Novel DARPin-based CD33 engagers

Abstract

The present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33. 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 Patent Application No. 63 / 158,539, filed March 9, 2021, U.S. Patent No. 63 / 172,818, filed April 9, 2021, and U.S. Patent Application No. 63 / 265,179, filed December 9, 2021. The disclosures of these patent applications are incorporated by reference herein in their entireties for all purposes.

[0002] The present invention relates to recombinant binding proteins comprising an ankyrin repeat domain, which has binding specificity for human CD33. Additionally, 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. Therapeutic resistance remains the leading cause of AML-related deaths (Winer and Stone, Ther Adv Hematol; 2019; vol. 10). Although standard protocols using chemotherapy remain the primary therapeutic approach applied 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] CD33 is an attractive target for the treatment of cancer, especially AML, since it is expressed on approximately 80-90% of AML blasts and leukemic stem cells (Ehninger et al., Blood Cancer Journal, Vol. 4, No. e218, 2014). CD33 has also been clinically validated as a target for AML treatment, where anti-CD33 antibodies have either been used as monotherapy or conjugated with cytotoxic agents (Winer & Stone, Ther Adv Hematol; 2019; Vol. 10). However, these drugs have shown either significant adverse effects or low efficacy. For example, treatment with gemtuzumab ozogamicin, a humanized anti-CD33 monoclonal antibody conjugated to the cytotoxic agent calicheamicin, resulted in significant hematologic and hepatotoxicity.

[0005] CAR-T cell therapy is an approach that has strongly influenced the management of lymphoid malignancies. There has been great interest in applying this technology to AML as well, but in practice, this has proven difficult. With respect to monoclonal antibodies, CD33 is considered to be the most promising target for CAR-T cell therapy in AML. However, preclinical models for this approach have shown a wide range of side effects (on-target / off-tumor toxicity) against non-AML cells, and cytokine release syndrome (CRS) is another recognized side effect.

[0006] T cell-directed killing of tumor cells using bispecific antibodies is another recent therapeutic tool that has been utilized to treat various cancer types, including AML. These T cell engager (TCE) bispecific antibodies contain two distinct variable regions, one that binds to the T cell receptor complex subunit CD3 and the other that binds to a tumor cell surface antigen. Binding of TCE to these two targets provides a functional connection between the cells, resulting in T cell activation and cytotoxic activity against tumor cells, circumventing normal TCR-MHC interactions (Ellerman, Methods; vol. 154:102-117 (2019)). AMG330 is a bispecific antibody against CD3 and CD33 that can trigger T cell cytotoxicity against AML cells.

[0007] However, both multispecific and bispecific antibody therapies present several drawbacks such as high production costs and carry the risk of causing certain severe side effects such as cytokine release syndrome (CRS) (Shimabukuro-Vornhagen et al., J Immunother Cancer; Vol. 6(Issue 1):56 (2018); Labrjin et al., Nat Rev Drug Discov; Vol. 18(Issue 8):585-608 (2019)), and / or on-target / off-tumor toxicity (Weiner et al., Cancer Res.; Vol. 55(Issue 20):4586-4593 (1995); Weiner et al., Cancer Immunol. Immunother.; Vol. 42(Issue 3):141-150 (1996)). Antibody-based T cell engagers often display over 1000-fold higher affinity for CD3 compared to natural TCR-MHC interactions (Wu et al., Pharmacol Ther. 182:161-75 (2018); WO 2014 / 167022; Junntila et al., Cancer Res. 19:5561-71 (2014); Yang et al., J Immunol. Aug. 15(137):1097-100 (1986)). This high affinity correlates with lower efficiency in terms of T cell activation and tumor cell killing (Bortoletto et al., Eur. J. Immunol. 32(11):3102-3107 (2002); Ellerman, Methods 154:102-117 (2019); Mandikian et al., Mol. Cancer Ther. 17(4):776, LP-785 (2018); Vafa et al., Frontiers in Oncology 10:446 (2020)). Furthermore, downregulation of tumor surface markers targeted by TCE may result in tumor resistance to TCE treatment.

[0008] Acute myeloid leukemia (AML), as described above, is a type of cancer that in many ways exemplifies the challenges of cancer therapy and the shortcomings of currently available cancer therapies. For AML, there remains a high medical need due to the high mortality rate, and treatment of relapsed or refractory AML remains difficult to cure.

[0009] Thus, there remains a need for novel CD33-specific binding proteins with beneficial properties. Such binding proteins may be useful in therapeutic and diagnostic approaches for the treatment and characterization of diseases, including cancers such as AML. In particular, there is a need for novel CD33-specific binding proteins that can function to specifically target CD33 on cancer cells and that can also be easily combined with other functional moieties, such as one or more binding moieties. Summary of the Invention

[0010] The present invention relates to recombinant binding proteins comprising an ankyrin repeat domain, which has binding specificity for human CD33. Additionally, 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).

[0011] The recombinant binding proteins of the invention specifically bind or target the tumor-associated antigen (TAA) CD33. Such binding proteins of the invention may serve as tools or building blocks for generating new therapeutic or diagnostic agents. Also disclosed herein are recombinant binding proteins in which a CD33-specific ankyrin repeat domain is combined with one or more other functional moieties in one molecule. Such other functional moieties include binding moieties with binding specificity for targets expressed on immune cells, half-life extending moieties, binding moieties with binding specificity for another tumor-associated antigen, and / or cytotoxic agents. Thus, the recombinant binding proteins of the invention with binding specificity for CD33 are useful for generating novel therapeutic molecules that may provide improved toxicity profiles and / or therapeutic windows compared to current therapeutic modalities.

[0012] Based on the disclosure provided herein, those of ordinary skill in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by embodiment (E) below.

[0013] 1. In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33, and which comprises an ankyrin repeat module having an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 29 to 51 and SEQ ID NOs: 79 to 81, and (2) sequences in which up to 9 amino acids in any of SEQ ID NOs: 29 to 51 and SEQ ID NOs: 79 to 81 are replaced by another amino acid.

[0014] 2. In a second embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33, and which comprises an amino acid sequence having at least 85% amino acid sequence identity with any one of SEQ ID NOs: 1 to 16 and SEQ ID NOs: 77 to 78.

[0015] 3. In a third embodiment, the present invention provides an ankyrin repeat domain that binds to human CD33 in PBS with a dissociation constant (K D ), optionally with a K between about 0.1 nM and about 100 nM D The present invention relates to a recombinant binding protein according to any one of embodiments 1 or 2, which binds to

[0016] 4. In a fourth embodiment, the present invention relates to an ankyrin repeat domain having an EC value ranging from about 0.1 nM to about 10 nM. 50 The present invention relates to a recombinant binding protein according to any one of embodiments 1 to 3, which binds to human CD33 at

[0017] 5. In a fifth embodiment, the present invention relates to a recombinant binding protein according to any one of embodiments 1 to 4, further comprising a binding moiety having binding specificity for a target expressed on an immune cell.

[0018] 6. In a sixth embodiment, the present invention relates to a recombinant binding protein according to embodiment 5, wherein said immune cell is a T cell and said target expressed on an immune cell is CD3.

[0019] 7. In a seventh embodiment, the present invention relates to a recombinant binding protein according to any one of embodiments 5 to 6, wherein said binding moiety having binding specificity for a target expressed on an immune cell is an ankyrin repeat domain.

[0020] 8. In an eighth embodiment, the present invention relates to a recombinant binding protein according to any one 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.

[0021] 9. In a ninth embodiment, the present invention relates to a recombinant binding protein according to any one of embodiments 5 to 7, wherein said 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 said 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.

[0022] 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.

[0023] 11. In an eleventh embodiment, the present invention relates to a recombinant binding protein according to any one of embodiments 5 to 10, wherein said ankyrin repeat domain with binding specificity for human CD33 and said binding moiety with binding specificity for a target expressed on an immune cell are covalently linked to a peptide linker.

[0024] 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.

[0025] 13. In the 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.

[0026] 14. In the fourteenth embodiment, the present invention relates to a recombinant binding protein according to any one of embodiments 1 to 13, wherein said binding protein further comprises a half-life extending moiety.

[0027] 15. In the fifteenth embodiment, the present 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.

[0028] 16. In the sixteenth embodiment, the present invention relates to a recombinant binding protein according to embodiment 15, wherein 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.

[0029] 17. In the seventeenth embodiment, the present invention relates to a recombinant binding protein according to embodiments 15 and 16, wherein said 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.

[0030] 18. In the eighteenth embodiment, the present invention relates to a recombinant binding protein according to any one of embodiments 1 to 17, wherein the binding protein further comprises at least one binding moiety having binding specificity for a target expressed in tumor cells, and wherein the target expressed in tumor cells is different from human CD33.

[0031] 19. In a nineteenth embodiment, the present invention relates to a nucleic acid encoding a recombinant binding protein according to any one of embodiments 1 to 18.

[0032] 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 pharma- ceutically acceptable carrier and / or diluent.

[0033] 21. In the 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.

[0034] 22. In the twenty-second embodiment, the present invention relates to the method according to embodiment 21, wherein the immune cells are T cells.

[0035] 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 treating 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.

[0036] 24. In the 24th embodiment, the invention relates to the method according to embodiment 23, wherein the medical condition is cancer.

[0037] 25. In the twenty-fifth embodiment, the invention relates to the method according to embodiment 23, wherein the medical condition is a cancer characterized by a liquid tumor.

[0038] 26. In the 26th embodiment, the invention relates to the method according to embodiment 23, wherein the medical condition is leukemia.

[0039] 27. In the twenty-seventh embodiment, the invention relates to the method according to embodiment 23, wherein the medical condition is acute myeloid leukemia.

[0040] 28. In the 28th 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 therapy.

[0041] 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 characterized by a liquid tumor.

[0042] 30. In a thirtieth embodiment, the present invention relates to a recombinant binding protein or a pharmaceutical composition for use according to embodiment 29, wherein the cancer is leukemia, optionally wherein the cancer is acute myeloid leukemia. [Brief description of the drawings]

[0043] [Figure 1A] Surface plasmon resonance (SPR) analysis of ankyrin repeat protein binding to human CD33. Figure 1A. SPR analysis of DARPin® protein #29; Figure 1B. SPR analysis of DARPin® protein #30. Different concentrations of purified ankyrin repeat proteins were applied to a GLC chip with immobilized human CD33 for on- and off-rate measurements. The resulting SPR trace analysis was used to analyze and determine the binding of ankyrin repeat proteins to CD33. RU, resonance units; s, time (seconds). [Figure 1B] Surface plasmon resonance (SPR) analysis of ankyrin repeat protein binding to human CD33. Figure 1A. SPR analysis of DARPin® protein #29; Figure 1B. SPR analysis of DARPin® protein #30. Different concentrations of purified ankyrin repeat proteins were applied to a GLC chip with immobilized human CD33 for on- and off-rate measurements. The resulting SPR trace analysis was used to analyze and determine the binding of ankyrin repeat proteins to CD33. RU, resonance units; s, time (seconds). [Diagram 2] Binding of exemplary binding proteins of the invention to CD33-expressing tumor cells. Concentration-dependent binding curves of DARPin® Protein #2, DARPin® Protein #29, and DARPin® Protein #30 (all single domain format) are shown. [Diagram 3] Short-term T cell activation as determined by the activation marker CD25. Pan-T effector 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. T cell activation induced by selected ankyrin repeat proteins DARPin® Protein #33, DARPin® Protein #31 and DARPin® Protein #32 is shown. [Figure 4] Tumor cell killing assessed by a cytotoxicity assay measuring LDH release. Pan-T effector cells and Molm-13 target cells were incubated at an E:T ratio of 5:1 and tumor cell killing assessed by FACS after 24 hours of co-culture in the presence of serial dilutions of the indicated molecules. Shown is tumor cell killing by T cells triggered by DARPin® protein #33, DARPin® protein #31, and DARPin® protein #32. [Diagram 5] Binding of CD33-specific ankyrin repeat proteins to full-length and truncated CD33 targets determined by HTRF. All three tested ankyrin repeat proteins, DARPin® protein #1, DARPin® protein #9 or DARPin® protein #14, bind to full-length CD33, but only DARPin® protein #9 binds to truncated CD33. Signals are shown as percentages (background corrected and normalized to the maximum HTRF signal observed for each protein). HTRF signals in AU are shown as numbers. [Figure 6]Binding competition ELISA. Biotinylated human CD33 target was pre-incubated with or without competitor (DARPin® Protein #1, DARPin® Protein #9, or DARPin® Protein #14). Bound target was then tested against immobilized AMG330. For DARPin® Protein #1, partial competition with AMG330 was observed. DARPin® Protein #14 shows complete competition with AMG330, whereas DARPin® Protein #9 shows no competition with AMG330. [Figure 7A] Figure 7A. Tumor growth over time in mice (n=5 mice per donor / 2 hPBMC donors used) injected intraperitoneally with hPBMCs, xenografted subcutaneously with MOLM-13 tumor cells 2 days after hPBMC injection, and treated with PBS 1x (closed circles) or DARPin® Protein #29 in multidomain format at 0.5 mg / kg (closed squares). Treatment began 4 days after tumor cell xenografting. Data are presented as mean + SEM. Figure 7B. Evaluation of tumor volume 17 days after tumor cell xenografting in mice described in Figure 7A. [Figure 7B] Figure 7A. Tumor growth over time in mice (n=5 mice per donor / 2 hPBMC donors used) injected intraperitoneally with hPBMCs, xenografted subcutaneously with MOLM-13 tumor cells 2 days after hPBMC injection, and treated with PBS 1x (closed circles) or DARPin® Protein #29 in multidomain format at 0.5 mg / kg (closed squares). Treatment began 4 days after tumor cell xenografting. Data are presented as mean + SEM. Figure 7B. Evaluation of tumor volume 17 days after tumor cell xenografting in mice described in Figure 7A. [Figure 8A]Figure 8A. Potency titration curves of DARPin® protein #29 in multidomain format with wild-type or CD33 knockout Molm-13 tumor cells (T cell activation). EC50 values ​​are shown in pM. Figure 8B. Potency titration curves of DARPin® protein #30 in multidomain format with wild-type or CD33 knockout Molm-13 tumor cells (T cell activation). EC50 values ​​are shown in pM. [Figure 8B] Figure 8A. Potency titration curves of DARPin® protein #29 in multidomain format with wild-type or CD33 knockout Molm-13 tumor cells (T cell activation). EC50 values ​​are shown in pM. Figure 8B. Potency titration curves of DARPin® protein #30 in multidomain format with wild-type or CD33 knockout Molm-13 tumor cells (T cell activation). EC50 values ​​are shown in pM. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0044] Disclosed herein is a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity for human CD33. Also disclosed are nucleic acids encoding the recombinant binding protein, pharmaceutical compositions comprising the binding protein or nucleic acid, and methods of using the binding protein, nucleic acid, or pharmaceutical composition.

[0045] Ankyrin repeat domain The described recombinant binding proteins or binding domains thereof, comprising designed ankyrin repeat motifs or modules, are also referred to herein as DARPin® proteins (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. In general, DARPin® proteins comprise at least one ankyrin repeat domain, and may comprise 2, 3, 4, 5 or more ankyrin repeat domains.

[0046] 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.

[0047] 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 class of binding molecules that have the potential to overcome the limitations of monoclonal antibodies, thus enabling novel therapeutic approaches. Such ankyrin repeat proteins may comprise a single designed ankyrin repeat domain or a combination of two or more designed ankyrin repeat domains with the same or different target specificities (Stumpp et al., Drug Discov. Today 13,695-701, 2008; US Pat. No. 9,458,211). Ankyrin repeat proteins containing only a single designed ankyrin repeat domain are small proteins (14 kDa) that can be selected to bind to a given target protein with high affinity and specificity. These features, and the possibility to combine two or more designed ankyrin repeat domains in 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, e.g. cytotoxic agents or half-life extenders, enabling completely new drug formats.

[0048] 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 generally have low immunogenic potential and no or only minor off-target effects. DARPin® candidates also show favorable development properties, including rapid, low-cost, and high-yield production, as well as a shelf life of up to several years at 4°C. Taken together, designed ankyrin repeat proteins are an example of the next generation of protein therapeutics with the potential to exceed existing antibody drugs.

[0049] DARPin® is a trademark owned by Molecular Partners AG, Switzerland.

[0050] As mentioned above, CD33 is an attractive therapeutic target for the treatment of certain cancers, in particular AML. The recombinant binding proteins described herein contain ankyrin repeat domains and ankyrin repeat modules that specifically bind to human CD33.

[0051] In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33, and which comprises an ankyrin repeat module having an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 29 to 51 and SEQ ID NOs: 79 to 81, and (2) sequences in which up to 9 amino acids in any of SEQ ID NOs: 29 to 51 and SEQ ID NOs: 79 to 81 are replaced by other amino acids.

[0052] In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33, and which comprises an ankyrin repeat module having an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 29 to 51 and SEQ ID NOs: 79 to 81, and (2) sequences in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 29 to 51 and SEQ ID NOs: 79 to 81 is replaced by another amino acid.

[0053] In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33, and which comprises an ankyrin repeat module having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29 to 51 and 79 to 81.

[0054] In another embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33, and which comprises an amino acid sequence having at least about 85% amino acid sequence identity with any one of SEQ ID NOs: 1 to 16 and SEQ ID NOs: 77 to 78.

[0055] In a further embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33, and which 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 to 16 and SEQ ID NOs: 77 to 78.

[0056] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33, and which comprises an amino acid sequence having at least about 85% amino acid sequence identity with SEQ ID NO:1, 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 even 100% amino acid sequence identity.

[0057] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33, and which 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 even 100% amino acid sequence identity, to SEQ ID NO:2.

[0058] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33, and which 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.

[0059] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33, and which 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.

[0060] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33, and which 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.

[0061] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33, and which 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 even 100% amino acid sequence identity, to SEQ ID NO: 6.

[0062] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33, and which 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 even 100% amino acid sequence identity, to SEQ ID NO: 7.

[0063] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33, and which 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.

[0064] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33, and which 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 even 100% amino acid sequence identity, to SEQ ID NO:9.

[0065] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33, and which 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.

[0066] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33, and which 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 even 100% amino acid sequence identity, to SEQ ID NO: 11.

[0067] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33, and which 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 even 100% amino acid sequence identity, to SEQ ID NO: 12.

[0068] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33, and which 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 even 100% amino acid sequence identity, to SEQ ID NO: 13.

[0069] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33, and which 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 even 100% amino acid sequence identity, to SEQ ID NO: 14.

[0070] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33, and which 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 even 100% amino acid sequence identity, to SEQ ID NO: 15.

[0071] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33, and which 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 even 100% amino acid sequence identity, to SEQ ID NO: 16.

[0072] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33, and which 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: 77.

[0073] In one embodiment, the invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33, and which 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: 78.

[0074] In a further embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, which has binding specificity for human CD33, and which comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 16 and SEQ ID NOs: 77 to 78.

[0075] In a further aspect, the invention relates to a recombinant binding protein as described above, further comprising at least one binding moiety having 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 having binding specificity for a target expressed on an immune cell for use in the present invention include antibodies, surrogate scaffolds, and polypeptides.

[0076] Antibodies include any polypeptide or protein that comprises an antigen-binding domain derived from an antibody or immunoglobulin molecule. The antigen-binding domain may be derived, for example, from 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 moiety described herein to comprise a human or humanized antigen-binding domain. Antibodies can be obtained using techniques well known in the art.

[0077] In one embodiment, the binding moiety that has binding specificity for a target expressed on an immune cell is an antibody.

[0078] In one embodiment, the binding moiety with 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 mammalian family Camelidae, 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, in the region of about 15 kDa.

[0079] In one embodiment, the binding moiety with binding specificity for a target expressed on an immune cell is a shark antibody domain. Like camelid nanobodies, shark antibody domains are also devoid of light chains.

[0080] Alternative scaffolds include any polypeptide or protein that can bind an antigen (such as a drug molecule) and that includes a binding domain that is not derived from an antibody or immunoglobulin molecule. The binding domain of the alternative scaffold may include 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), finomers, 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.

[0081] In one embodiment, the binding moiety with binding specificity for a target expressed on an immune cell is a surrogate scaffold. In one embodiment, the binding moiety with 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 finomer, a knottin, a Kunitz domain, or an antigen binding domain derived from or related to a T cell receptor (TCR).

[0082] Adnectins are originally derived from the tenth extracellular domain of human fibronectin type III protein (10Fn3). Fibronectin type III domains have seven or eight β-strands distributed between two β-sheets that themselves pack together to form the core of the protein and further contain loops (similar to CDRs) that connect the beta-strands to each other and are solvent exposed. There are at least three such loops on each edge of the β-sheet sandwich, the edges being the boundaries of the protein perpendicular to the direction of the β-strands (see US Pat. No. 6,818,418). Because of this structure, the non-antibody scaffolds mimic antigen-binding properties similar to the nature and affinity of the antigen-binding properties of antibodies. These scaffolds can be used in in vitro loop randomization and shuffling approaches that are similar to the process of affinity maturation of antibodies in vivo.

[0083] Affibody affinity ligands are composed of a three-helix bundle based on a scaffold of 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 an affibody library with a large number of ligand variants (see, e.g., U.S. Pat. No. 5,831,012). Affibody molecules mimic antibodies, but are much smaller, with a molecular weight of about 6 kDa compared to about 150 kDa for antibodies. Despite the difference in size, the binding site of affibody molecules bears similarity to that of antibodies.

[0084] Affilins are synthetic antibody mimics structurally derived from human ubiquitin (and historically from gamma-B crystallin). Affilins consist of two identical domains with a predominantly beta-sheet structure and a total molecular weight of approximately 20 kDa. Affilins contain several surface-exposed amino acids suitable for modification. Affilins resemble antibodies in their affinity and specificity for antigens, but not in structure.

[0085] Affimers are a type of peptide aptamer with a structure known as SQT (Stefin A quadruple mutant-Tracy). Aptamers and affimers are short peptides in which both the N-terminus and C-terminus of the binding peptide are embedded in the inert scaffold, causing them to bind with affinity to inert, rigid protein scaffolds due to structural constraints.

[0086] Affitins are variants of the DNA-binding protein Sac7d that have been engineered to obtain specific binding affinities. Sac7d, originally from the hyperthermophilic archaeon Sulfolobus acidocaldarius, binds DNA and prevents its thermal denaturation. Affitins are commercially known as Nanofitins.

[0087] Alpha bodies are small (approximately 10 kDa) proteins engineered to bind a variety of antigens and are therefore antibody mimics. Alpha body scaffolds are computationally designed based on coiled-coil structures. A standard alpha body scaffold contains three alpha helices, each composed of four heptad repeats (stretches of seven residues), linked via glycine / serine-rich linkers. The standard heptad sequence is "IAAIQKQ". The ability of alpha bodies to target extracellular and intracellular proteins, combined with their high binding affinity, may enable them to bind targets that cannot be reached by antibodies.

[0088] 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.

[0089] Armadillo repeat protein-based scaffolds are abundant in eukaryotes and are involved in a wide range of biological processes, especially those related to nuclear transport. Armadillo repeat protein-based scaffolds usually consist of 3-5 internal repeats and two capping elements. They also have a tandem extended superhelical structure, which is capable of binding the corresponding peptide ligand in an extended conformation.

[0090] Atrimer is a scaffold derived from a trimeric plasma protein known as tetranectin and belongs to a family of C-type lectins consisting of three identical units. The structure of the C-type lectin domain (CTLD) within tetranectin has five flexible loops that mediate interactions with targeting molecules.

[0091] Avimers are derived from naturally occurring A domain-containing proteins such as HER3 and consist of multiple different "A domain" monomers (2-10) linked via amino acid linkers. For example, the methodologies 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.

[0092] Fynomer is a small globular protein (~7 kDa) evolved from amino acids 83-145 of the Src homology domain 3 (SH3) of 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.

[0093] Knottins, also known as cysteine ​​knot miniproteins, are typically 30 amino acid long proteins that contain three antiparallel β-sheets and a constraining loop held together by disulfide bonds that create a cysteine ​​knot, which confers high thermal stability, making knottins attractive antibody mimics.

[0094] Kunitz domain peptides or Kunitz domain inhibitors are a class of protease inhibitors that have 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.

[0095] 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).

[0096] In a preferred embodiment, the binding moiety with binding specificity for a target expressed on immune cells is an ankyrin repeat domain.

[0097] 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.

[0098] Thus, in a preferred embodiment, the present invention relates to a recombinant binding protein comprising: (i) a first ankyrin repeat domain, which has binding specificity for human CD33, the first ankyrin repeat domain comprising an ankyrin repeat module having an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 29 to 51 and SEQ ID NOs: 79 to 81, and (2) sequences in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2 or up to 1 amino acid in any of SEQ ID NOs: 29 to 51 and SEQ ID NOs: 79 to 81 are replaced by another amino acid; and (ii) a second ankyrin repeat domain, which has binding specificity for CD3, more preferably for human CD3.

[0099] In another embodiment, the present invention relates to a method for the preparation of a medicament for ... The present invention relates to a recombinant binding protein comprising a second ankyrin repeat domain having binding specificity for human CD3, the second ankyrin repeat domain comprising 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.

[0100] In a further embodiment, the invention relates to a method for the preparation of a medicament for the treatment ... Nos. 55-59 and a second ankyrin repeat domain comprising 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.

[0101] 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 CD33, and which comprises an ankyrin repeat module having an amino acid sequence selected from the group consisting of: (1) SEQ ID NOs: 29 to 51 and SEQ ID NOs: 79 to 81, and (2) sequences in which up to 9, up to 8, up to 7, up to 6, up to 5, up to 4, up to 3, up to 2, or up to 1 amino acid in any of SEQ ID NOs: 29 to 51 is replaced by another amino acid; and (ii) a second ankyrin repeat domain, which has binding specificity for human CD3, and which comprises an amino acid sequence of any one of SEQ ID NOs: 55 to 59.

[0102] 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 CD33 and which comprises an ankyrin repeat module having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29 to 51 and SEQ ID NOs: 79 to 81, and (ii) a second ankyrin repeat domain, which has binding specificity for human CD3 and which comprises one of the amino acid sequences of SEQ ID NOs: 55 to 59.

[0103] In a preferred embodiment, the present invention relates to a recombinant binding protein comprising: (i) a first ankyrin repeat domain, which has binding specificity for human CD33, the first ankyrin repeat domain comprising 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 even 100% amino acid sequence identity, to any one of SEQ ID NOs: 1 to 16 and SEQ ID NOs: 77 to 78; and (ii) a second ankyrin repeat domain, which has binding specificity for CD3, more preferably human CD3.

[0104] In another embodiment, the present invention relates to a first ankyrin repeat domain comprising: (i) a first ankyrin repeat domain having binding specificity for human CD33, the first ankyrin repeat domain comprising 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 to 16 and SEQ ID NOs: 77 to 78. and (ii) a second ankyrin repeat domain, wherein the second ankyrin repeat domain has binding specificity for human CD3, and the second ankyrin repeat domain 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 to 59.

[0105] In one embodiment, the present invention relates to a method for the treatment of osteosarcoma comprising: (i) a first ankyrin repeat domain, which has binding specificity for human CD33, and which comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 16 and SEQ ID NOs: 77 to 78; and (ii) a second ankyrin repeat domain, which has binding specificity for human CD3, and which comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 55 to 59. and a second ankyrin repeat domain comprising 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.

[0106] In one embodiment, the present invention provides a method for the preparation of a human CD33-binding protein comprising: (i) a first ankyrin repeat domain, the first ankyrin repeat domain having binding specificity for human CD33, the first ankyrin repeat domain 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%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at least about 121%, at least about 122%, at least about 123%, at least about 124%, at least about 125%, at least about 126%, at least about 127%, at least about 128%, at least about 129%, at least about 130%, at least about 131%, at least about 132%, at least about 133%, at least about 134%, at least about 135%, at least about 136%, at least about 137%, at least about 138%, at least about 139%, at least about 140%, at least about 141%, at least about and (ii) a second ankyrin repeat domain, wherein the second ankyrin repeat domain has binding specificity for human CD3 and comprises the amino acid sequence of any one of SEQ ID NOs: 55 to 59.

[0107] 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 CD33 and comprises any one of the amino acid sequences of SEQ ID NOs: 1 to 16 and 77 to 78, and (ii) a second ankyrin repeat domain, which has binding specificity for human CD3 and comprises any one of the amino acid sequences of SEQ ID NOs: 55 to 59.

[0108] The present invention further relates to a recombinant binding protein comprising: (i) a first ankyrin repeat domain, which has binding specificity for human CD33; and (ii) a second ankyrin repeat domain, which has binding specificity for human CD3, wherein the recombinant binding protein 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: 17 to 28 and SEQ ID NOs: 82 to 84.

[0109] In one embodiment, the invention relates to a recombinant binding protein comprising (i) a first ankyrin repeat domain, which has binding specificity for human CD33, and (ii) a second ankyrin repeat domain, which has binding specificity for human CD3, wherein the recombinant binding protein comprises an amino acid sequence having at least about 85% amino acid sequence identity with any one of SEQ ID NOs: 17 to 28 and 82 to 84.

[0110] 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).

[0111] In some embodiments, the recombinant binding proteins described herein comprise an ankyrin repeat domain that specifically binds to serum albumin (preferably, such as 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, for example, 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, a second and a 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.

[0112] In some embodiments, the half-life extending moieties described herein comprise a serum albumin-specific ankyrin repeat domain, wherein the ankyrin repeat domain of the binding moiety comprises 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 about 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.

[0113] In some embodiments, the serum albumin binding domain is located at the N-terminus of the recombinant binding protein of the present 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 present invention.

[0114] 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), IgA2(α2). In some embodiments, the Fc domain is a human IgG1 Fc domain.

[0115] In some embodiments, the Fc domain comprises an uninterrupted native sequence of the Fc domain (i.e., wild-type sequence). In some embodiments, the immunoglobulin Fc domain comprises a mutant Fc domain that results in an 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., International Patent Publication No. WO2005 / 063815) and / or to increase homogeneity during production of the recombinant binding protein. In some embodiments, the Fc domain is a human IgG1 Fc domain 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 a 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).

[0116] Further binding moieties with binding specificity for tumor-associated antigens In a further aspect, the present invention relates to a recombinant binding protein as described above, further comprising at least one binding moiety having binding specificity for a tumor associated antigen (TAA) different from CD33. In one embodiment, the one or more TAAs different from CD33 are TAA co-expressed with CD33 in cells derived from the same cancer. In a further embodiment, the one or more TAAs different from CD33 are TAA co-expressed with CD33 in cancer characterized by liquid tumors. In a preferred embodiment, the one or more TAAs different from CD33 are TAA co-expressed with CD33 in leukemia, such as TAA co-expressed with CD33 in AML cancer cells. Examples of binding moieties having binding specificity for a tumor associated antigen (TAA) different from CD33 for use in the present invention include antibodies, alternative scaffolds, and polypeptides. Many TAAs are known in the art, including TAAs expressed in AML cancer cells.

[0117] replacement In some embodiments, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2 or no more than 1 substitution is made in any ankyrin repeat module of a recombinant binding protein of the invention for the sequences of SEQ ID NOs: 29-51 and SEQ ID NOs: 79-81. In some embodiments, no more than 5 substitutions are made for the sequences of SEQ ID NOs: 29-51 and SEQ ID NOs: 79-81. In some embodiments, no more than 4 substitutions are made for the sequences of SEQ ID NOs: 29-51 and SEQ ID NOs: 79-81. In some embodiments, no more than 3 substitutions are made for the sequences of SEQ ID NOs: 29-51 and SEQ ID NOs: 79-81. In some embodiments, no more than 2 substitutions are made for the sequences of SEQ ID NOs: 29-51 and SEQ ID NOs: 79-81. In some embodiments, no more than 1 substitution is made for the sequences of SEQ ID NOs: 29-51 and SEQ ID NOs: 79-81.

[0118] In some embodiments, no more than 15%, no more than 14%, no more than 13%, no more than 12%, no more than 11%, no more than 10%, no more than 9%, no more than 8%, or no more than 7%, no more than 6%, no more than 5%, no more than 4%, no more than 3%, no more than 2%, or no more than 1% of the amino acid sequence of any ankyrin repeat domain of the recombinant binding protein of the invention is modified by substitutions to the sequences of SEQ ID NOs: 1-16 and SEQ ID NOs: 77-78. In some embodiments, no more than 10% of the amino acid sequence is modified by substitutions to the sequences of SEQ ID NOs: 1-16 and SEQ ID NOs: 77-78. In some embodiments, no more than 8% of the amino acid sequence is modified by substitutions to the sequences of SEQ ID NOs: 1-16 and SEQ ID NOs: 77-78. In some embodiments, no more than 6% of the amino acid sequence is modified by substitutions to the sequences of SEQ ID NOs: 1-16 and SEQ ID NOs: 77-78. In some embodiments, no more than 4% of the amino acid sequence is modified by substitutions to the sequences of SEQ ID NOs: 1 to 16 and SEQ ID NOs: 77 to 78. In some embodiments, no more than 2% of the amino acid sequence is modified by substitutions to the sequences of SEQ ID NOs: 1 to 16 and SEQ ID NOs: 77 to 78.

[0119] In some embodiments, the amino acid substitution(s) made to a binding agent is / are less than the K of the unsubstituted binding agent. D Compared with the value, K D For example, in some embodiments, the amino acid substitution(s) does not change the K value for CD33 of a binding agent comprising any of the sequences of SEQ ID NOs: 1-16, 29-51, and 77-81. D Compared with the value, K 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.

[0120] In certain embodiments, the substitutions are conservative substitutions according to 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.

[0121] [Table 1]

[0122] In certain embodiments, the substitutions are made within the structural core residues of the ankyrin repeat domain. In other embodiments, the substitutions are made outside the structural core residues of the ankyrin repeat domain. By way of 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, the substitutions are made in the residue designated as "x". In another embodiment, the substitutions are made in the residues not designated as "x".

[0123] 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: 1-16, 52-59, and 77-78, optionally in which A at the penultimate position is replaced by L and / or A at the final position is replaced by N, or optionally in which L at the penultimate position is replaced by A and / or N at the final position is replaced by A. In exemplary embodiments, each ankyrin repeat domain comprises an amino acid sequence that is at least 90% identical to any of SEQ ID NOs: 1-16, 52-58 and 77-78, optionally in which A at the penultimate position is replaced by L and / or A at the last position is replaced by 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).

[0124] In addition, each ankyrin repeat domain contained in the recombinant binding protein of the invention may optionally contain 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 to 16, 52 to 59, and 77 to 78, and further comprises GS (e.g., as in SEQ ID NO: 64) at its N-terminus or only G or S instead of GS.

[0125] binding affinity In certain embodiments, the affinity between the recombinant binding protein and its target (i.e., human CD33) is K 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 -9M~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 10-7 M~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 It's M.

[0126] In exemplary embodiments, the recombinant binding protein has a K of about 900 nM, about 800 nM, about 700 nM, about 600 nM, about 500 nM, about 400 nM, about 300 nM, about 250 nM, about 200 nM, 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 CD33 with a K value of 100 nM or less. D In another exemplary embodiment, the recombinant binding protein binds to CD33 with a K value of 10 nM or less. D Binds to CD33 at 100 ng / mL.

[0127] In one aspect, the recombinant binding protein has an EC of less than about 5000, about 4000, about 3000, about 2000, about 1000, about 900, about 700, 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 CD33 with an EC 50 in another embodiment, the binding protein binds to human CD33 on T cells with an EC 50in another embodiment, the binding protein binds to human CD33 on T cells with an EC 50 in another embodiment, the binding protein binds to human CD33 on T cells with an EC 50 in another embodiment, the binding protein binds to human CD33 on T cells with an EC 50 in another embodiment, the binding protein binds to human CD33 on T cells with an EC 50 in another embodiment, the binding protein binds to human CD33 on T cells with an EC 50 in another embodiment, the binding protein binds to human CD33 on T cells with an EC 50 In another embodiment, the binding protein binds to human CD33 on T cells with an EC 50 in another embodiment, the binding protein binds to human CD33 on T cells with an EC 50 in another embodiment, the binding protein binds to human CD33 on T cells with an EC 50 in another embodiment, the binding protein binds to human CD33 on T cells with an EC 50 in another embodiment, the binding protein binds to human CD33 on T cells with an EC 50 in another embodiment, the binding protein binds to human CD33 on T cells with an EC 50 in another embodiment, the binding protein binds to human CD33 on T cells with an EC 50 in another embodiment, the binding protein binds to human CD33 on T cells with an EC 50 in another embodiment, the binding protein binds to human CD33 on T cells with an EC 50 in another embodiment, the binding protein binds to human CD33 on T cells with an EC 50 in another embodiment, the binding protein binds to human CD33 on T cells with an EC50 in another embodiment, the binding protein binds to CD33 with an EC 50 in another embodiment, the binding protein binds to CD33 with an EC 50 in another embodiment, the binding protein binds to CD33 with an EC 50 in another embodiment, the binding protein binds to CD33 with an EC 50 in another embodiment, the binding protein binds to CD33 with an EC 50 in another embodiment, the binding protein binds to CD33 with an EC 50 in another embodiment, the binding protein binds to CD33 with an EC 50 in another embodiment, the binding protein binds to CD33 with an EC 50 in a further aspect, the binding protein binds to CD33 with an EC 50 and binds to CD33.

[0128] Additional Polypeptides In one embodiment, the recombinant binding protein of the 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 the amino acid sequence improves the physicochemical behavior of the polypeptide / protein, or the amino acid sequence has an effector function. The individual polypeptide tags of the recombinant binding protein may be connected to other sites of the recombinant binding protein, either directly or via a peptide linker. Polypeptide tags are well known in the art and are fully available to the skilled artisan. Examples of polypeptide tags are small polypeptide sequences, such as His-tags, HA-tags, myc-tags, FLAG-tags or Strep-tags, or polypeptides such as enzymes (e.g. alkaline phosphatase), which allow detection of the polypeptide / protein, or polypeptides that can be used for targeting (such as immunoglobulins or fragments thereof) and / or as effector molecules.

[0129] In one embodiment, the recombinant binding protein of the 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-proteinaceous compound or a polymer such as polyethylene glycol, or a protein domain and a biologically active molecule, a protein domain and a localizer, or two sequence tags. Peptide linkers are known to those skilled in the art. A list of examples is provided in the specification of published patent application WO2002 / 020565. In one embodiment, a peptide linker for use in the invention has a length of 1 to 50 amino acids. In another embodiment, a peptide linker for use in the invention has a length of about 5 to about 40 amino acids. In another embodiment, a peptide linker for use in the invention has a length of about 10 to about 30 amino acids.

[0130] Specific examples of peptide linkers are glycine-serine-linkers and variable length proline-threonine-rich linkers. In the context of the present invention, a proline-threonine-rich linker comprises 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 NO:65 and SEQ ID NO:66.

[0131] N- and C-terminal capping sequences The ankyrin repeat domain of the recombinant binding protein disclosed herein may comprise an N- or C-terminal capping sequence. Capping sequence refers to an additional polypeptide sequence fused to the N- or C-terminus of the ankyrin repeat motif(s), where said capping sequence forms strict tertiary interactions (i.e. tertiary structure interactions) with the ankyrin repeat motif(s), thereby providing a cap that shields the hydrophobic core of the flanking ankyrin repeat domain from exposure to the solvent.

[0132] 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 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: 69-72, and examples of ankyrin C-terminal capping modules (i.e. C-terminal capping repeats) include SEQ ID NOs: 73-76.

[0133] 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.

[0134] Compositions, Uses and Methods of Treatment In one aspect the present invention relates to a pharmaceutical composition comprising a recombinant binding protein and / or a designed ankyrin repeat domain of the invention and / or a nucleic acid encoding a recombinant binding protein and / or a designed ankyrin repeat domain of the invention and optionally a pharma- ceutically acceptable carrier and / or diluent.

[0135] 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 invention, and optionally a pharma- ceutically acceptable carrier and / or diluent.

[0136] Pharmaceutically acceptable carriers and / or diluents are known to those of skill in the art and are described in more detail below.

[0137] The pharmaceutical composition comprises a recombinant binding protein, and / or a designed ankyrin repeat domain, and / or a nucleic acid as described herein, preferably a recombinant binding protein and / or a nucleic acid, and a pharma- ceutically acceptable carrier, excipient, or stabilizer, e.g. as described in "Remington's Pharmaceutical Sciences", 16th ed., Osol, A. Ed. (1980).

[0138] 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 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 acids, polyglycolic acids, polymeric amino acids, and amino acid copolymers. The pharmaceutical composition may also be a combination product that includes additional active ingredients, 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 easily accomplished by filtration through sterile filtration membranes.

[0139] In one aspect, a pharmaceutical composition comprises at least one recombinant binding protein as described herein, and a detergent, such as a non-ionic detergent, a buffer, such as a phosphate buffer, and a sugar, such as sucrose. In one aspect, such a composition comprises a recombinant binding protein as described above and PBS.

[0140] In another aspect, the present invention provides a method for tumor-localized 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.

[0141] In another aspect, the invention provides 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 of the invention, a nucleic acid of the invention, or a pharmaceutical composition of the invention.

[0142] In another aspect, the invention provides a method for treating a medical condition, the method comprising administering to a patient in need of treating the medical condition a therapeutically effective amount of a recombinant binding protein of the invention, further comprising a binding agent having binding specificity for a disease associated antigen, a nucleic acid encoding the recombinant binding protein, or a pharmaceutical composition comprising the binding protein.

[0143] In one aspect, the present invention relates to a pharmaceutical composition, a recombinant binding protein, or a nucleic acid according to the present invention for use in the treatment of a disease. For that purpose, the pharmaceutical composition, the nucleic acid, or the recombinant binding protein according to the present invention is administered in a therapeutically effective amount to a patient in need thereof. Administration can include topical administration, oral administration, and parenteral administration. A typical route of administration is parenteral administration. In parental administration, the pharmaceutical composition of the present invention is formulated in a unit dose injectable form, such as a solution, suspension, or emulsion, together with a pharma- ceutically acceptable excipient as defined above. The dose and the method of administration vary according to the individual and the disease to be treated.

[0144] Furthermore, any of the pharmaceutical compositions, nucleic acids or recombinant proteins described above are contemplated for use in the treatment of disorders.

[0145] 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 drip infusion.

[0146] In one aspect, the 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 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 invention relates to a process for the manufacture of a medicament for the treatment of a disease, wherein a recombinant binding protein of the invention, a nucleic acid molecule of the invention, or a pharmaceutical composition of the invention is an active ingredient of the medicament. In one aspect, the 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.

[0147] In one aspect, the present invention further provides the use of such recombinant binding proteins for treating a medical condition in a subject in need thereof.

[0148] As used herein, the medical condition or disease is cancer, preferably a liquid tumor, more preferably leukemia, even more preferably acute myeloid leukemia (AML).

[0149] The use of the recombinant binding protein of the invention, the nucleic acid of the invention, or the pharmaceutical composition of the invention can also be 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 at the same time and one of the two is also administered at a later stage).

[0150] 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 CD33, e.g., SEQ ID NOs: 1-16 and SEQ ID NOs: 77-78, and / or a nucleic acid encoding a recombinant protein of the present invention comprising an ankyrin repeat domain having binding specificity for CD33, e.g., SEQ ID NOs: 1-16 and SEQ ID NOs: 77-78, and / or a pharmaceutical composition comprising a recombinant protein of the present invention comprising an ankyrin repeat domain having binding specificity for CD33, e.g., SEQ ID NOs: 1-16 and SEQ ID NOs: 77-78. 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: 1-59 and SEQ ID NOs: 77-81, and / or a nucleic acid encoding the recombinant protein, and / or a pharmaceutical composition comprising the recombinant protein.

[0151] 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, for example a recombinant protein comprising the amino acid sequence of any one of SEQ ID NOs: 1-59 and SEQ ID NOs: 77-84, comprising the steps of (i) expressing the recombinant binding protein in a suitable host cell (e.g., in 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.

[0152] The present invention is not limited to the specific embodiments described in the Examples. This specification makes reference to a number of amino acid sequences, nucleic acid sequences, and SEQ ID NOs, which are disclosed in the accompanying Sequence Listing, which is incorporated herein by reference in its entirety.

[0153] 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. Furthermore, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, the nomenclature used in connection with the techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry described herein are those well known and commonly used in the art.

[0154] The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms unless otherwise indicated. When an embodiment of the invention describes a feature as "comprising," the embodiment also contemplates that the feature "consists of" or "consists essentially of." Any and all examples provided herein, or the use of illustrative language (e.g., "such as"), are intended merely to better illustrate the disclosure and do not impose limitations on the scope of the disclosure unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure. Other than in the illustrative examples, or unless otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood to be modified in all instances by the term "about" as that term would be interpreted by one of ordinary skill in the art. As used herein, the term "about" corresponds to ±10% of a given numerical value, unless otherwise indicated.

[0155] The recitation of ranges of values ​​herein, unless otherwise indicated herein, is merely intended to serve as a shorthand method of referring individually to each separate value and each endpoint falling within the range, and each separate value and endpoint is incorporated herein as if it were individually recited herein.

[0156] In the context of the present invention, the term "protein" refers to a molecule that comprises a polypeptide, at least a portion of which has or can acquire a defined three-dimensional sequence by forming secondary, tertiary, or quaternary structures within a single polypeptide chain and / or between multiple polypeptide chains. When a protein comprises more than one polypeptide chain, the individual polypeptide chains can be linked by non-covalent or covalent bonds, for example, by disulfide bonds between two polypeptides. The portions of a protein that individually have or can acquire a defined three-dimensional sequence by forming secondary and / or tertiary structures are referred to as "protein domains". Such protein domains are well known to those skilled in the art.

[0157] The term "recombinant" as used in recombinant proteins and polypeptides means that the protein or polypeptide is produced by the use of recombinant DNA technology well known to those skilled in the art. For example, a recombinant DNA molecule (e.g., produced by gene synthesis) encoding a polypeptide can be cloned into a bacterial expression plasmid (e.g., pQE30, QIAgen), yeast expression plasmid, mammalian expression plasmid, or plant expression plasmid, or DNA that allows for in vitro expression. For example, when such a recombinant bacterial expression plasmid is inserted into a suitable bacterium (e.g., E. coli), these bacteria can produce the polypeptide(s) encoded by the recombinant DNA. The polypeptide or protein produced accordingly is called a recombinant polypeptide or protein.

[0158] In the context of the present invention, the term "binding protein" refers to a protein that comprises 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 CD33.

[0159] Additionally, any such binding proteins may include additional polypeptides known to those of skill 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 include localizer molecules.

[0160] 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.

[0161] As used herein, the term "target" refers to an individual molecule, such as a nucleic acid molecule, a polypeptide or protein, a carbohydrate, or any other naturally occurring molecule, including any part of such an individual molecule, or a complex of two or more of such molecules, or a whole cell or tissue sample, or any non-natural compound.Preferably, the target is CD33.More preferably, the target is human CD33.

[0162] In the context of the present invention, the term "polypeptide" refers to a molecule that consists of a chain of multiple, i.e., two or more, amino acids linked by peptide bonds. Preferably, a polypeptide consists of more than eight amino acids linked by peptide bonds. The term "polypeptide" also includes multiple chains of amino acids linked by cysteine ​​S-S bridges. Polypeptides are well known to those skilled in the art.

[0163] Patent application WO2002 / 020565 and Forrer et al., 2003 (Forrer, P., Stumpp, MT, Binz, HK, Pluckthun, A., 2003. FEBS Letters 539, 2-6) contain a general description of the characteristics of repeat proteins and of the characteristics, techniques and applications of repeat domains. The term "repeat protein" refers to a protein comprising one or more repeat domains. Preferably, a repeat protein comprises one, two, three, four, five or six repeat domains. Furthermore, the repeat protein may comprise additional non-repeat protein domains, polypeptide tags and / or peptide linkers. The repeat domain may be a binding domain.

[0164] The term "repeat domain" refers to a protein domain comprising two or more consecutive repeat modules as structural units, which repeat modules have 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 the skilled person from the examples of ankyrin repeat domains (WO 2002 / 020565), leucine-rich repeat domains (WO 2002 / 020565), tetratricopeptide repeat domains (Main, ER, Xiong, Y., Cocco, MJ, D'Andrea, L., Regan, L., Structure 11(5), 497-508, 2003) and armadillo repeat domains (WO 2009 / 040338). It is further known to those skilled in the art that such repeated domains are distinct from proteins that contain repeated amino acid sequences, all of which can form individual domains (e.g., the FN3 domain of fibronectin).

[0165] 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 extension 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).

[0166] The term "designed" as used in designed repeat proteins, designed repeat domains etc. refers to the property that such repeat proteins and repeat domains, respectively, are artificial and do not occur in nature. The binding proteins of the 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.

[0167] The term "target interaction residues" refers to amino acid residues of a repeat module that contribute to direct interaction with the target.

[0168] 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 influences on the polypeptide backbone structure found in α-helices or β-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 the skilled person in structural biology and / or bioinformatics.

[0169] 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, such as 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 a homologous repeat module with the same target specificity.

[0170] Thus, the term "ankyrin repeat module" refers to a repeat module derived from the repeat unit of ankyrin repeat protein originally occurring in nature. Ankyrin repeat proteins are well known to those skilled in the art. Designed ankyrin repeat proteins have been described previously, see, for example, WO 2002 / 020565, WO 2010 / 060748, WO 2011 / 135067, WO 2012 / 069654, WO 2012 / 069655, WO 2014 / 001442, WO 2014 / 191574, WO 2014 / 083208, WO 2016 / 156596 and WO 2018 / 054971, all of which are incorporated by reference in their entirety. Typically, ankyrin repeat modules contain approximately 31-33 amino acid residues that form two alpha helices separated by a loop.

[0171] A repeat module may comprise positions with amino acid residues that are not randomized in the library for the purpose of selecting a target-specific repeat domain ("non-randomized positions") and positions with amino acid residues that are randomized in the library for the purpose of selecting a target-specific repeat domain ("randomized positions"). Non-randomized positions include framework residues. Randomized positions include target interaction residues. "Randomized" means, for example, that more than one amino acid is allowed at an amino acid position of a repeat module, that any of the regular 20 naturally occurring amino acids are allowed, or that amino acids other than cysteine ​​or most of the 20 naturally occurring amino acids are allowed, such as amino acids other than glycine, cysteine, and proline.

[0172] The term "repeat sequence motif" refers to an amino acid sequence deduced from one or more repeat modules. Preferably, the repeat modules are derived from repeat domains with binding specificity for the same target. Such a repeat sequence motif comprises framework residue positions and target interaction residue positions. The framework residue positions correspond to the positions of the framework residues of a repeat module. Similarly, the target interaction residue positions correspond to the positions of the target interaction residues of a repeat module. A repeat sequence motif comprises non-randomized positions and randomized positions.

[0173] The term "repeat unit" refers to an amino acid sequence that contains 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 the folding of the protein. Examples of such repeat units include leucine-rich repeat units, ankyrin repeat units, armadillo repeat units, tetratricopeptide repeat units, HEAT repeat units, and leucine-rich variant repeat units.

[0174] The terms "having binding specificity for a target," "specifically binds to a target," "binds to a target with high specificity," "specific for a target" or "target specificity," or "specifically binds," and the like, 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 in PBS for the target ("K D ") is at least 10 times larger than the corresponding dissociation constant for MBP. 2 times, more preferably at least 10 3 times, more preferably at least 10 4 times, or more preferably at least 10 5 Methods for measuring the dissociation constant of a protein-protein interaction, such as surface plasmon resonance (SPR) based techniques (e.g., SPR equilibrium analysis) or isothermal titration calorimetry (ITC), are well known to those of skill in the art. The K D Measurements of K may vary when measured under different conditions (e.g., salt concentration, pH). D The measurement of the value is preferably carried out using a standardized solution of the protein and a standardized buffer, such as PBS. The dissociation constant (K DA typical and preferred determination of the equilibrium dissociation constant (K ) by surface plasmon resonance (SPR) analysis is described in Example 2. 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 is at least 2-fold background signal or noise, more typically more than 10-fold background signal. More specifically, binding agents have an equilibrium dissociation constant (K ) that is greater than or equal to 10-fold the equilibrium dissociation constant (K ). D A target is said to "specifically bind" when its β-actin value is <1 μM, e.g., <500 nM, <100 nM, <10 nM, <1 nM, <100 pM, or <10 pM.

[0175] The term "binding agent" or "binding moiety" refers to any molecule capable of specifically binding to a target molecule. The term "binding agent" includes, for example, antibodies, antibody fragments, aptamers, peptides (e.g., Williams et al., J Biol Chem 266:5182-5190 (1991)), surrogate scaffolds, antibody mimetics, repeat proteins, e.g., 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, e.g., to include non-naturally occurring residues and / or to lack natural residues.

[0176] 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.

[0177] Preferably, the clearance and / or exposure and / or terminal half-life are evaluated in a mammal, more preferably in a mouse and / or a cynomolgus monkey, more preferably in a cynomolgus monkey. Preferably, when the clearance and / or exposure and / or terminal half-life are measured in a mouse, the evaluation is performed taking into account data up to 48 hours after injection. More preferably, the evaluation of the terminal half-life in a mouse is calculated from 24 hours to 48 hours. Preferably, when the clearance and / or exposure and / or terminal half-life are measured in a cynomolgus monkey, the evaluation is performed taking into account data up to 7 days after injection. More preferably, the evaluation of the terminal half-life in a cynomolgus monkey is calculated from day 1 to day 5. The skilled person can further identify effects such as target-mediated clearance and take them into account when calculating the 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 applied to a mammal after reaching pseudo-equilibrium (e.g., calculated from 24 hours 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 excrete 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., same mg / kg dose) or equimolar doses (i.e., same mol / kg dose), more preferably equimolar doses (i.e., same mol / kg dose). Those skilled in the art will appreciate 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 dosage 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, more preferably about 0.5 to about 10 mg / kg.

[0178] 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 as three pairs (εγ, εδ, ζζ). The CD3 complex functions as a T cell coreceptor that non-covalently associates with the T cell receptor. It may refer to any form of CD3, as well as variants, isoforms and species homologues thereof 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 human. 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 all mammalian species of native sequence CD3, e.g., human, canine, feline, equine, and bovine. The amino acid sequences of human CD3 gamma, delta and zeta are shown in the NCBI (www.ncbi.nlm.nih.gov / ) reference sequences NP_000064.1, NP_000723.1, and NP_932170.1, respectively.

[0179] As used herein, the term "CD3-expressing cell" refers to any cell that expresses CD3 (cluster of differentiation 3) on the cell surface, including, but not limited to, T cells, such as cytotoxic T cells (CD8+ T cells) and T helper cells (CD4+ T cells).

[0180] The term "CD33" refers to the myeloid cell surface antigen CD33, which is a sialic acid-binding immunoglobulin-like lectin (Siglec) that mediates cell-cell interactions and plays a role in maintaining immune cells in a quiescent state. The amino acid sequence of human CD33 (hCD33) is shown in UniProt (www.uniprot.org) reference number P20138.

[0181] The term "tumor-localized activation of T cells" means that T cells are preferentially activated in tumor tissue compared to non-tumor tissue.

[0182] Furthermore, the term "peptide" also encompasses peptides modified, for example by glycosylation, and proteins comprising two or more polypeptide chains, each of 4 to 600 amino acids in length, such as disulfide-linked proteins, such as insulin and immunoglobulins. The term "chemical or biochemical agent" is intended to include any compound of natural origin or synthetic that can be administered to a recipient. In a preferred embodiment, the localizer is a target-specific ankyrin repeat domain.

[0183] 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 neoplastic diseases. A "medical condition" may be one characterized by inappropriate cell proliferation. A medical condition may be a hyperproliferative condition. The present invention particularly relates to a method 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 as described above. In a preferred embodiment, the medical condition is a neoplastic disease. The term "neoplastic disease" as used herein refers to an abnormal state or condition of cells or tissues characterized by rapidly proliferating cell proliferation or tumors. In one embodiment, the medical condition is a malignant neoplastic disease. In one embodiment, the medical condition is a cancer, preferably a leukemia, more preferably an acute myeloid leukemia. The term "therapeutically effective amount" means an amount sufficient to produce a desired effect in a patient.

[0184] 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, 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.

[0185] The terms "cancer" and "cancerous" are used herein to refer to or describe a physiological condition in a mammal that is typically characterized by unregulated cell proliferation. Cancer includes solid and liquid tumors, as well as primary and metastatic tumors. A "tumor" contains one or more cancerous cells. A solid tumor typically also contains a 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. 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 intestine cancer, The cancer types include DLBCL, follicular lymphoma, Hodgkin's lymphoma (HL), mantle 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 (SLL), malignant mesothelioma, colorectal cancer, or gastric cancer. EXAMPLES

[0186] 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.

[0187] material Chemicals were purchased from Sigma-Aldrich (USA). Oligonucleotides were purchased from Microsynth (Switzerland). DNA polymerases, restriction enzymes, and buffers were purchased from New England Biolabs (USA) or Fermentas / Thermo Fisher Scientific (USA) unless otherwise stated. Inducible E. coli expression strains were used for cloning and protein production, e.g., E. coli XL1-blue (Stratagene, USA) or BL21 (Novagen, USA).

[0188] 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).

[0189] Designed ankyrin repeat protein library Methods for generating designed ankyrin repeat protein libraries are described, for example, in US Pat. No. 7,417,130; Binz et al., J. Mol. Biol. 332:489-503, 2003; Binz et al. (2004), cited above. By such methods, designed ankyrin repeat protein libraries with randomized ankyrin repeat modules and / or randomized capping modules can be constructed. For example, such libraries may thus be assembled based on a fixed N-terminal capping module (e.g. N-terminal capping module of SEQ ID NO: 69, 70 or 71) or a randomized N-terminal capping module (e.g. according to SEQ ID NO: 72) and a fixed C-terminal capping module (e.g. C-terminal capping module of SEQ ID NO: 73, 74 or 75) or a randomized C-terminal capping module (e.g. according to SEQ ID NO: 76). Preferably, such libraries are assembled in such a way that they do not have any of the amino acids C, G, M, N (before a G residue) and P in the randomized positions of the repeat or capping module.

[0190] Furthermore, such randomized modules in such libraries may contain additional polypeptide loop insertions with randomized amino acid positions. Examples of such polypeptide loop insertions are complement determining region (CDR) loop libraries of antibodies or de novo generated peptide libraries. For example, such loop insertions could be designed using the structure of the N-terminal ankyrin repeat domain of human ribonuclease L as guidance (Tanaka, N., Nakanishi, M, Kusakabe, Y, Goto, Y., Kitade, Y, Nakamura, KT, EMBO J..23(30), 3929-3938, 2004). Similar to this ankyrin repeat domain in which 10 amino acids are inserted into a β-turn located near the boundary of two ankyrin repeats, ankyrin repeat protein libraries may contain randomized loops (with fixed and randomized positions) of variable length (e.g., 1-20 amino acids) inserted into one or more β-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 NO: 1), 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: 69-71 provide examples of N-terminal capping modules comprising a RILLAA, RILLKA or RELLKA motif, and SEQ ID NOs: 73-75 provide examples of C-terminal capping modules comprising a KLN, KLA or KAA motif.

[0191] The design of such ankyrin repeat protein libraries can be guided by known structures of ankyrin repeat domains interacting with targets. Examples of such structures, identified by unique Protein Data Bank (PDB) accession or identification codes (PDB-IDs), are 1WDY, 3V31, 3V30, 3V2X, 3V2O, 3UXG, 3TWQ-3TWX, 1N11, 1S70, and 2ZGD.

[0192] 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 N2C and N3C describe the number of randomized repeat modules present between the N- and C-terminal capping modules.

[0193] The nomenclature used to define the repeat units and positions within the modules is based on Binz et al. (2004) citation above, with the modification that the boundaries between ankyrin repeat modules and ankyrin repeat units are shifted by one amino acid position. For example, position 1 of the ankyrin repeat module of Binz et al. (2004) citation above corresponds to position 2 of the ankyrin repeat module of the present disclosure, and consequently position 33 of the ankyrin repeat module of Binz et al. (2004) citation above corresponds to position 1 of the following ankyrin repeat module of the present disclosure.

[0194] Example 1: Selection of binding proteins containing ankyrin repeat domains with binding specificity for CD33 Using ribosome display (Hanes, J. and Pluckthun, A., PNAS 94:4937-42 (1997)), a number of ankyrin repeat proteins with binding specificity for human CD33 (hCD33) (UniProt ID P20138) were selected from a DARPin® library similar to that described by Binz et al. (2004) cited above. Binding of selected clones to recombinant human CD33 targets (full length extracellular domain (ECD) of CD33 and splice variants of ECD of CD33) was assessed by homogeneous time-resolved fluorometry (HTRF) of crude extracts, showing that several hundred hCD33-specific binding proteins were successfully selected. For example, the ankyrin repeat domains of SEQ ID NOs: 61, 3, 5, 7-9, and 14-16 constitute the amino acid sequences of selected binding proteins containing ankyrin repeat domains with binding specificity for hCD33.

[0195] Selection of CD33-specific ankyrin repeat proteins by ribosome display Selection of hCD33-specific ankyrin repeat proteins was performed by ribosome display (Hanes and Pluckthun, loc. cit.) using the biotinylated extracellular domain (ECD) of human CD33 as target protein, a library of ankyrin repeat proteins as described above, and established protocols (see, e.g., Zahnd, C., Amstutz, P., and Pluckthun, A., Nat. Methods, vol. 4, pp. 69-79, 2007). The CD33 target (Evitria) contained a C-terminal Fc tag and an Avi tag and was biotinylated using the enzyme BirA-GST. Two different forms of CD33 were used for the selection: the full-length ECD of CD33 (residues 18-259) (SEQ ID NO: 60), which contains both the variable and constant domains of CD33, and a splice variant of the ECD of CD33 (residues 140-259) (SEQ ID NO: 61), which contains only the constant domain CD33. A total of four rounds of standard ribosome selection were employed, with decreasing target concentration and increasing washing stringency to increase selection pressure from round 1 to round 4 (Binz et al., 2004, cited above). A deselection strategy was applied in each round by using streptavidin and neutravidin beads in conjunction with biotinylated non-CD33 Fc domains. The number of reverse transcription (RT)-PCR cycles after each selection round was constantly decreased from 45 to 28, adjusting for yield, to enrich for binders.

[0196] To enrich for high affinity CD33-specific ankyrin repeat proteins, the output from the fourth round of standard ribosome display selection (supra) was subjected to off-rate selection rounds with increasing selection stringency (Zahnd (2007) supra). A final standard selection round was performed after the off-rate selection round to amplify and recover the off-rate selected binding proteins. In these last two selection rounds, the number of RT-PCR cycles was 30 and 35, respectively.

[0197] A total of three different selection approaches were performed as above with the following differences: in the first approach, selection was performed only against the ECD of the full-length CD33 protein; in the second approach, targeting of full-length CD33 ECD and splice variant ECD was alternated in each round; in the third approach, a competitive elution step was applied using the conditions of the first approach by adding HIM-3-4 CD33 binding antibody (BD Pharmingen™). From each approach, binders were generated against full-length CD33 and / or its splice variants.

[0198] Selected clones specifically bind human CD33 as demonstrated by crude extract HTRF. Individual selected ankyrin repeat proteins that specifically bind hCD33 in solution were identified by 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 pQE30 (Qiagen) expression vector (pMPDV045) containing a C-terminal CD3-specific ankyrin repeat domain followed by a Flag tag, 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 plated (each clone in a single well) into 96-well plates containing 160 μl 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 min of incubation at 37° C. and 700 rpm, expression was induced with IPTG (final concentration 0.5 mM) and continued for 4 h. 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 h at room temperature with shaking (900 rpm). Then 160 μL of PBS was added and cell debris was removed by centrifugation (3220 g for 15 min).

[0199] Extracts of each lysed clone were applied to wells of a 384-well plate as a 1:1000 dilution (final concentration) together with 6 nM (final concentration) biotinylated hCD33 (full length or splice variant of ECD CD33), 1:400 (final concentration) anti-strep-Tb HTRF antibody-FRET receptor conjugate (Cisbio), and 1:400 (final concentration) anti-6His-D2 antibody FRET receptor conjugate (Cisbio) in PBSTB (PBS supplemented with 0.1% Tween20® and 0.2% (w / v) BSA, pH 7.4) and incubated for 60 min at room temperature. HTRF was read on a Tecan M1000 using an excitation wavelength of 340 nm and an emission filter of 665±10 nm. Such screening of hundreds of clones by crude cell extract HTRF revealed an ankyrin repeat domain specific to hCD33. The amino acid sequences of selected ankyrin repeat domains that specifically bind to hCD33 are provided in SEQ ID NOs: 1, 3, 5, 7-9, and 14-16.

[0200] [Table 2]

[0201] These DARPin® proteins optionally additionally comprise a G, S, or GS sequence at their N-terminus.

[0202] Engineering additional ankyrin repeat proteins with binding specificity for hCD33

[0203] [Table 3]

[0204] SEQ ID NOs: 2, 4, 6, and 10-13 are ankyrin repeat proteins with binding specificity for hCD33 that were engineered based on the sequences of DARPin® Protein #1 (SEQ ID NO: 1), DARPin® Protein #3 (SEQ ID NO: 3), DARPin® Protein #5 (SEQ ID NO: 5), and DARPin® Protein #9 (SEQ ID NO: 9), respectively.

[0205] For SEQ ID NO:1 and SEQ ID NO:3, the sequences were modified to reduce the number of aromatic residues and to change the surface charge. In both N-terminal capping modules, the RILLAA motif was replaced with RILLKA and the aspartic acid (position 18) was replaced with leucine. In both C-terminal capping modules, the glutamic acid (position 18) was replaced with glutamic acid. For SEQ ID NO:3, an additional phenylalanine (position 14) was replaced with valine in the N-terminal capping module. For SEQ ID NO:1, an additional tryptophan (position 7) was replaced with valine in the N-terminal capping module and the EDIA motif in the second internal repeat (positions 18-21) was replaced with LEIV. The engineered variants did not alter T-cell killing (assessed in combination with other TAAs and CD3-binding ankyrin repeat domains) compared to the parental version by more than 2-fold as measured in a standard LDH killing assay after 48 hours of incubation using pan-T cells and MOLM-13 cells at a 5:1 ratio.

[0206] For SEQ ID NO: 9, the sequence was modified to reduce the number of aromatic residues, change the surface charge, and / or optimize the framework. In all N-terminal capping modules, the RILLAA motif was replaced by RELLKA. In three mutants, either a valine (position 12) in the second internal repeat was removed to obtain a standard ankyrin repeat protein framework as described in Binz et al. (2004) Nature Biotechnology, or a glycine (position 18) in the C-terminal capping repeat was replaced by glutamic acid, or a combination of therapies was introduced. In one mutant, a tyrosine (position 7) in the N-terminal capping module was replaced by a valine. The engineered mutants maintained binding to hCD33 as measured by HTRF and cell binding to MOLM13 cells as measured by FACS.

[0207] For SEQ ID NO:5, a serine (position 6) was replaced with a glycine in the N-terminal capping module. The engineered variant did not alter T cell killing (assessed in combination with the CD3-binding ankyrin repeat domain) compared to the parental version by more than 2-fold as measured in a standard LDH killing assay after 48 hours of incubation using pan-T cells and MOLM-13 cells at a 5:1 ratio.

[0208] Expression of CD33-specific ankyrin repeat proteins For further analysis, selected clones showing specific human CD33 binding in crude cell extract HTRF as described above were expressed in E. coli cells with a His-tag (SEQ ID NO: 85) fused to their N-terminus for easy purification. The expressed proteins were purified according to standard protocols. 0.11 ml of stationary overnight culture (TB, 1% glucose, 50 mg / l ampicillin; 37°C) was used to inoculate 0.99 mL culture (TB, 50 mg / l ampicillin, 37°C) in 96 deep-well plates. After 2 h 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 h. Cells were harvested and pellets were frozen overnight at -20°C before being 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 h with shaking (900 rpm). 60 μL of low-salt sodium phosphate buffer was then added and cell debris was removed by centrifugation (3220 g for 15 min). In total, eight individual expressions were pooled and then cell debris was removed by centrifugation (3,200 g for 60 min at 4°C). Supernatants were filtered using MultiScreen filter plates (Millipore) before purification using 96-well Thermo HisPur cobalt spin plates and the protein solution was rebuffered in PBS using 96-well Thermo Zeba spin desalting plates. The purified protein was soluble and monomeric in PBS using a standard Sephadex 150 / 5 column on an Agilent 1200 HPLC system.

[0209] Generation of affinity matured CD33-specific binding proteins In the further development of the initially identified CD33-specific binding protein, new variants with very high affinity and / or very low off-rates from the target protein were generated using affinity maturation. Thereby, one initially identified CD33-specific binding protein DARPin® protein #2 (the "parent" binding protein) was selected as a suitable starting point for affinity maturation. The affinity maturation procedure involved saturation mutagenesis of each randomized position of the ankyrin repeat domain used as a starting point. The sequences generated by the affinity maturation procedure were screened for lower off-rates by competitive HTRF. Briefly, single amino acid point mutation variants were generated by standard QuikChange PCR on the parent plasmid (pMCHE1190) using primers with a single NNK degenerate codon to introduce all 20 amino acids at potential binding positions. Crude extracts (CE) of ankyrin repeat proteins containing an N-terminal His tag (SEQ ID NO: 85) were incubated with biotinylated target, after which excess non-flag-tagged parent CD33-specific binding protein was added and the HTRF signal was measured over time. Beneficial mutations identified based on a higher HTRF signal than the parental clones were combined in the binding protein by protein engineering. In this way, affinity matured protein #29 and DARPin® protein #30 were generated from DARPin® protein #2.

[0210] Such affinity matured CD33 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: 85) followed by the CD33 specific binding domain of SEQ ID NO: 77 or 78, a peptide linker (SEQ ID NO: 65) and a C-terminal CD3 binding domain of SEQ ID NO: 57. These constructs in T cell engager format were expressed in E. coli cells and purified using their His tag according to standard protocols. Proteins were tested in dose-dependent in vitro T cell activation and tumor cell killing assays using primary T cells isolated from healthy donor PBMCs as effector cells (E) and Molm-13-N1 tumor cells (E:T ratio of 5:1) as target cells (T). Assay incubation of co-cultures for 48 hours and analysis by flow cytometry and LDH release. All constructs were expressed in E. coli cells and purified using their His tag according to standard protocols.

[0211] Example 2: Dissociation constants (K) of recombinant ankyrin repeat proteins with binding specificity for human CD33 by surface plasmon resonance (SPR) analysis D ) Determination The binding affinity of the purified ankyrin repeat proteins on the recombinant human CD33 target was analyzed using a ProteOn instrument (BioRad) and measured according to standard procedures known to those skilled in the art. To this end, the DARPin® proteins of the invention #1, #3, #5, #7-#9 and #14-#16 were subcloned and expressed as described above in derivatives of the pQE30 (Qiagen) expression vector, resulting in constructs containing an N-terminal His tag and a CD33-specific ankyrin repeat domain followed by one of the five CD3-specific ankyrin repeat domains listed in the sequence listing.

[0212] Briefly, SPR measurements were performed using a ProteOn XPR36 instrument (BioRad). The running buffer was PBS pH 7.4 (PBST) containing 0.005% Tween 20®. The bio.hCD33 full-length target (SEQ ID NO: 60) was immobilized on a NLC chip (BioRad) to a level of 540 RU, and the spliced ​​variant (SEQ ID NO: 61) was immobilized to a level of 560 RU. The interaction of 96-well purified CD33-specific binding proteins in TCE format to full-length and spliced ​​variants of CD33 ECD was measured by injecting 50 nM binding protein with 120 s association and 1200 s dissociation using a constant flow rate of 100 μL / min. The target was regenerated between individual measurements with 2 M MgCl2. The signal was double-referenced against a running buffer (PBST)-treated control lane.

[0213] Table 2a shows the K values ​​obtained for DARPin® proteins #1, #3, #5, #7-9, and #14-16 in the TCE format (i.e., formatted with the CD3-specific ankyrin repeat domain). D The dissociation constant (K D ) was calculated from the estimated on-rates and off-rates using standard procedures known to those skilled in the art. K D Values ​​were determined by single trace SPR to be in the range of 0.47-17 nM (see Table 2a). Values ​​in Table 2a are the average of multiple replicates.

[0214] [Table 4]

[0215] Furthermore, the binding affinity of two additional purified ankyrin repeat proteins to the recombinant human CD33 target was measured and analyzed using the same procedure as above for DARPin proteins #1, #3, #6, #7-#9, and #14-#16. Briefly, bio.hCD33 was coated onto a NLC chip (BioRad) to a level of 1180 RU. DARPin® protein #29 and DARPin® protein #30 were applied as analytes at a single concentration of 100 nM with a constant flow rate of 100 uL / min, an association phase of 120 s and a dissociation phase of 1200 s. The target was regenerated with 4 M MgCl2. The signal was double-referenced against a control lane treated with running buffer L1 and A6 (PBST PBS pH 7.4 containing 0.005% Tween 20®). A 1:1 Langmuir model was used for fitting. The K obtained in this study D The values ​​are summarized in Table 2b.

[0216] Table 2b shows the K of the CD33-specific ankyrin repeat proteins of the invention binding to the bio.hCD33 full-length target. D The value is shown. D Values ​​were calculated from estimated on- and off-rates using standard procedures known to those skilled in the art. Values ​​in Table 2b are the average of multiple replicates.

[0217] [Table 5]

[0218] Figure 1 (A-B) shows surface plasmon resonance (SPR) analysis of ankyrin repeat protein binding to human CD33. Figure 1A shows the SPR analysis of DARPin® protein #29 and Figure 1B shows the SPR analysis of DARPin® protein #30. Example 3: Pharmacokinetic analysis of CD33-specific ankyrin repeat proteins in female BALB / c mice To determine whether the CD33-specific ankyrin repeat domains of the present invention can have a suitable serum half-life in vivo to be useful for the development of therapeutic agents, the pharmacokinetic profiles of DARPin® protein #2, DARPin® protein #29, and DARPin® protein #30 were analyzed in mice.To this end, the DARPin constructs are subcloned and expressed as described above into a derivative of pQE30 (Qiagen) expression vector, which encodes an N-terminal His tag (SEQ ID NO: 85), an HSA-binding ankyrin repeat domain (SEQ ID NO: 53) for half-life extension, a peptide linker (such as SEQ ID NO: 65), and one of the CD33-specific binding domains at the C-terminus.For example, an expression vector encoding the following ankyrin repeat proteins is constructed:

[0219] In vivo administration and sampling DARPin® Protein #2, DARPin® Protein #29 and DARPin® Protein #30, formatted with the above-mentioned human serum albumin-specific ankyrin repeat domain (SEQ ID NO: 53), were administered as a single intravenous bolus injection into the tail vein of six mice for each ankyrin repeat fusion protein. The target dose level is 1 mg / kg with an application volume of 5 mL / kg. The ankyrin repeat fusion proteins are formulated in phosphate buffered saline (PBS) solution.

[0220] Mice are divided into two groups with equal number of animals. Four serum samples are taken from each mouse. Blood samples for pharmacokinetic studies are taken 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 taken.

[0221] ELISA-based bioassay for measuring ankyrin repeat proteins in serum samples Coat NUNC Maxisorb ELISA plates overnight at 4°C with 100 μL per well of 10 nM polyclonal goat anti-rabbit IgG antibody (Ab18) in PBS. After washing 5 times with 300 μL per well PBST (PBS supplemented with 0.1% Tween 20), block wells for 1 h at room temperature (RT) with 300 μl PBST supplemented with 0.25% casein (PBST-C) on a Heidolph Titramax 1000 shaker (450 rpm). Wash plates as above. Coat with 100 μL of 5 nmol / L rabbit anti-DARPin in PBST-C. (登録商標) Add 1-1-1 antibody and incubate plate with orbital shaking (450 rpm) for 1 hour at room temperature (22° C.). Wash plate as above.

[0222] Apply 100 µL of diluted serum samples (1:20 to 1:312500 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 room temperature with shaking at 450 rpm. Wash the plates as above.

[0223] Wells are then incubated with 100 μL of mouse anti-RGS-His-HRP IgG (Ab06, 1:2000 in PBST-C) and incubated for 1 h at room temperature at 450 rpm. Plates are washed as above. ELISA is developed using 100 μL / well of TMB substrate solution for 5 min 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.

[0224] Pharmacokinetic analysis Pharmacokinetic data analysis is performed at Molecular Partners using the WinNonlin program, version 7.0, 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). Pharmacokinetic parameters are calculated as follows: AUCinf, AUClast, AUC_%extrapol, Cmax, Tmax, Cl_pred, Vss_pred, t1 / 2.

[0225] 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 to the last sampling point (Tlast) and extrapolation to infinity assuming a monoexponential terminal phase decay. The extrapolation to infinity is performed using Clast / λz, where λz denotes the terminal rate constant estimated by log-linear regression and Clast denotes the concentration estimated at Tlast by terminal log-linear regression. The total serum clearance (Cl_pred) and the apparent terminal half-life are calculated as follows: Cl_pred = intravenous 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 of ​​the first instant of the drug concentration-time curve extrapolated to infinity using the same extrapolation procedure as described for the calculation of AUCinf. To calculate PK parameters based on concentrations given in nmol / L doses, the dose values ​​given as 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 three ankyrin repeat proteins of the invention, DARPin® protein #2, DARPin® protein #29 and DARPin® protein #30, formatted with the human serum albumin-specific ankyrin repeat domain as described above.

[0226] [Table 6]

[0227] In conclusion, the CD33-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 adequate serum half-life in vivo to be useful for the development of therapeutic agents.

[0228] Example 4: Determination of binding of ankyrin repeat proteins according to the invention to CD33-expressing tumor cells The binding of the binding proteins of the invention to CD33 expressed on the surface of cells was analyzed by fluorescence-activated cell sorting (FACS) flow cytometry. For this purpose, CD33-expressing tumor cells (Molm-13 N1) were seeded at 100'000 cells per well in a 96-well plate. DARPin protein #2, DARPin protein #29 and DARPin protein #30 in monospecific format were titrated at a dilution ratio of 1:5, starting at 2000 nM. Tumor cells were resuspended with the diluted DARPin proteins and incubated for 60 min at 4° C. The assay was carried out in PBS containing 2% fetal bovine serum and without 20 μM 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. An incubation step of at least 30 min at 4° C. followed. Afterwards, cells were washed with PBS and 2 μg / mL of anti-rabbit secondary goat antibody labeled with Alexa Fluor 488 antibody (ThermoFisher) was added. The same incubation conditions were applied. Finally, 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 by Attune NXT (ThermoFisher) using FlowJo software for analysis and GraphPad Prism 8 for data plotting (FIG. 2 shows the binding curves of DARPin® protein #2, DARPin® protein #29 and DARPin® protein 30 to CD33-expressing tumor cells). Table 4 shows the quantification of the binding of three exemplary ankyrin repeat proteins to CD33 expressed on cells, represented by their EC50 values.

[0229] [Table 7]

[0230] In conclusion, the CD33-specific binding proteins of the present invention bind to CD33 expressed on the surface of cells with an EC50 of about 2 nM or less.

[0231] Example 5: Evaluation of the specificity and potency of CD33-specific ankyrin repeat proteins of the invention by target-specific short-term T cell activation assays. The specificity and potency of the above-mentioned exemplary CD33-specific ankyrin repeat proteins of the invention were evaluated in an in vitro short-term T cell activation assay by FACS measurement of the CD25 activation marker on CD8+ T cells. The tested proteins, DARPin Protein #2, DARPin Protein #29 and DARPin Protein #30, were evaluated in a bispecific T cell engager format. Such T cell engager proteins contain, in addition to the above-mentioned CD33-specific ankyrin repeat domain, a CD3-specific binding domain (SEQ ID NO: 57) and are designated DARPin Protein #33, DARPin Protein #31 and DARPin Protein #32, respectively.

[0232] Thus, 100,000 purified pan-T effector cells and 20,000 Molm-13 target cells per well were co-incubated in duplicate with serial dilutions of selected DARPin proteins in the presence of 600 μM human serum albumin for 48 h at 37 °C (E:T ratio 5:1). After 48 h, cells were washed and stained with 1:1,000 Live / Dead Green (ThermoFisher), 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 an Attune NxT (ThermoFisher) machine. T cell activation was assessed by measuring CD25+ cells on Live / Dead negative and CD8+ gated T cells. FACS data was analyzed using FlowJo software and data was plotted using GraphPad Prism8 (3-PL-fit). Figure 3 shows short-term T cell activation triggered by DARPin protein #33, DARPin protein #31 and DARPin protein #32 as measured by the activation marker CD25. All CD33-specific binding proteins of the invention tested were able to bind to CD733 expressed on tumor cells in a T cell engager format and activate T cells.

[0233] Example 6: Evaluation of the specificity and potency of CD33-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 CD33-specific ankyrin repeat proteins of the invention, DARPin Protein #2, DARPin Protein #29 and DARPin Protein #30 in a T cell engager format (i.e. DARPin Protein #33, DARPin Protein #31 and DARPin Protein #32, respectively) was also evaluated using an in vitro short-term cytotoxicity assay measuring LDH release. For this purpose, 100,000 purified pan-T effector cells and 20,000 Molm-13 target cells / 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 for 48 h at 37° C. (E:T ratio 5:1). After 48 h of incubation, the 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, incubation 30 min). Absorbance was measured at 492-620 nm using a TECAN infinite M1000Pro reader. OD values ​​were plotted using GraphPad Prism8.

[0234] Figure 4 shows tumor cell killing induced by DARPin protein #33, DARPin protein #31 and DARPin protein #32. All CD33-specific binding proteins of the invention tested were able to bind to CD33 expressed on tumor cells in a T cell engager format and activate T cells that then kill the tumor cells.

[0235] Example 7: Determination of the CD33 epitope bound by the CD33-specific binding protein of the present invention The CD33 epitope bound by the CD33-specific ankyrin repeat protein of the invention was investigated using HTRF and a competitive ELISA against an analogue of the benchmark control molecule AMG330 (CD3-CD33 specific BiTE® purchased from Evitria).

[0236] Competition of the monovalent CD33-specific binding proteins DARPin® Protein #1, DARPin® Protein #9, and DARPin® Protein #14, as well as the AMG330 analogue, for binding to human CD33 (purchased from Evitria) was assessed by HTRF and competition ELISA.

[0237] First, binding experiments were performed by HTRF to full length (V / C2 domain) and truncated CD33 (C2 domain) using crude extracts of the three CD33 specific binding proteins DARPin® Protein #1, DARPin® Protein #9 and DARPin® Protein #14. Binding to the CD33 target was detected using Strep-Tb and anti-His-D2 HTRF reagents. Briefly, crude extracts of DARPin® Protein #1, DARPin® Protein #9 and DARPin® Protein #14 were diluted 1:250 (final 1:1000) in PBSTB and added to a 384-well plate (PerkinElmer, 6008280). Biotinylated target_hCD33-Fc(kih)-Avi (full length CD33; purchased from Evitria) and hCD33-C2-Fc(kih)-Avi (truncated CD33; purchased from Evitria) were then each diluted to 8 nM (6 nM final) and mixed with HTRF reagents Mab Anti-6His-d2 and Strep-Tb, both diluted 1:200 (1:400 final). As shown in FIG. 5, all three CD33-specific binding proteins DARPin® Protein #1, DARPin® Protein #9, and DARPin® Protein #14 show binding to full length CD33, but only DARPin® Protein #9 binds to the truncated CD33 target. These data indicate that DARPin® Protein #9 binds to the C2 domain, while DARPin® Protein #91 and DARPin® Protein #14 bind to the V domain of CD33.

[0238] Next, a competitive ELISA experiment was performed using the CD33-binding benchmark molecule AMG330 as well. AMG330 analogs, which bind to the top of the V domain as shown by Friedrich et al. (2014), were immobilized on a microplate. Biotinylated CD33 target and a 50-fold excess of DARPin® Protein #1, DARPin® Protein #9, and DARPin® Protein #14 were pre-incubated for 2 hours, after which binding to AMG330 was measured directly via streptavidin covalently linked to peroxidase. Briefly, Nunc MaxiSorp 96-well plates were coated overnight with 10 nM DARPin® Protein #1, DARPin® Protein #9, and DARPin® Protein #14, and 5 nM AMG330 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 4 hours 15 minutes at 450 rpm. Meanwhile, 500 nM of competitors DARPin® Protein #1, DARPin® Protein #9 and DARPin® Protein #14 were pre-incubated with 20 nM of bio.hCD33 in a 1:1 ratio for 2 hours at room temperature. Bio.hCD33 without competitor was included as a positive control. The pre-incubated samples were then added to the coated MaxiSorp plates and incubated for 30 minutes at room temperature and 450 rpm. The plates were washed three times with PBST before detecting the targets with streptavidin-POD antibody (Roche, Cat. No.: 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 buffer (PBS) values. GraphPad Prism was used for analysis.

[0239] As expected, the C2 domain-binding DARPin® protein #9 does not show a similar competitive binding with AMG330. For both V domain-binding molecules DARPin® protein #1 or DARPin® protein #14, competition resulted in a complete or partial inhibition of the ELISA signal, respectively. Thus, DARPin® protein #1 binds to the V domain and shows partial competition with AMG330 and DARPin® protein #14. In addition, DARPin® protein #14 also binds to the V domain and competes completely for binding with AMG330, whereas DARPin® protein #9 binds to the C2 domain, thereby allowing simultaneous binding of AMG330 to CD33 (Figure 6).

[0240] Example 8: In vivo efficacy evaluation of exemplary multidomain TCE-binding proteins, including DARPin® Protein #29, in PBMC humanized mouse and MOLM-13 tumor models DARPin® Protein #29, formatted as a multidomain T-cell engager binding protein, additionally containing two ankyrin repeat domains with binding specificity for human serum albumin, two ankyrin repeat domains with binding specificity for tumor-associated antigen 1 (TAA1) and tumor-associated antigen 2 (TAA2), respectively, and one ankyrin repeat domain with binding specificity for CD3, was tested in a peripheral blood mononuclear cell (PBMC) humanized mouse model harboring 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 under standardized environmental conditions in standard rodent microisolator cages (room temperature of 20±1°C, relative humidity of 50±10% and a 12-h light-dark cycle). Mice were fed irradiated food and bedding and 0.22 um filtered drinking water. All experiments were performed with approval from the cantonal and federal veterinary authorities in accordance with the Swiss Animal Protection Act.

[0241] Mice were injected with PBMCs (5 × 10 6 hPBMCs (1000 PBMCs) were injected intraperitoneally 2 days prior to cancer cell xenografting. MOLM-13 cells were xenografted subcutaneously (sc) into the right flank of the mice. Two hPBMC donors were used. Treatments were injected intravenously (iv) 4 days after cancer cell implantation. Treatments were administered as follows: -DARPin® protein #29 in multidomain TCE format or vehicle was administered iv at 0.5 mg / kg, 3 times a week for 2 weeks 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 .

[0242] As can be seen in Figures 7 (A-B), DARPin® Protein #29 in the multidomain TCE format showed good efficacy in terms of inhibiting tumor growth and tumor volume over the entire duration of the experiment (Figure 7A) and 17 days after the first injection (Figure 7B).

[0243] Example 9: In vitro efficacy evaluation of exemplary multidomain TCE-binding proteins, including DARPin® Protein #29 or DARPin® Protein #30, using MOLM13 wild-type and MOLM13 CRISPR CD33 knockout (KO) target cells co-cultured with human pan-T cells DARPin® Protein #29 and DARPin® Protein #30, each formatted as a multidomain T cell engager binding protein additionally containing two ankyrin repeat domains with binding specificity for human serum albumin, two ankyrin repeat domains with binding specificity for tumor-associated antigen 1 (TAA1) and tumor-associated antigen 2 (TAA2), respectively, and one ankyrin repeat domain with binding specificity for CD3, were tested in an in vitro short-term T cell activation assay by measuring the CD25 activation marker on CD8+ T cells by FACS. In this assay, pan-T cells were co-cultured with target cells, whereby the target cells were either (1) Molm-13 tumor cells with wild-type target expression of CD33, TAA1, and TAA2, or (2) Molm-13 tumor cells in which expression of CD33 (but not TAA1 and TAA2) had been eliminated by CRISPR knockout (KO) technology (Figures 8A and 8B).

[0244] For this purpose, 100,000 purified pan-T effector cells and 20,000 target cells per well were co-incubated in duplicate with serial dilutions of the respective 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-CD 25 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 is analyzed using FlowJo software and data are plotted using GraphPad Prism8 (3-PL-fit).

[0245] The results demonstrate that both exemplary multidomain T cell engager binding proteins comprising either DARPin® protein #29 (Figure 8A) or DARPin® protein #30 (Figure 8B) were able to potently activate T cells in the presence of Molm-13 tumor cells with wild-type expression (curve 1; EC50 value of DARPin® protein #29 TCE: 5.71 pM; EC50 value of DARPin® protein #30 TCE: 5.60 pM). Furthermore, the results also demonstrate that when expression of CD33 on Molm-13 tumor cells was eliminated, T cell activation was significantly reduced (curve 2; EC50 value of DARPin® protein #29 TCE: 27.36 pM; EC50 value of DARPin® protein #30 TCE: 47.00 pM). This provides evidence that DARPin® protein #29 (Figure 8A) and DARPin® protein #30 (Figure 8B) were functional in the context of exemplary multidomain T cell engager binding proteins and contributed significantly to the overall efficacy of the multispecific T cell engager proteins due to their ability to specifically bind CD33 on target cells.

[0246] This specification is most fully understood in light of the teachings of the references cited within the specification. The embodiments within this specification provide examples of embodiments of the invention and should not be construed as limiting the scope of the invention. Those skilled in the art will readily recognize that many other embodiments are encompassed by the present invention. All publications, patents, and GenBank sequences cited in this disclosure are incorporated by reference in their entirety. To the extent that the material incorporated by reference contradicts or is inconsistent with this specification, this specification takes precedence over any such material. The citation of a reference herein is not an admission that such reference is prior art to the present invention.

[0247] 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.

[0248] [Table 8-1]

[0249] [Table 8-2]

[0250] [Table 8-3]

[0251] [Table 8-4]

[0252] [Table 8-5]

[0253] [Table 8-6]

Claims

1. A recombinant binding protein comprising an ankyrin repeat domain, said ankyrin repeat domain having binding specificity for human CD33, 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 to 16 and 77 to 78.

2. The ankyrin repeat domain binds to human CD33 in PBS with a dissociation constant (K D ), optionally with a K between about 0.1 nM and about 100 nM D and / or The recombinant binding protein of claim 1, wherein the ankyrin repeat domain binds to human CD33 with an EC 50 ranging from about 0.1 nM to about 10 nM.

3. further comprising a binding moiety having binding specificity for a target expressed on an immune cell, 3. The recombinant binding protein of claim 1 or 2, wherein the immune cell is a T cell and the target expressed on the immune cell is CD3.

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. or 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 wherein the ankyrin repeat domain having binding specificity for human CD3 comprises an amino acid sequence that has at least 95% amino acid sequence identity to any one of SEQ ID NOs: 55 to 59; or The recombinant binding protein according to claim 3, wherein said ankyrin repeat domain having 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 CD33 and the binding moiety having binding specificity for a target expressed on an immune cell are covalently linked to a peptide linker, Optionally, the peptide linker is a proline-threonine rich peptide linker; 4. The recombinant binding protein of claim 3, wherein the amino acid sequence of the peptide linker optionally 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. or wherein the ankyrin repeat domain having binding specificity for human serum albumin comprises an amino acid sequence which is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 52 to 54; or The recombinant binding protein according to claim 7, wherein said 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.

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 CD33.

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

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

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

13. 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.

14. 14. The pharmaceutical composition of claim 13, wherein the cancer is leukemia, optionally wherein the cancer is acute myeloid leukemia.

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