered saline (PBS), DARPin® protein-specific tumor cell binding was detected by adding an unlabeled primary anti-rabbit DARPin® antibody (anti-rabbit 1-1-1 antibody, CePower) at 2 μg / ml. This was followed by an incubation step of at least 30 min at 4°C. Afterwards, the cells were washed with PBS, and a secondary goat anti-rabbit antibody labeled with Alexa Fluor 488 antibody (ThermoFisher) was added at 2 μg / ml. The same incubation conditions were applied. Finally, the cells were washed twice and resuspended in Cytofix fixation buffer (BD Biosciences) for 15 min at room temperature (RT). The median fluorescence intensity (MFI) of Alexa Fluor 488 DARPin® protein-labeled tumor cells was measured with Attune NXT (ThermoFisher) using FlowJo software for analysis and GraphPad Prism 8 for data plotting. Figure 2 shows the binding curves of DARPin® Protein #2, DARPin® Protein #9, DARPin® Protein #24, and DARPin® Protein 25 to CD70-expressing tumor cells. Table 4 shows the quantification of binding of four exemplary ankyrin repeat proteins to CD70 expressed on cells, expressed as EC50 values. [Table 5]

[0212] In conclusion, the CD70-specific binding proteins of the present invention bind to CD70 expressed on the surface of cells with an EC50 of about 500 nM or less.

[0213] Example 5: Evaluation of the specificity and potency of CD70-specific ankyrin repeat proteins of the invention in a T cell engager format using a target-specific short-term T cell activation assay The specificity and potency of the exemplary CD70-specific ankyrin repeat proteins of the invention described above were evaluated in an in vitro short-term T cell activation assay by FACS measuring CD25 as an activation marker on CD8+ T cells. The tested proteins, DARPin Protein #2, DARPin Protein #9, DARPin Protein #24, and DARPin Protein #25, were evaluated in a bispecific T cell engager format. Such T cell engager proteins contain a CD3-specific binding domain (SEQ ID NO: 57) in addition to the CD70-specific ankyrin repeat domain mentioned, and are designated DARPin Protein #28, DARPin Protein #29, DARPin Protein #26, and DARPin Protein #27, respectively.

[0214] Therefore, 100,000 purified pan-effector T cells and 20,000 Molm-13 target cells per well were co-incubated in duplicate with serial dilutions of selected DARPin proteins (E:T ratio 5:1) in the presence of 600 μM human serum albumin for 48 h at 37 °C. After 48 h, cells were washed and stained with 1:1,000 Live / Dead Green (Thermo Fisher), 1:400 mouse anti-human CD8 Pacific Blue (BD), and 1:100 mouse anti-human CD25 PerCP-Cy5.5 (eBiosciences) antibodies for 30 min at 4 °C. After washing and fixation, cells were analyzed using an Attune NxT (ThermoFisher) instrument. T cell activation was assessed by measuring CD25+ cells on Live / Dead-negative and CD8+-gated T cells. FACS data were analyzed using FlowJo software and plotted using GraphPad Prism 8 (3-PL-fit). Figure 3 shows short-term T cell activation induced by DARPin Protein #28, DARPin Protein #29, DARPin Protein #26, and DARPin Protein #27, as measured by the activation marker CD25. All of the CD70-specific binding proteins of the invention tested in the T cell engager format were able to bind to CD70 expressed on tumor cells and activate T cells.

[0215] Example 6: Evaluation of the specificity and potency of CD70-specific ankyrin repeat proteins of the invention in a T cell engager format using a target-specific short-term tumor cell killing assay The specificity and potency of the CD70-specific ankyrin repeat proteins of the invention, DARPin Protein #2, DARPin Protein #9, DARPin Protein #24, and DARPin Protein #25 (i.e., DARPin Protein #28, DARPin Protein #29, DARPin Protein #26, and DARPin Protein #27, respectively), in a T cell engager format was also evaluated using an in vitro short-term cytotoxicity assay measuring LDH release. To this end, 100,000 purified pan-effector T cells and 20,000 Molm-13 target cells per well were co-incubated in duplicate with serial dilutions of the indicated T cell engager proteins in the presence of 600 μM human serum albumin at 37°C for 48 h (E:T ratio 5:1). After 48 hours of incubation, cells were spun down and 100 μl of supernatant from each well was analyzed for LDH release according to the manufacturer's protocol (LDH detection kit, Roche Applied Science, 30 minutes of incubation). Absorbance was measured at 492-620 nm using a TECAN infinite M1000Pro reader. OD values ​​were plotted using GraphPad Prism8.

[0216] Figure 4 shows tumor cell killing induced by DARPin Protein #28, DARPin Protein #29, DARPin Protein #26, and DARPin Protein #27. All of the CD70-specific binding proteins of the invention tested in a T cell engager format were able to bind to CD70 expressed on tumor cells and activate T cells that killed the tumor cells.

[0217] Example 7: Determination of the CD70 epitope bound by the CD70-specific binding protein of the present invention The CD70 epitopes bound by the CD70-specific ankyrin repeat proteins of the present invention were investigated using a competitive ELISA against the benchmark control molecule ARGX-110 analogue (CD70-specific antibody, purchased from Evitria) and a soluble form of the native CD70-binding receptor CD27 (CD27-Fc, AMD1218071, purchased from R&D Systems).

[0218] Competition between the CD70-specific binding protein DARPin® Protein #2 and human CD27 (a receptor for CD70) and the anti-CD70 antibody ARGX-110 analog for binding to human CD70 was assessed by competitive ELISA. In the first setup, DARPin® Protein #2 was immobilized on a microplate. After approximately 2 hours of preincubation with a biotinylated target (BiohCD70-Fc-trimer, provided by ACRO Biosystems) and one of the competitor molecules, binding of the biotinylated target to the immobilized DARPin® Protein #2 was measured directly via streptavidin covalently linked to peroxidase. In the second and third setups, either CD27-Fc or an ARGX-110 analog was immobilized on a microplate, and similar competitive ELISAs were performed.

[0219] Briefly, Nunc MaxiSorp 96-well plates were coated overnight with 10 nM DARPin® Protein #2, CD27-Fc, or 5 nM ARGX-110 analogs. The ELISA plates were washed three times and blocked with PBSTC (PBS containing 0.1% (v / v) Tween 20® and 0.25% casein) for 2 hours and 15 minutes at 450 rpm. Meanwhile, 1000 nM DARPin® Protein #2 and 500 nM ARGX-110 were preincubated with 20 nM bio.hCD70 at a 1:1 ratio for 2 hours at room temperature. Bio.hCD70 without competitor was included as a positive control. The preincubated samples were then added to the coated MaxiSorp plates and incubated for 30 minutes at room temperature and 450 rpm. After washing the plate three times with PBST, the target was detected with streptavidin-POD antibody (Roche, catalog number 11 089 153 001). For detection, freshly prepared TMB buffer (30 mM citrate buffer pH 4.1, 5% (v / v) TMB solution (Carl Roth GmbH) and 0.16% H2O2) was added, and the reaction was stopped with 1 M H2SO4. Absorbance was measured at OD450 and referenced to OD620 using a Sunrise microplate reader (Tecan). Data were analyzed by subtracting the buffer (PBS) values. GraphPad Prism was used for analysis.

[0220] When the human CD70 target was preincubated with a 50-fold molar excess of DARPin® Protein #2 (positive competition control) or a 25-fold molar excess of the ARGX-110 analog antibody, both DARPin® Protein #2 and the ARGX-110 analog were able to efficiently compete with the binding of plate-immobilized DARPin® Protein #2 to the hCD70 target, thereby reducing the binding of plate-immobilized DARPin® Protein #2 to hCD70 to baseline levels (Figure 5A). Similarly, efficient competition for binding to the hCD70 target was observed in settings 2 and 3, i.e., with plate-immobilized CD27 (Figure 5B) or plate-immobilized ARGX-110 analog (Figure 5C).

[0221] In conclusion, all CD70-binding molecules tested (DARPin® Protein #2, CD27, and ARGX-110) competed with each other for binding to CD70, indicating that they may bind to the same or overlapping epitopes on CD70.

[0222] Example 8: In vivo efficacy evaluation of exemplary multidomain TCE-binding proteins, including DARPin® Protein #24, in PBMC-humanized mouse and MOLM-13 tumor models DARPin® Protein #24, a multidomain T cell engager binding protein formatted with two ankyrin repeat domains with binding specificity for human serum albumin (TCA1 and TCA2), respectively, and one ankyrin repeat domain with binding specificity for CD3, was tested in a peripheral blood mononuclear cell (PBMC)-humanized mouse model bearing the tumor cell line MOLM-13. In vivo experiments were performed in 6-9 week-old female immunodeficient NXG mice (provided by Janvier Labs). Mice were maintained in standard rodent microisolator cages under standardized environmental conditions (room temperature of 20 ± 1°C, relative humidity of 50 ± 10%, and a 12-hour light-dark cycle). Mice were provided with irradiated food and bedding and 0.22 μm filtered drinking water. All experiments were performed in accordance with Swiss animal protection laws and with approval from cantonal and federal veterinary authorities.

[0223] Two days before cancer cell xenotransplantation, hPBMCs (5 × 10 prepared from buffy coats of two different donors) were cultured. 6 Mice were intraperitoneally injected with 1000 hPBMCs (1000 PBMCs). MOLM-13 cells were xenografted subcutaneously (sc) into the right flank of the mice. Two hPBMC donors were used. Treatments were administered intravenously (iv) starting 4 days after cancer cell implantation. Treatments were administered as follows: DARPin® Protein #24 in multidomain TCE format or vehicle was administered iv at 0.5 mg / kg, three times a week for two weeks.

[0224] Tumor size was assessed by caliper measurement. Tumor volume was calculated using the following formula: tumor volume (mm 3 ) = 0.5 x length x width 2 .

[0225] As can be seen in Figure 6 (A-B), DARPin® Protein #24 in multidomain TCE format showed good efficacy in inhibiting tumor growth and tumor volume over the entire duration of the experiment (Figure 6A) and 17 days after the first injection (Figure 6B).

[0226] Example 9: In vitro efficacy evaluation of exemplary multidomain TCE-binding proteins, including DARPin® Protein #24, using MOLM13 wild-type and MOLM13 CRISPR CD70 knockout (KO) target cells co-cultured with human pan-T cells DARPin® Protein #24, a multidomain T cell engager binding protein formatted with two ankyrin repeat domains with binding specificity for human serum albumin (TCA1 and TAA2), respectively, and one ankyrin repeat domain with binding specificity for CD3, was tested in an in vitro short-term T cell activation assay by FACS measuring the CD25 activation marker on CD8+ T cells. In this assay, pan-T cells were cocultured with target cells, either (1) Molm-13 tumor cells with wild-type target expression of CD70, TAA1, and TAA2, or (2) Molm-13 tumor cells in which expression of CD70 (but not TAA1 and TAA2) had been eliminated by CRISPR knockout (KO) technology (Figure 7).

[0227] To this end, 100,000 purified pan-effector T cells and 20,000 target cells per well were co-incubated in duplicate with serial dilutions of multidomain T cell engager binding proteins (E:T ratio 5:1) in the presence of 20 μM human serum albumin for 48 h at 37°C. After 48 h, cells were washed and stained with 1:1,000 Live / Dead Green (Thermo Fisher), 1:400 mouse anti-human CD8 Pacific Blue (BD), and 1:100 mouse anti-human CD25 PerCP-Cy5.5 (eBiosciences) antibodies for 30 min at 4°C. After washing and fixation, cells were analyzed on a FACS Canto II (BD) machine. T cell activation was assessed by measuring CD25+ cells on Live / Dead-negative and CD8+-gated T cells. FACS data were analyzed using FlowJo software and data were plotted using GraphPad Prism8 (3-PL-fit).

[0228] The results demonstrate that an exemplary multidomain T cell engager binding protein comprising DARPin® protein #24 was able to potently activate T cells in the presence of Molm-13 tumor cells with wild-type expression ( FIG. 7 , curve 1; EC value for DARPin® protein #24 TCE: 5.71 pM). The results further demonstrate that when expression of CD70 on Molm-13 tumor cells was eliminated, T cell activation was significantly reduced ( FIG. 7 , curve 2; EC value for DARPin® protein #24 TCE: 20.70 pM). This provides evidence that DARPin® protein #24 was functional in the context of an exemplary multidomain T cell engager binding protein and contributed significantly to the overall efficacy of the multispecific T cell engager protein by its ability to specifically bind CD70 on target cells.

[0229] The specification is most thoroughly understood in light of the teachings of the references cited within the specification. The embodiments within the specification provide illustrations of embodiments of the invention and should not be construed as limiting the scope of the invention. One of ordinary skill in the art will readily recognize that many other embodiments are encompassed by the present invention. All publications, patents, and GenBank sequences cited in this disclosure are incorporated by reference in their entirety. In the event that material incorporated by reference contradicts or is inconsistent with the present specification, the present specification will take precedence over any such material. The citation of any reference herein is not an admission that such reference is prior art to the present invention.

[0230] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein which equivalents are intended to be encompassed by the following claims.

[0231] array [Table 6-1] [Table 6-2] [Table 6-3] [Table 6-4] [Table 6-5] [Table 6-6]

Claims

1. A recombinant binding protein comprising an ankyrin repeat domain, said ankyrin repeat domain having binding specificity for human CD70, said ankyrin repeat domain comprising an amino acid sequence having at least 85% amino acid sequence identity with any one of SEQ ID NOs: 1-12 and 71-72.

2. The ankyrin repeat domain has a dissociation constant (K D ) which binds to human CD70; and / or The recombinant binding protein of claim 1, wherein the ankyrin repeat domain binds to human CD70 with an EC 50 ranging from about 0.2 to about 500 nM.

3. further comprising a binding moiety having binding specificity for a target expressed on an immune cell; Optionally, said immune cell is a T cell and said target expressed on an immune cell is CD3; 3. The recombinant binding protein according to claim 1 or 2, wherein the binding moiety, optionally having binding specificity for a target expressed on an immune cell, is an ankyrin repeat domain.

4. 4. The recombinant binding protein of claim 3, wherein the binding moiety having binding specificity for a target expressed on an immune cell is an ankyrin repeat domain having binding specificity for human CD3.

5. wherein the binding moiety having binding specificity for a target expressed on an immune cell is an ankyrin repeat domain having binding specificity for human CD3, and the ankyrin repeat domain having binding specificity for human CD3 comprises an amino acid sequence having at least 95% amino acid sequence identity with any one of SEQ ID NOs: 55 to 59, 4. The recombinant binding protein according to claim 3, wherein said ankyrin repeat domain, optionally with binding specificity for human CD3, comprises the amino acid sequence of any one of SEQ ID NOs: 55 to 59.

6. the ankyrin repeat domain having binding specificity for human CD70 and the binding moiety having binding specificity for a target expressed on an immune cell are covalently linked by a peptide linker, Optionally, the peptide linker is a proline-threonine rich peptide linker; 4. The recombinant binding protein of claim 3, optionally wherein the amino acid sequence of the peptide linker has a length of 1 to 50 amino acids.

7. the binding protein further comprises a half-life extending moiety; 2. The recombinant binding protein of claim 1, optionally wherein the half-life extending moiety is an ankyrin repeat domain having binding specificity for human serum albumin.

8. said ankyrin repeat domain having binding specificity for human serum albumin comprises an amino acid sequence which is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 52 to 54; 8. The recombinant binding protein according to claim 7, wherein the ankyrin repeat domain, optionally having binding specificity for human serum albumin, comprises the amino acid sequence of any one of SEQ ID NOs: 52 to 54.

9. The recombinant binding protein of claim 1, wherein the binding protein further comprises at least one binding moiety having binding specificity for a target expressed in a tumor cell, the target expressed in a tumor cell being different from human CD70.

10. A nucleic acid encoding the recombinant binding protein of claim 1.

11. A pharmaceutical composition comprising the recombinant binding protein of claim 1 or the nucleic acid of claim 10 and a pharma- ceutically acceptable carrier and / or diluent.

12. 12. The pharmaceutical composition of claim 11, for use in activating immune cells in tumor tissue of a human patient, optionally wherein the immune cells are T cells.

13. 12. A pharmaceutical composition according to claim 11 for use in therapy.

14. 12. A pharmaceutical composition according to claim 11 for use in the treatment of cancer, optionally for use in the treatment of cancer characterised by liquid tumours.

15. 15. The pharmaceutical composition of claim 14, wherein the cancer is leukemia, optionally wherein the cancer is acute myeloid leukemia.

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