New DARPin-based CD33 Engineer

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 of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 158,539 filed on 9 March 2021, U.S. Patent No. 63 / 172,818 filed on 9 April 2021, and U.S. Patent Application No. 63 / 265,179 filed on 9 December 2021. The disclosures of these patent applications are incorporated herein by reference in their entirety for all purposes.

[0002] The present invention relates to recombinant binding proteins comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to human CD33. Furthermore, 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, for example, acute myeloid leukemia (AML). [Background technology]

[0003] Acute myeloid leukemia (AML) is a heterogeneous and complex malignant disease characterized by rapid cell proliferation, an invasive clinical course, and generally high mortality. Treatment resistance remains a major cause of AML-related deaths (Winer and Stone, "Ther Adv Hematol"; 2019; Vol. 10). While standard protocols using chemotherapy remain the primary treatment approach applied globally, recent advances in immunotherapy offer 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] Since CD33 is expressed in approximately 80-90% of AML blast cells and leukemic stem cells, CD33 is an attractive target for the treatment of cancer, particularly AML (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 are used either 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 with the cytotoxic agent calitiamycin, resulted in significant hematological and hepatotoxicity.

[0005] CAR-T cell therapy has been a highly influential approach in the management of lymphoid malignancies. While there is considerable interest in applying this technology to AML, it has proven challenging in practice. Regarding monoclonal antibodies, CD33 is considered the most promising target for CAR-T cell therapy in AML. However, preclinical models of this approach have shown widespread side effects (on-target / off-tumor toxicity) in non-AML cells, with cytokine release syndrome (CRS) being another recognized side effect.

[0006] T-cell targeted cytotoxicity of tumor cells using bispecific antibodies is another recent therapeutic tool being used 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 tumor cell surface antigens. Binding of TCEs to these two targets provides a functional connection between cells, resulting in T-cell activation and cytotoxic activity against tumor cells, while evading normal TCR-MHC interactions (Ellerman, "Methods"; Vol. 154: pp. 102-117 (2019)). AMG330 is a bispecific antibody against CD3 and CD33 that can induce T-cell cytotoxicity against AML cells.

[0007] However, both multispecific and bispecific antibody therapies present several drawbacks, such as high manufacturing costs, and carry the risk of causing certain serious side effects, including cytokine release syndrome (CRS) (Shimabukuro-Vornhagen et al., "J Immunother Cancer"; Vol. 6 (No. 1): p. 56 (2018); Labrjin et al., "Nat Rev Drug Discov"; Vol. 18 (No. 8): pp. 585-608 (2019)), and / or on-target / off-tumor toxicity (Weiner et al., "Cancer Res."; Vol. 55 (No. 20): pp. 4586-4593 (1995); Weiner et al., "Cancer Immunol.Immunother."; Vol. 42 (No. 3); pp. 141-150 (1996)). Antibody-based T cell engagers often show more than 1000 times higher affinity for CD3 compared to natural TCR-MHC interactions ((Wu et al., "Pharmacol Ther"; Vol. 182: pp. 161-175 (2018); International Publication No. 2014 / 167022; Junntila et al., "Cancer Res."; Vol. 19: pp. 5561-571 (2014); Yang et al., "J Immunol Aug.; Vol. 15 (No. 137): pp. 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." Vol. 32; No. 11: pp. 3102-3107 (2002); Ellerman, "Methods"; Vol. 154: pp. 102-117 (2019); Mandikian et al., "Mol. Cancer Ther."; Vol. 17 (No. 4): p. 776, LP-785 (2018); Vafa et al., "Frontiers in Oncology"; Vol. 10: p. 446 (2020)). Furthermore, downregulation of tumor surface markers targeted by TCE may lead to tumor resistance to TCE treatment.

[0008] Acute myeloid leukemia (AML), as mentioned above, is a type of cancer that exemplifies in many ways the challenges of cancer therapy and the shortcomings of currently available cancer therapies. AML remains a medically urgent issue due to its high mortality rate, and the treatment of relapsed or refractory AML remains challenging.

[0009] Therefore, there is still a need for novel CD33-specific binding proteins with beneficial properties. Such binding proteins could be useful in therapeutic and diagnostic approaches for treating and characterizing 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 can also be readily combined with other functional moieties, such as one or more binding sites. [Overview of the project]

[0010] The present invention relates to recombinant binding proteins comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to human CD33. Furthermore, 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, for example, acute myeloid leukemia (AML).

[0011] The recombinant binding proteins of the present invention either specifically bind to or target tumor-associated antigen (TAA) CD33. Such binding proteins of the present invention may serve as tools or components for generating novel 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 a single molecule. Such other functional moieties include binding moieties having binding specificity to a target expressed on immune cells, half-life extension moieties, binding moieties having binding specificity to another tumor-associated antigen, and / or cytotoxic agents. Thus, recombinant binding proteins of the present invention having binding specificity to CD33 are useful in generating novel therapeutic molecules that may provide an improved toxicity profile and / or therapeutic concentration range compared to current forms of therapy.

[0012] Based on the disclosures provided herein, those skilled in the art will recognize many equivalents to the particular embodiments of the invention described herein, or can confirm them by routine experimentation alone. Such equivalents are intended to be encompassed by the following embodiment (E).

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

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

[0015] 3. In a third embodiment, the present invention provides an ankyrin repeat domain to human CD33 in PBS with a dissociation constant (K) less than approximately 100 nM. D ) and, at your discretion, select K between approximately 0.1 nM and approximately 100 nM. D This relates to a recombinant binding protein according to either Embodiment 1 or 2, which binds via [a specific mechanism].

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

[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 to a target expressed on immune cells.

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

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

[0020] 8. In the eighth embodiment, the present invention relates to a recombinant binding protein according to any one of embodiments 5 to 7, wherein the binding portion having binding specificity to a target expressed on immune cells is an ankyrin repeat domain having binding specificity to human CD3.

[0021] 9. In the ninth embodiment, the present invention relates to a recombinant binding protein according to any one of embodiments 5 to 7, wherein the binding portion having binding specificity to a target expressed on immune cells is an ankyrin repeat domain having binding specificity to human CD3, and the ankyrin repeat domain having binding specificity to human CD3 includes an amino acid sequence that is at least 85% identical to any one of sequence numbers 55 to 59.

[0022] 10. In the tenth embodiment, the present invention relates to the recombinant binding protein according to Embodiment 9, wherein the ankyrin repeat domain having binding specificity to human CD3 comprises one of the amino acid sequences of SEQ ID NOs. 55 to 59.

[0023] 11. In the eleventh embodiment, the present invention relates to a recombinant binding protein according to any one of embodiments 5 to 10, wherein the ankyrin repeat domain having binding specificity to human CD33 and the binding portion having binding specificity to a target expressed on immune cells are covalently bonded to a peptide linker.

[0024] 12. In a twelfth embodiment, the present invention relates to the recombinant binding protein according to Embodiment 11, wherein the peptide linker is a proline-threonine-rich peptide linker.

[0025] 13. In the 13th embodiment, the present invention relates to the 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 14th embodiment, the present invention relates to a recombinant binding protein according to any one of Embodiments 1 to 13, wherein the binding protein further comprises a half-life extension portion.

[0027] 15. In a 15th embodiment, the present invention relates to the recombinant binding protein according to Embodiment 14, wherein the half-life extension portion is an ankyrin repeat domain having binding specificity to human serum albumin.

[0028] 16. In the 16th embodiment, the present invention relates to the recombinant binding protein according to Embodiment 15, wherein the ankyrin repeat domain having binding specificity to human serum albumin comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs. 52 to 54.

[0029] 17. In the 17th embodiment, the present invention relates to the recombinant binding protein according to Embodiments 15 and 16, wherein the ankyrin repeat domain having binding specificity to human serum albumin comprises one of the amino acid sequences of SEQ ID NOs. 52 to 54.

[0030] 18. In the 18th 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 to a target expressed in tumor cells, and the target expressed in tumor cells is different from human CD33.

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

[0032] 20. In the 20th embodiment, the present invention relates to a pharmaceutical composition comprising a recombinant binding protein described in any one of embodiments 1 to 18 or a nucleic acid described in embodiment 19, and a pharmaceutically acceptable carrier and / or diluent.

[0033] 21. In the 21st embodiment, the present invention relates to a method for activating immune cells in tumor tissue of a human patient, comprising the step of administering to the patient a recombinant binding protein described in any one of embodiments 1 to 18, a nucleic acid described in embodiment 19, or a pharmaceutical composition described in embodiment 20.

[0034] 22. In a 22nd embodiment, the present invention relates to the method of Embodiment 21, wherein the immune cell is a T cell.

[0035] 23. In the 23rd embodiment, the present invention relates to a method for treating a medical condition, comprising the step of administering to a patient in need of treatment for the medical condition a therapeutically effective amount of a 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 present invention relates to the method of Embodiment 23, wherein the medical condition is cancer.

[0037] 25. In the 25th embodiment, the present invention relates to the method of Embodiment 23, wherein the medical condition is cancer characterized by a humoral tumor.

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

[0039] 27. In the 27th embodiment, the present invention relates to the method of 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, a nucleic acid of embodiment 19, or a pharmaceutical composition of embodiment 20 for therapeutic use.

[0041] 29. In the 29th embodiment, the present invention relates to a recombinant binding protein according to any one of Embodiments 1 to 18, a nucleic acid according to Embodiment 19, or a pharmaceutical composition according to Embodiment 20, for use in the treatment of cancer, optionally for use in the treatment of cancer characterized by humoral tumors.

[0042] 30. In the 30th embodiment, the present invention relates to a recombinant binding protein or pharmaceutical composition for use as described in Embodiment 29, wherein the cancer is leukemia, and optionally, the cancer is acute myeloid leukemia. [Brief explanation of the drawing]

[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. Purified ankyrin repeat proteins at various concentrations were applied to GLC chips with immobilized human CD33 for on-rate 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. Purified ankyrin repeat proteins at various concentrations were applied to GLC chips with immobilized human CD33 for on-rate 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 2] Binding of exemplary binding proteins of the present invention to CD33-expressing tumor cells. Concentration-dependent binding curves for DARPin® protein #2, DARPin® protein #29, and DARPin® protein #30 (all in single-domain format) are shown. [Figure 3] This describes short-term T cell activation determined by the activation marker CD25. Pan-T effector cells and Molm-13 target cells were incubated in a 5:1 E:T ratio, and T cell activation was evaluated by FACS after 24 hours of co-culture in serial dilutions of the indicated molecules. Activated T cells were gated as viable CD8+ / CD25+ cells. The results show T cell activation induced by selected ankyrin repeat proteins DARPin® protein #33, DARPin® protein #31, and DARPin® protein #32. [Figure 4] Tumor cell killing was assessed by a cytotoxic assay measuring LDH release. Pan-T effector cells and Molm-13 target cells were incubated in a 5:1 E:T ratio, and tumor cell killing was assessed by FACS after 24 hours of co-culture in serial dilutions of the indicated molecules. This shows tumor cell killing by T cells induced by DARPin® protein #33, DARPin® protein #31, and DARPin® protein #32. [Figure 5] Binding of CD33-specific ankyrin repeat proteins to full-length and cleaved CD33 targets as determined by HTRF. All three ankyrin repeat proteins tested—DARPin® protein #1, DARPin® protein #9, or DARPin® protein #14—bound to full-length CD33, but only DARPin® protein #9 bound to cleaved 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 targets were pre-incubated with or without competing substances (DARPin® protein #1, DARPin® protein #9, or DARPin® protein #14). The binding targets were then tested against immobilized AMG330. Partial competition with AMG330 was observed for DARPin® protein #1. DARPin® protein #14 showed complete competition with AMG330, while DARPin® protein #9 showed no competition with AMG330. [Figure 7A] Figure 7A. Tumor growth over time in mice (n=5 mice per donor / 2 hPBMC donors used) that received intraperitoneal injection of hPBMCs, subcutaneous xenograft of MOLM-13 tumor cells 2 days after hPBMC injection, and treatment with DARPin® protein #29 (black square) in PBS 1× (black circle) or 0.5 mg / kg multi-domain format. Treatment was initiated 4 days after tumor cell xenografting. Data are shown as mean + SEM. Figure 7B. Evaluation of tumor volume 17 days after tumor cell xenografting in the mice described in Figure 7A. [Figure 7B] Figure 7A. Tumor growth over time in mice (n=5 mice per donor / 2 hPBMC donors used) that received intraperitoneal injection of hPBMCs, subcutaneous xenograft of MOLM-13 tumor cells 2 days after hPBMC injection, and treatment with DARPin® protein #29 (black square) in PBS 1× (black circle) or 0.5 mg / kg multi-domain format. Treatment was initiated 4 days after tumor cell xenografting. Data are shown as mean + SEM. Figure 7B. Evaluation of tumor volume 17 days after tumor cell xenografting in the mice described in Figure 7A. [Figure 8A]Figure 8A. Efficacy titration curve (T cell activation) of multi-domain format DARPin® protein #29 using wild-type or CD33 knockout Molm-13 tumor cells. EC50 values ​​are shown in pM. Figure 8B. Efficacy titration curve (T cell activation) of multi-domain format DARPin® protein #30 using wild-type or CD33 knockout Molm-13 tumor cells. EC50 values ​​are shown in pM. [Figure 8B] Figure 8A. Efficacy titration curve (T cell activation) of multi-domain format DARPin® protein #29 using wild-type or CD33 knockout Molm-13 tumor cells. EC50 values ​​are shown in pM. Figure 8B. Efficacy titration curve (T cell activation) of multi-domain format DARPin® protein #30 using wild-type or CD33 knockout Molm-13 tumor cells. EC50 values ​​are shown in pM. [Modes for carrying out the invention]

[0044] This specification discloses recombinant binding proteins comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to human CD33. Also disclosed are nucleic acids encoding recombinant binding proteins, binding proteins, or pharmaceutical compositions comprising nucleic acids, and methods using binding proteins, nucleic acids, or pharmaceutical compositions.

[0045] Ankyrin repeating domain The described recombinant binding proteins or their binding domains, which include the designed ankyrin repeat motif or module, are also referred to herein as DARPin® proteins (Stumpp et al., "Curr Opin Drug Discov Devel." Vol. 10 (No. 2): pp. 153-159 (2007); and Binz et al., "Nature Biotech." Vol. 22 (No. 5): pp. 575-582 (2004)). DARPin® proteins can be considered antibody mimetic molecules with high specificity and high binding affinity to target proteins. Generally, DARPin® proteins contain at least one ankyrin repeat domain, and may contain two, three, four, five, 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 a target. The structural core comprises conserved amino acid residues, while the target-binding surface comprises different amino acid residues depending on the target.

[0047] The designed ankyrin repeat protein library (International Publication No. 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 and thus enable novel therapeutic approaches. Such ankyrin repeat proteins may contain a single designed ankyrin repeat domain or a combination of two or more designed ankyrin repeat domains having the same or different target specificity (Stumpp et al., "Drug Discov. Today 13,695-701, 2008; U.S. Patent No. 9,458,211"). Ankyrin repeat proteins containing only a single designed ankyrin repeat domain are small proteins (14 kDa) that can be selected to bind to a given target protein with high affinity and specificity. These features, and the possibility of combining two or more designed ankyrin repeat domains in a single protein, make designed ankyrin repeat proteins ideal agonist, antagonist, and / or inhibitor candidates. Furthermore, such ankyrin repeat proteins can be engineered to carry various effector functions, such as cytotoxic agents or half-life extenders, enabling entirely novel drug formats.

[0048] The engineered ankyrin repeat proteins can also target epitopes that are not readily accessible with monoclonal antibodies. A further advantage of the described engineered ankyrin repeat proteins is that they generally have low immunogenicity and no or minimal off-target effects. The DARPin® candidate also exhibits favorable development characteristics, including rapid, low-cost, and high-yield production, as well as a shelf life of up to several years at 4°C. Taken together, engineered ankyrin repeat proteins represent an example of next-generation protein therapies with the potential to surpass existing antibody drugs.

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

[0050] As described above, CD33 is an attractive therapeutic target for the treatment of certain cancers, particularly AML. The recombinant binding proteins described herein include an ankyrin repeat domain and an ankyrin repeat module that specifically bind to human CD33.

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

[0052] In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to human CD33, and the ankyrin repeat domain comprises an ankyrin repeat module having an amino acid sequence selected from the group consisting of (1) sequences of SEQ ID NOs. 29-51 and SEQ ID NOs. 79-81, 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. 29-51 and SEQ ID NOs. 79-81 are substituted with other amino acids.

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

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

[0055] In a further embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to human CD33, and the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity with any one of SEQ ID NOs: 1-16 and SEQ ID NOs: 77-78, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0056] In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to human CD33, and the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity with SEQ ID NO: 1, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0057] In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to human CD33, and the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity with SEQ ID NO: 2, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0058] In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to human CD33, and the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity with SEQ ID NO: 3, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0059] In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to human CD33, and the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity with SEQ ID NO: 4, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0060] In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to human CD33, and the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity with SEQ ID NO: 5, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0061] In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to human CD33, and the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity with SEQ ID NO: 6, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0062] In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to human CD33, and the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity with SEQ ID NO: 7, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0063] In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to human CD33, and the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity with SEQ ID NO: 8, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0064] In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to human CD33, and the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity with SEQ ID NO: 9, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0065] In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to human CD33, and the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity with SEQ ID NO: 10, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0066] In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to human CD33, and the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity with SEQ ID NO: 11, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0067] In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to human CD33, and the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity with SEQ ID NO: 12, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0068] In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to human CD33, and the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity with SEQ ID NO: 13, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0069] In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to human CD33, and the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity with SEQ ID NO: 14, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0070] In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to human CD33, and the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity with SEQ ID NO: 15, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0071] In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to human CD33, and the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity with SEQ ID NO: 16, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0072] In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to human CD33, and the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity with SEQ ID NO: 77, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0073] In one embodiment, the present invention relates to a recombinant binding protein comprising an ankyrin repeat domain, wherein the ankyrin repeat domain has binding specificity to human CD33, and the ankyrin repeat domain comprises an amino acid sequence having at least about 85% amino acid sequence identity with SEQ ID NO: 78, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

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

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

[0076] Antibodies comprise any polypeptide or protein containing an antigen-binding domain derived from an antibody or immunoglobulin molecule. Antigen-binding domains may be derived from, for example, monoclonal antibodies, polyclonal antibodies, recombinant antibodies, human antibodies, humanized antibodies, and single-domain antibodies, such as heavy-chain variable domains (VH), light-chain variable domains (VL), and variable domains (VHH) of human or camelid origin. In some cases, it is beneficial that the antigen-binding domain originates from the same species in which the binding portion is ultimately used. For example, for use in humans, it may be beneficial that the antigen-binding domain of the binding portion described herein contains a human or humanized antigen-binding domain. Antibodies can be obtained using techniques well known in the art.

[0077] In one embodiment, the binding portion having binding specificity to a target expressed on immune cells is an antibody.

[0078] In one embodiment, the binding moiety having binding specificity to a target expressed on immune cells is a camelid nanobody. Camelid nanobodies (also known as camelid single-domain antibodies or VHHs) are derived from mammals of the camelid family, such as llamas, camels, and alpacas. Unlike other antibodies, camelid antibodies lack a light chain and consist 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 portion having binding specificity to a target expressed on immune cells is a shark antibody domain. Similar to camelid nanobodies, the shark antibody domain also lacks a light chain.

[0080] An alternative scaffold includes any polypeptide or protein that can bind to an antigen (such as a drug molecule) and contains a binding domain that does not originate from an antibody or immunoglobulin molecule. The binding domain of an alternative scaffold may include, or be derived from, a variety of different polypeptide or protein structures. Examples of alternative scaffolds include, but are not limited to, adnectin (monobody), afibody, affin, afimer and aptamer, afitin, alphabody, antikalin, armadillo repeat protein-based scaffolds, atrimers, avimers, ankyrin repeat protein-based scaffolds (e.g., DARPin® protein), finomers, Notchin, and Knitz 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 having binding specificity to a target expressed on an immune cell is an alternative scaffold. In one embodiment, the binding moiety having binding specificity to a target expressed on an immune cell includes adnectin, monobody, afibody, affilin, affimer, aptamer, afitin, alphabody, anticarin, repeat protein domain, armadillo repeat domain, atrimer, avimer, ankyrin repeat domain, finomer, Nottin, Knitz domain, or antigen-binding domains derived from or related to the T cell receptor (TCR).

[0082] Adnectin is originally derived from the 10th extracellular domain of human fibronectin type III protein (10Fn3). The fibronectin type III domain has 7 or 8 β-chains, which are distributed between two β-sheets, and the β-sheets themselves pack together to form the protein core, further containing loops (similar to CDRs), which connect the β-chains to each other and expose the solvent. There are at least three such loops at each edge of the β-sheet sandwich, and the edges are protein boundaries perpendicular to the direction of the β-chains (see U.S. Patent No. 6818418). Due to this structure, this non-antibody scaffold mimics antigen-binding properties similar to the nature and affinity of antibody antigen-binding properties. These scaffolds can be used in in vitro loop randomization and shuffling techniques that mimic the process of antibody affinity maturation in vivo.

[0083] Affibody affinity ligands consist of a 3-helix bundle based on one scaffold of the IgG-binding domain of protein A, a surface protein derived from the bacterium Staphylococcus aureus. This scaffold domain consists of 58 amino acids, 13 of which are randomized to create an affibody library with numerous ligand variants (see, for example, U.S. Patent No. 5,831,012). Although affibody molecules mimic antibodies, they are considerably smaller, with a molecular weight of approximately 6 kDa compared to approximately 150 kDa for antibodies. Despite the size difference, the binding sites of affibody molecules are similar to those of antibodies.

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

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

[0086] Afitin is a variant of the DNA-binding protein Sac7d that has been engineered to achieve specific binding affinity. Sac7d originally comes from the hyperthermophilic archaeon Sulfolobus acidocaldarius and binds to DNA to prevent its thermal denaturation. Afitin is commercially known as Nanofitin.

[0087] Alpha bodies are small (approximately 10 kDa) proteins engineered to bind to various antigens, and are therefore antibody mimetic. Alpha body scaffolds are computer-designed based on a coiled-coil structure. A standard alpha body scaffold contains three α-helices, each consisting of four heptad repeats (7-residue stretches) linked via a high-glycine / serine linker. The standard heptad sequence is "IAAIQKQ". The ability of alpha bodies to target extracellular and intracellular proteins, combined with their high binding affinity, can allow them to bind to targets that antibodies cannot reach.

[0088] Antikarin is a group of binding proteins that possess a robust and conserved β-barrel structure, found in lipocalin. Lipocalin is 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, particularly those related to nuclear transport. Armadillo repeat protein-based scaffolds typically consist of 3–5 internal repeats and 2 capping elements. They also possess a tandem-extended superhelix structure, which allows them to bind to corresponding peptide ligands in an extended three-dimensional structure.

[0090] Atrimers are scaffolds derived from trimer plasma proteins known as tetranectin and belong to the family of C-type lectins, which consist 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 natural A-domain-containing proteins such as HER3 and consist of numerous different "A-domain" monomers (2-10) linked via amino acid linkers. For example, avimers capable of binding to target antigens can be produced using methodologies described in U.S. Patent Publications 2004 / 0175756, 2005 / 0053973, 2005 / 0048512, and 2006 / 0008844.

[0092] Finomers are small, globular proteins (approximately 7 kDa) that evolved from amino acids 83-145 of the Src homologous domain 3 (SH3) of human Fyn tyrosine kinase. Due to their high thermal stability, cysteine-free scaffolding, and human origin, finomers are attractive binding molecules that reduce potential immunogenicity.

[0093] Nottin, also known as the cysteine ​​knot miniprotein, is typically a 30-amino acid-long protein containing three antiparallel β-sheets and a constraining loop bound by disulfide bonds that create a cysteine ​​knot. These disulfide bonds provide high thermal stability, making Nottin an attractive antibody mimetic.

[0094] Knitz domain peptides, or Knitz domain inhibitors, are a type of protease inhibitor that have an irregular secondary structure consisting of approximately 60 amino acids with three disulfide bonds and three loops, which can be mutated without destabilizing the structural framework.

[0095] In one embodiment, the binding portion having binding specificity to a target expressed on immune cells is a polypeptide or protein containing an antigen-binding domain derived from a T cell receptor (TCR).

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

[0097] There are no particular restrictions on the properties of the target expressed on the immune cells. In one embodiment, the target is expressed on immune cells that are T cells, and the target expressed on these immune cells is CD3.

[0098] Accordingly, in a preferred embodiment, the present invention relates to a recombinant binding protein comprising: (i) a first ankyrin repeat domain having binding specificity to human CD33, and the first ankyrin repeat domain having an ankyrin repeat module having an amino acid sequence selected from the group consisting of (1) sequences in SEQ ID NOs: 29-51 and SEQ ID NOs: 79-81, 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 is substituted by another amino acid; and (ii) a second ankyrin repeat domain having binding specificity to CD3, more preferably to human CD3.

[0099] In another embodiment, the present invention comprises (i) a first ankyrin repeat domain, the first ankyrin repeat domain having binding specificity to human CD33, and the first ankyrin repeat domain having an ankyrin repeat module having an amino acid sequence selected from the group consisting of (1) sequences in SEQ ID NOs: 29-51 and 79-81, and (2) sequences in any of SEQ ID NOs: 29-51 and 79-81 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 substituted by another amino acid, and (ii) a second ankyrin repeat module The present invention relates to a recombinant binding protein comprising a second ankyrin repeat domain having binding specificity to human CD3, wherein the second ankyrin repeat domain contains an amino acid sequence having at least about 85% amino acid sequence identity with any one of SEQ ID NOs. 55 to 59, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0100] In further embodiments, the present invention includes (i) a first ankyrin repeat domain, the first ankyrin repeat domain having binding specificity to human CD33, and the first ankyrin repeat domain comprising an ankyrin repeat module having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-51 and SEQ ID NOs: 79-81, and (ii) a second ankyrin repeat domain, the second ankyrin repeat domain having binding specificity to human CD3, and the second ankyrin repeat domain comprising a sequence number The present invention relates to a recombinant binding protein comprising one of numbers 55-59 and a second ankyrin repeat domain having an amino acid sequence having at least about 85% amino acid sequence identity, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0101] In another embodiment, the present invention relates to a recombinant binding protein comprising: (i) a first ankyrin repeat domain, the first ankyrin repeat domain having binding specificity to human CD33, and the first ankyrin repeat domain having an ankyrin repeat module having an amino acid sequence selected from the group consisting of (1) sequences of SEQ ID NOs: 29-51 and SEQ ID NOs: 79-81, 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: 29-51, 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 is substituted by another amino acid; and (ii) a second ankyrin repeat domain, the second ankyrin repeat domain having binding specificity to human CD3, and the second ankyrin repeat domain having an amino acid sequence in any one of SEQ ID NOs: 55-59.

[0102] In another embodiment, the present invention relates to a recombinant binding protein comprising: (i) a first ankyrin repeat domain, the first ankyrin repeat domain having binding specificity to human CD33, and the first ankyrin repeat domain comprising an ankyrin repeat module having an amino acid sequence selected from the group consisting of SEQ ID NOs: 29-51 and SEQ ID NOs: 79-81; and (ii) a second ankyrin repeat domain, the second ankyrin repeat domain having binding specificity to human CD3, and the second ankyrin repeat domain comprising any one of the amino acid sequences of SEQ ID NOs: 55-59.

[0103] In preferred embodiments, the present invention relates to a recombinant binding protein comprising: (i) a first ankyrin repeat domain having binding specificity to human CD33, and the first ankyrin repeat domain having at least about 85% amino acid sequence identity with any one of SEQ ID NOs: 1-16 and SEQ ID NOs: 77-78, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%; and (ii) a second ankyrin repeat domain having binding specificity to CD3, more preferably to human CD3.

[0104] In another embodiment, the present invention relates to (i) a first ankyrin repeat domain, the first ankyrin repeat domain having binding specificity to human CD33, and the first ankyrin repeat domain comprising an amino acid sequence having at least about 85% amino acid sequence identity with any one of SEQ ID NOs: 1-16 and SEQ ID NOs: 77-78, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%. The present invention relates to a recombinant binding protein comprising (ii) a main and a second ankyrin repeat domain, the second ankyrin repeat domain having binding specificity to human CD3, and the second ankyrin repeat domain having at least about 85% sequence identity with any one of SEQ ID NOs. 55 to 59, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0105] In one embodiment, the present invention comprises (i) a first ankyrin repeat domain, the first ankyrin repeat domain having binding specificity to human CD33, and the first ankyrin repeat domain comprising any one amino acid sequence of SEQ ID NOs: 1-16 and SEQ ID NOs: 77-78, and (ii) a second ankyrin repeat domain, the second ankyrin repeat domain having binding specificity to human CD3, and the second ankyrin repeat domain comprising any one of SEQ ID NOs: 55-59 The present invention relates to a recombinant binding protein comprising one of the following and a second ankyrin repeat domain having an amino acid sequence having at least approximately 85% sequence identity, for example, at least approximately 86%, at least approximately 87%, at least approximately 88%, at least approximately 89%, at least approximately 90%, at least approximately 91%, at least approximately 92%, at least approximately 93%, at least approximately 94%, at least approximately 95%, at least approximately 96%, at least approximately 97%, at least approximately 98%, at least approximately 99%, or 100%.

[0106] In one embodiment, the present invention relates to (i) a first ankyrin repeat domain, wherein the first ankyrin repeat domain has binding specificity to human CD33, and the first ankyrin repeat domain has at least about 85% amino acid sequence identity with any one of SEQ ID NOs: 1-16 and SEQ ID NOs: 77-78, 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%, and at least The present invention relates to a recombinant binding protein comprising: (ii) a first ankyrin repeat domain having an amino acid sequence having amino acid sequence identity of at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%; and (ii) a second ankyrin repeat domain having binding specificity to human CD3, and the second ankyrin repeat domain having one amino acid sequence of sequence numbers 55 to 59.

[0107] In further embodiments, the present invention relates to a recombinant binding protein comprising: (i) a first ankyrin repeat domain having binding specificity to human CD33, and the first ankyrin repeat domain comprising any one of the amino acid sequences of SEQ ID NOs: 1-16 and SEQ ID NOs: 77-78; and (ii) a second ankyrin repeat domain having binding specificity to human CD3, and the second ankyrin repeat domain comprising any one of the amino acid sequences of SEQ ID NOs: 55-59.

[0108] The present invention further relates to a recombinant binding protein comprising: (i) a first ankyrin repeat domain having binding specificity to human CD33; and (ii) a second ankyrin repeat domain having binding specificity to 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. 17-28 and SEQ ID NOs. 82-84, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100%.

[0109] In one embodiment, the present invention relates to a recombinant binding protein comprising: (i) a first ankyrin repeat domain having binding specificity to human CD33; and (ii) a second ankyrin repeat domain having binding specificity to 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 sequence numbers 17-28 and sequences 82-84.

[0110] Half-life extension portion The "half-life extension portion" extends the in vivo serum half-life of the recombinant binding protein described herein compared to the same protein without the half-life extension portion. Examples of half-life extension portions include, but are not limited to, polyhistidine, Glu-Glu, glutathione S-transferase (GST), thioredoxin, protein A, protein G, immunoglobulin domain, maltose-binding protein (MBP), human serum albumin (HSA)-binding domain, or polyethylene glycol (PEG).

[0111] In some embodiments, the recombinant binding proteins described herein include an ankyrin repeat domain that specifically binds to serum albumin (preferably human serum albumin, etc.), also referred herein to as the “serum albumin-binding domain.” The recombinant binding proteins described herein may also include two or more serum albumin-binding domains, for example, two or three serum albumin-binding domains. Thus, the recombinant binding proteins described herein may include first and second serum albumin-binding domains, or first, second, and third serum albumin-binding domains. 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 extension portion described herein includes a serum albumin-specific ankyrin repeat domain, wherein the binding ankyrin repeat domain contains 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. In exemplary embodiments, the half-life extension portion described herein contains an amino acid sequence that is at least about 90% identical to any one of SEQ ID NOs. In one embodiment, the half-life extension portion described herein includes 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 extension portion described herein includes 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 extension portion includes an immunoglobulin domain. In some embodiments, the immunoglobulin domain includes an Fc domain. In some embodiments, the Fc domain is derived from 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 one of the known heavy chain isotypes or subtypes IgG1(γ1), IgG2(γ2), IgG3(γ3), IgG4(γ4), IgA1(α1), or IgA2(α2). In some embodiments, the Fc domain is the Fc domain of human IgG1.

[0115] In some embodiments, the Fc domain includes an uninterrupted native sequence of the Fc domain (i.e., the wild-type sequence). In some embodiments, the immunoglobulin Fc domain includes a mutant Fc domain that results in altered biological activity. For example, at least one point mutation or deletion may be introduced into the Fc domain to reduce or eliminate effector activity (e.g., International Patent Publication WO2005 / 063815) and / or to increase homogeneity during the production of recombinant binding proteins. In some embodiments, the Fc domain is the Fc domain of human IgG1 and includes one or more of the following effector null substitutions: L234A, L235A, and G237A (Eu-numbered). In some embodiments, the Fc domain does not include lysine located at the C-terminal position of human IgG1 (i.e., K447, Eu-numbered). The absence of lysine may increase homogeneity during the production of recombinant binding proteins. In some embodiments, the Fc domain includes lysine located at the C-terminal position (K447, Eu-numbered).

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

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

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

[0119] In some embodiments, the amino acid substitution(s) performed on the binder is the K of the unsubstituted binder. D Compared to the value, K D The value should not be changed by more than approximately 1000 times, more than approximately 100 times, or more than approximately 10 times. For example, in some embodiments, the amino acid substitution(s) is a K of the CD33 of a binder containing any of the sequences of SEQ ID NOs: 1-16, SEQ ID NOs: 29-51, or SEQ ID NOs: 77-81. D Compared to the value, K D Do not change the value by more than approximately 1000 times, 300 times, 100 times, 50 times, 25 times, 10 times, or 5 times.

[0120] In certain embodiments, the substitutions are conservative substitutions as shown in Table 1. In certain embodiments, the substitutions are made outside the structural core residues of the ankyrin repeat domain, for example, within the beta loops connecting the alpha helices.

[0121] [Table 1]

[0122] In certain embodiments, substitutions are made within the structural core residues of the ankyrin repeat domain. In other embodiments, substitutions are made outside the structural core residues of the ankyrin repeat domain. For example, the ankyrin domain may include the consensus sequence: xDxxGxTPLHLAxxxGxxxlVxVLLxxGADVNA (SEQ ID NO: 62), where "x" represents any amino acid (preferably not cysteine, glycine, or proline), or it may include xDxxGxTPLHLAAxxGHLEIVEVLLKzGADVNA (SEQ ID NO: 63), where "x" represents any amino acid (preferably not cysteine, glycine, or proline), and "z" is selected from the group consisting of asparagine, histidine, or tyrosine. In one embodiment, substitutions are made at the residue designated as "x". In another embodiment, substitutions are made at the residue not designated as "x".

[0123] In addition, the second-to-last position of any ankyrin repeat domain of the recombinant binding protein of the present invention may be "A" or "L", and / or the last position may be "A" or "N". Therefore, 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: SEQ ID NOs: 1-16, SEQ ID NOs: 52-59, and SEQ ID NOs: 77-78, where optionally, the second-to-last A is replaced with L and / or the last A is replaced with N, or optionally, the second-to-last L is replaced with A and / or the last N is replaced with 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, wherein the second-to-last A is substituted with L and / or the last A is substituted with N. Furthermore, any ankyrin repeat domain sequence contained in the binding protein of the present invention may optionally contain G, S, or GS at its N-terminus (see below).

[0124] In addition, each ankyrin repeat domain included in the recombinant binding protein of the present invention may optionally include a "G", "S", or "GS" sequence at its N-terminus. Thus, in some embodiments, each ankyrin repeat domain has 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, SEQ ID NOs: 52-59, and SEQ ID NOs: 77-78, and further includes GS (e.g., as in SEQ ID NO: 64) at its N-terminus, or includes 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 described with respect to K D Exemplary embodiments, 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​​​​​​​​​​​​​​​​​​​​​​​​​​​​​M~approximately 10 -15 M, approximately 10 -10 M~approximately 10 -15 M, approximately 10 -5 M~approximately 10 -14 M, approximately 10 -6 M~approximately 10 -14 M, approximately 10 -7 M~approximately 10 -14 M, approximately 10 -8 M~approximately 10 -14 M, approximately 10 -9 M~approximately 10 -14 M, approximately 10 -10 M~approximately 10 -14 M, approximately 10 -5 M~approximately 10 -13 M, approximately 10 -6 M~approximately 10 -13 M, approximately 10 -7 M~approximately 10 -13 M, approximately 10 -8 M~approximately 10 -13 M, approximately 10 -9 M~approximately 10 -13 M, approximately 10 -10 M~approximately 10 -13 M, approximately 10 -5 M~approximately 10 -12 M, approximately 10 -6 M~approximately 10 -12 M, approximately 10 -7 M~approximately 10 -12 M, approximately 10 -8 M~approximately 10 -12 M, approximately 10 -9 M~approximately 10 -12 M, approximately 10 -10 M~approximately 10 -12 M, approximately 10 -5 M~approximately 10 -11 M, approximately 10 -6 M~approximately 10 -11 M, approximately 10 -7 M~approximately 10 -11 M, approximately 10 -8 M~approximately 10 -11 M, approximately 10 -9 M~approximately 10 -11 M, approximately 10 -10 M~approximately 10 -11 M, approximately 10 -5 M~approximately 10 -10 M, approximately 10 -6 M~approximately 10 -10 M, approximately 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 M.

[0126] In an exemplary embodiment, the recombinant binding protein binds to human CD33 with a K D value 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 one exemplary embodiment, the recombinant binding protein binds to CD33 with a K D value of 100 nM or less. In another exemplary embodiment, the recombinant binding protein binds to CD33 with a K

[0127] In one aspect, the recombinant binding protein binds to human CD33 with an EC 50 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. Thus, in one aspect, the binding protein binds to human CD33 on T cells with an EC 50 of less than about 5 μM; in another aspect, the binding protein binds to human CD33 on T cells with an EC 50In another embodiment, the binding protein has an EC of less than approximately 3 μM. 50 In another embodiment, the binding protein has an EC of less than approximately 2 μM. 50 In another embodiment, the binding protein has an EC of less than approximately 1 μM. 50 In another embodiment, the binding protein has an EC of less than approximately 900 nM. 50 In another embodiment, the binding protein has an EC of less than approximately 800 nM. 50 In another embodiment, the binding protein has an EC of less than approximately 600 nM. 50 It then binds to human CD33 on T cells. In another embodiment, the binding protein has an EC of less than approximately 700 nM. 50 In another embodiment, the binding protein has an EC of less than approximately 500 nM. 50 In another embodiment, the binding protein has an EC of less than approximately 400 nM. 50 In another embodiment, the binding protein has an EC of less than approximately 300 nM. 50 In another embodiment, the binding protein has an EC of less than approximately 200 nM. 50 In another embodiment, the binding protein has an EC of less than approximately 100 nM. 50 In another embodiment, the binding protein has an EC of less than approximately 70 nM. 50 In another embodiment, the binding protein has an EC of less than approximately 60 nM. 50 In another embodiment, the binding protein has an EC of less than approximately 50 nM. 50 In another embodiment, the binding protein has an EC of less than approximately 40 nM. 50 In another embodiment, the binding protein has an EC of less than approximately 30 nM.50 In another embodiment, the binding protein has an EC of less than approximately 20 nM. 50 In another embodiment, the binding protein has an EC of less than approximately 15 nM. 50 In another embodiment, the binding protein has an EC of less than approximately 10 nM. 50 In another embodiment, the binding protein has an EC of less than approximately 7 nM. 50 In another embodiment, the binding protein has an EC of less than approximately 5 nM. 50 In another embodiment, the binding protein has an EC of less than approximately 3 nM. 50 In another embodiment, the binding protein has an EC of less than approximately 1 nM. 50 In another embodiment, the binding protein has an EC of less than approximately 0.5 nM. 50 In a further embodiment, the binding protein has an EC of less than approximately 0.1 nM. 50 Then it is coupled to CD33.

[0128] Additional polypeptides In one embodiment, the recombinant binding protein of the present invention further comprises a polypeptide tag. The polypeptide tag is an amino acid sequence bound to a polypeptide / protein, where the amino acid sequence is useful for the purification, detection, or targeting of the polypeptide / protein, or the amino acid sequence improves the physicochemical behavior of the polypeptide / protein, or the amino acid sequence has an effector function. Individual polypeptide tags of the recombinant binding protein may be attached to other sites of the recombinant binding protein directly or via a peptide linker. Polypeptide tags are well known in the art and are readily available to those skilled in the art. Examples of polypeptide tags include small polypeptide sequences, such as His tags, HA tags, myc tags, FLAG tags, or Strep tags, or polypeptides such as enzymes (e.g., alkaline phosphatase), which enable the detection of the polypeptide / protein, or polypeptides that can be used for targeting (such as immunoglobulins or their fragments) and / or polypeptides that can be used as effector molecules.

[0129] In one embodiment, the recombinant binding protein of the present invention further comprises a peptide linker. The peptide linker is an amino acid sequence capable of linking, for example, two protein domains, a polypeptide tag and a protein domain, a protein domain and a non-protein compound or a polymer such as polyethylene glycol, a protein domain and a biologically active molecule, a protein domain and a localizer, or two sequence tags. Peptide linkers are known to those skilled in the art. A list of examples is provided in the specification of Japanese Patent Application Publication No. WO2002 / 020565. In one embodiment, the peptide linker for use in the present invention has a length of 1 to 50 amino acids. In another embodiment, the peptide linker for use in the present invention has a length of about 5 to about 40 amino acids. In yet another embodiment, the peptide linker for use in the present invention has a length of about 10 to about 30 amino acids.

[0130] Specific examples of peptide linkers include glycine-serine linkers and variable-length proline-threonine-rich linkers. In the context of the present invention, a proline-threonine-rich linker contains at least about 20% proline residues and at least about 20% threonine residues in its amino acid sequence. Examples of glycine-serine linkers are the amino acid sequences GS and SEQ ID NO: 67, and examples of proline-threonine-rich linkers are the amino acid sequences of SEQ ID NOs: 65 and 66.

[0131] N-terminal and C-terminal capping sequences The ankyrin repeat domains of the recombinant binding proteins disclosed herein may include an N-terminal or C-terminal capping sequence. The capping sequence refers to an additional polypeptide sequence fused to the N-terminus or C-terminus of the ankyrin repeat motif(s), wherein the capping sequence forms a tight tertiary interaction (i.e., a tertiary structural interaction) with the ankyrin repeat motif(s), thereby providing a cap that shields the hydrophobic core of the lateral ankyrin repeat domain from exposure to the solvent.

[0132] The N-terminal and / or C-terminal capping sequences may originate from capping units or other structural units found in naturally occurring repeat proteins adjacent to repeat units. Examples of capping sequences are described in International Publications 2002 / 020565 and 2012 / 069655, U.S. Patent Application Publication 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 Sequence IDs 69-72, and examples of ankyrin C-terminal capping modules (i.e., C-terminal capping repeats) include Sequence IDs 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 nucleic acid of the present invention. Nucleic acids are well known to those skilled in the art. In the examples, nucleic acids were used to produce the designed ankyrin repeat domain or recombinant binding protein of the present invention in E. coli.

[0134] Composition, use, and treatment method In one embodiment, the present invention relates to a pharmaceutical composition comprising the recombinant binding protein and / or designed ankyrin repeat domain of the present invention, and / or a nucleic acid encoding the recombinant binding protein and / or designed ankyrin repeat domain of the present invention, and optionally a pharmaceutically acceptable carrier and / or diluent.

[0135] In one embodiment, the present invention relates to a pharmaceutical composition comprising a recombinant binding protein, or a nucleic acid encoding the recombinant binding protein of the present invention, and optionally a pharmaceutically acceptable carrier and / or diluent.

[0136] Pharmacopoeia-acceptable carriers and / or diluents are known to those skilled in the art and are described in more detail below.

[0137] The pharmaceutical composition comprises recombinant binding proteins and / or designed ankyrin repeat domains and / or nucleic acids as described herein, preferably recombinant binding proteins and / or nucleic acids, and pharmaceutically acceptable carriers, excipients, or stabilizers, such as those described in "Remington's Pharmaceutical Sciences," 16th edition, edited by Osol, A. (1980).

[0138] Suitable carriers, diluents, excipients, or stabilizers known to those skilled in the art include, for example, physiological saline, Ringer's solution, dextrose solution, Hanks' solution, fixative oil, ethyl oleate, 5% dextrose physiological saline, substances that enhance isotonicity and chemical stability, buffers, and preservatives. Other suitable carriers include any carrier that does not induce the production of antibodies harmful to the individual administered the composition, such as proteins, polysaccharides, polylactic acid, polyglycolic acid, polymeric amino acids, and amino acid copolymers. The pharmaceutical composition may also be a combination containing additional active ingredients such as anticancer agents or anti-angiogenic agents, or further bioactive compounds. Compositions used for in vivo administration must be sterile or sterilized. This can be easily accomplished by filtration through a sterile filtration membrane.

[0139] In one embodiment, the pharmaceutical composition comprises at least one recombinant binding protein as described herein, as well as a detergent such as a nonionic detergent, a buffer such as a phosphate buffer, and a sugar such as sucrose. In one embodiment, such a composition comprises the recombinant binding protein and PBS as described above.

[0140] In another embodiment, the present invention provides a method for tumor-local activation of T cells in a mammal, including a human, comprising the step of administering to the mammal a recombinant protein of the present invention, a nucleic acid of the present invention, or a pharmaceutical composition of the present invention.

[0141] In another aspect, the present invention provides a method for treating a medical condition, comprising the step of administering a therapeutically effective amount of the recombinant binding protein of the present invention, the nucleic acid of the present invention, or the pharmaceutical composition of the present invention to a patient who is in need of treatment for the medical condition.

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

[0143] In one embodiment, the present invention relates to a pharmaceutical composition, recombinant binding protein, or nucleic acid according to the present invention for use in the treatment of a disease. For this purpose, the pharmaceutical composition, nucleic acid, or recombinant binding protein according to the present invention is administered in a therapeutically effective dose to a patient in need thereof. Dosage methods include topical administration, oral administration, and parenteral administration. The typical route of administration is parenteral administration. In parental administration, the pharmaceutical composition of the present invention is formulated in a unit dose injectable form such as a solution, suspension, or emulsion, together with pharmaceutically acceptable excipients as defined above. The dose and method of administration vary depending on the individual being treated and the disease.

[0144] Furthermore, any of the aforementioned pharmaceutical compositions, nucleic acids, or recombinant proteins are considered to be for use in the treatment of disorders.

[0145] In one embodiment, the recombinant binding protein or other such pharmaceutical composition described herein is administered intravenously. For parenteral use, the recombinant binding protein or the pharmaceutical composition may be administered in a therapeutically effective dose as a bolus injection or by slow intravenous infusion.

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

[0147] In one embodiment, the present invention further provides the use of such recombinant binding proteins for treating medical conditions in subjects requiring them.

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

[0149] The use of the recombinant binding protein, nucleic acid, or pharmaceutical composition of the present invention may also be in combination with one or more other therapies known in the art. The term “in combination with ~” as used herein means co-administration carried out under a given dosing schedule. This includes simultaneous administration of different compounds and administration of different compounds at different times (for example, administering compound A once and compound B several times thereafter, or vice versa, or administering both compounds simultaneously and then administering one of them again at a later stage).

[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 the recombinant protein of the present invention and / or the nucleic acid of the present invention and / or the pharmaceutical composition of the present invention. In one aspect, the present invention relates to a kit comprising a recombinant protein comprising an ankyrin repeat domain having binding specificity to CD33, for example, SEQ ID NOs: 1-16 and SEQ ID NOs: 77-78, and / or a nucleic acid encoding the recombinant protein comprising an ankyrin repeat domain having binding specificity to CD33, for example, SEQ ID NOs: 1-16 and SEQ ID NOs: 77-78, and / or a pharmaceutical composition comprising an ankyrin repeat domain having binding specificity to CD33, for example, 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 recombinant proteins of the present invention. In one aspect, the present invention relates to a method for producing recombinant binding proteins, for example, recombinant proteins comprising one amino acid sequence of any one of SEQ ID NOs: 1-59 and SEQ ID NOs: 77-84, comprising: (i) expressing the recombinant binding protein in a suitable host cell (e.g., in bacteria); and (ii) purifying the recombinant binding protein (e.g., using chromatography). The method may include additional steps. Such a method for producing recombinant binding proteins of the present invention is described in Example 1.

[0152] The present invention is not limited to the specific embodiments described in the examples. This specification refers to many amino acid sequences, nucleic acid sequences, and sequence numbers disclosed in the accompanying sequence listings, which are incorporated herein by reference in their entirety.

[0153] definition Unless otherwise defined herein, all technical and scientific terms used herein have meanings that are generally understood by those skilled in the art. Furthermore, unless the context specifically requires otherwise, singular terms shall include plural forms and plural terms shall include singular forms. In general, the nomenclature used herein in relation to the techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry is well known and commonly used in the art.

[0154] The terms “comprising,” “having,” “including,” and “containing” should be interpreted as non-restrictive unless otherwise specified. Where a feature is described as “comprising” in an aspect of the present invention, the aspect may also describe that feature as “consisting of” or “essentially consisting of.” Any use of any example or illustrative wording provided herein (e.g., “etc.”) is intended solely to better illustrate the disclosure and, unless otherwise requested, does not impose any limitation on the scope of the disclosure. Nothing in this specification should be interpreted as indicating any unclaimed element as essential to the practice of the disclosure. Except for the examples provided herein, or unless otherwise indicated, all numbers representing quantities of components or reaction conditions used herein should be understood in all cases as being modified by the term “about,” so that the term “about” is interpreted by those skilled in the art. Where used herein, the term “about” means ±10% of a given number unless otherwise specified.

[0155] The enumeration of value ranges in this specification is intended merely as a simplified way of referring individually to each distinct value and each endpoint that falls within that range, unless otherwise indicated herein, and each distinct value and endpoint is incorporated herein as if it were individually enumerated.

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

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

[0158] In the context of the present invention, the term "binding protein" refers to a protein containing a binding domain. The binding protein may also contain two, three, four, five or more binding domains. Preferably, the binding protein is a recombinant binding protein. The binding protein of the present invention contains an ankyrin repeat domain having binding specificity to CD33.

[0159] Furthermore, any such binding protein may include additional polypeptides well known to those skilled in the art (e.g., polypeptide tags, peptide linkers, fusion to other protein domains with binding specificity, cytokines, hormones, or antagonists), or chemical modifications (e.g., coupling to polyethylene glycol, toxins (e.g., DM1 from immunogens), small molecules, antibiotics, etc.). The binding protein of the present invention may also include a localizer molecule.

[0160] The term "binding domain" refers to a protein domain that exhibits binding specificity to a target. Preferably, this binding domain is a recombinant binding domain.

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

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

[0163] Patent application publication 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, techniques, and applications of repeat proteins and repeat domains. The term “repeat protein” refers to a protein containing one or more repeat domains. Preferably, a repeat protein contains one, two, three, four, five, or six repeat domains. Furthermore, the repeat protein may also contain additional non-repeat protein domains, polypeptide tags, and / or peptide linkers. The repeat domains may be binding domains.

[0164] The term “repeatable domain” refers to a protein domain comprising two or more consecutive repeating modules as structural units, wherein these repeating modules have structural homology and sequence homology. Preferably, the repeating domain further comprises N-terminal and / or C-terminal capping modules. For clarity, the capping modules may be repeating modules. Such repeating domains, repeating modules, and capping modules, sequence motifs, and their structural and sequence homology are well known to those skilled in the art from examples of ankyrin repeating domains (International Publication No. 2002 / 020565), leucine-rich repeating domains (International Publication No. 2002 / 020565), tetratricopeptide repeating domains (Main, ER, Xiong, Y., Cocco, MJ, D'Andrea, L., Regan, L., Structure 11(5), 497~508, 2003), and armadillo repeating domains (International Publication No. 2009 / 040338). It is more well known to those skilled in the art that such repeating domains are different from proteins containing repeating amino acid sequences, and all repeating amino acid sequences can form individual domains (for example, the FN3 domain of fibronectin).

[0165] The term "ankyrin repeat domain" refers to a repeat domain containing two or more consecutive ankyrin repeat modules as structural units. Ankyrin repeat domains may 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, International Publication No. 2002 / 020565, International Publication No. 2016 / 156596, International Publication No. 2018 / 054971).

[0166] The term "designed" as used in terms such as designed repeat proteins and designed repeat domains refers to the characteristic that such repeat proteins and repeat domains are artificial and do not occur in nature. The binding proteins of the present invention design repeat proteins, which include at least one designed ankyrin repeat domain. Preferably, the designed repeat domain is a designed ankyrin repeat domain.

[0167] The term "target interaction residue" refers to amino acid residues in a repeating module that contribute to direct interaction with a target.

[0168] The term "framework residue" refers to an amino acid residue in a repeating module that contributes to the folding topology, i.e., to the folding of the repeating module or to interactions with adjacent modules. Such contributions may include interactions with other residues within the repeating module, influences on the polypeptide backbone structure observed in α-helices or β-sheets, or involvement in amino acid stretching to form linear polypeptides or loops. Such framework and target interaction residues can be identified by analysis of structural data obtained by physicochemical methods such as X-ray crystallography, NMR and / or CD spectroscopy, or by comparison with known relevant structural information well known to those skilled in the art in structural biology and / or bioinformatics.

[0169] The term "repetitive module" originally refers to the repeated amino acid sequence and structural unit of a designed repeat domain, derived from the repeat units of naturally occurring repeat proteins. Each repeat module contained within a repeat domain is derived from one or more repeat units of a naturally occurring family or subfamily of repeat proteins, such as the ankyrin repeat protein family. Furthermore, each repeat module contained within a repeat domain may include a "repetitive sequence motif" derived from a homologous repeat module having the same target specificity, as described in Example 1, for example.

[0170] Therefore, the term “ankyrin repeat module” refers to a repeat module derived from the repeat unit of the naturally occurring ankyrin repeat protein. The ankyrin repeat protein is well known to those skilled in the art. Designed ankyrin repeat proteins have been previously described, and please refer to, for example, International Publications 2002 / 020565, 2010 / 060748, 2011 / 135067, 2012 / 069654, 2012 / 069655, 2014 / 001442, 2014 / 191574, 2014 / 083208, 2016 / 156596, and 2018 / 054971, all of which are incorporated by reference in their entirety. Typically, an ankyrin repeat module contains approximately 31–33 amino acid residues that form two alpha helices separated by a loop.

[0171] The repeating module may include positions with amino acid residues that are not randomized in the library ("unrandomized positions") and positions with amino acid residues that are randomized in the library ("randomized positions") for the purpose of selecting target-specific repeating domains. Unrandomized positions include framework residues. Randomized positions include target interaction residues. "Randomized" means, for example, that two or more amino acids are permitted at an amino acid position in the repeating module, and any of the usual 20 naturally occurring amino acids are permitted, or most of the 20 naturally occurring amino acids are permitted, such as amino acids other than cysteine, or amino acids other than glycine, cysteine, and proline.

[0172] The term “repetitive sequence motif” refers to an amino acid sequence inferred from one or more repetitive modules. Preferably, the repetitive modules are derived from repetitive domains having binding specificity to the same target. Such a repetitive sequence motif includes framework residue locations and target interaction residue locations. The framework residue locations correspond to the positions of framework residues in the repetitive module. Similarly, the target interaction residue locations correspond to the positions of target interaction residues in the repetitive module. The repetitive sequence motif includes non-randomized and randomized locations.

[0173] The term “repeat unit” refers to an amino acid sequence containing one or more naturally occurring protein sequence motifs, which are found in multiple copies and exhibit a defined folding topology common to all such motifs that determine the protein's folding. Examples of such repeat units include leucine-rich repeat units, ankyrin repeat units, armadillo repeat units, tetratricopeptide repeat units, HEAT repeat units, and leucine-rich variant repeat units.

[0174] The terms "having binding specificity to the target," "binding specifically to the target," "binding to the target with high specificity," "specific to the target," or "target specificity," or "binding specifically," mean that the binding protein or binding domain binds to the target in PBS with a lower dissociation constant (i.e., with higher affinity) than it would to unrelated proteins such as E. coli maltose-binding protein (MBP). Preferably, the dissociation constant in PBS for the target is ("K"). D ) is at least 10 times greater than the corresponding dissociation constant for MBP. 2 times, more preferably at least 10 3 times, more preferably at least 10 4 double, or more preferably at least 10 5 It is twice as low. Methods for measuring the dissociation constant of protein-protein interactions, such as techniques based on surface plasmon resonance (SPR) (e.g., SPR equilibrium analysis) or isothermal titration calorimetry (ITC), are well known to those skilled in the art. The K of a particular protein-protein interaction D The measured values ​​may vary when measured under different conditions (e.g., salt concentration, pH). Therefore, K D The value is preferably measured using a standardized protein solution and a standardized buffer such as PBS. The dissociation constant (K) of the recombinant binding protein of the present invention having binding specificity to CD33. DA typical and preferred determination of ) by surface plasmon resonance (SPR) analysis is described in Example 2. Various assay formats can be used to select or characterize binding sites that specifically bind to the drug molecule of interest. For example, solid-phase ELISA immunoassay, 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 sites that specifically bind to target drug molecules. Typically, specific or selective binding is at least 2x background signal or noise, more typically more than 10x background signal. More specifically, the binding agent is the equilibrium dissociation constant (K D If the value is <1 μM, for example, <500 nM, <100 nM, <10 nM, <1 nM, <100 pM, or <10 pM, it is said to "specifically bind" to the target.

[0175] The terms “binding agent” or “binding site” refer to any molecule that can specifically bind to a target molecule. The term “binding agent” includes, for example, antibodies, antibody fragments, aptamers, peptides (e.g., Williams et al., “J Biol Chem,” Vol. 266: pp. 5182–5190 (1991)), alternative scaffolds, antibody mimetic bodies, repeat proteins, such as designed ankyrin repeat proteins, receptor proteins, and other naturally occurring interaction partners of any of the target molecules, and may include native proteins and proteins that have been modified or genetically engineered, for example, to include and / or lack native residues.

[0176] The term "PBS" refers to a phosphate-buffered aqueous solution containing 137 mM NaCl, 10 mM phosphate, and 2.7 mM KCl, with a pH of 7.4.

[0177] Preferably, clearance, and / or exposure, and / or terminal phase half-life are evaluated in mammals, more preferably mice and / or cynomolgus monkeys. Preferably, when clearance, and / or exposure, and / or terminal phase half-life are measured in mice, the evaluation is carried out considering data up to a maximum of 48 hours after injection. More preferably, the evaluation of terminal phase half-life in mice is calculated over 24 to 48 hours. Preferably, when clearance, and / or exposure, and / or terminal phase half-life are measured in cynomolgus monkeys, the evaluation is carried out considering data up to a maximum of 7 days after injection. More preferably, the evaluation of terminal phase half-life in cynomolgus monkeys is calculated over 1 to 5 days. Those skilled in the art may further identify effects such as target-mediated clearance and take them into consideration when calculating terminal phase half-life. The term "terminal phase half-life" for drugs such as recombinant binding proteins of the present invention refers to the time required to reach half the plasma concentration of the drug after it has reached spurious equilibrium in a mammal (for example, calculated as 24 to 48 hours in mice, or as 1 to 5 days in cynomolgus monkeys). Terminal phase half-life is not defined as the time required to excrete half of the dose of the drug administered to a mammal. The term terminal phase half-life is well known to those skilled in the art. Preferably, pharmacokinetic comparisons are made at any dose, more preferably equivalent doses (i.e., the same mg / kg dose) or equimolar doses (i.e., the same mol / kg dose), more preferably equimolar dosages (i.e., the same mol / kg dose). Those skilled in the art will understand that equivalent and / or equimolar dosages in animals involve experimental dose variability 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 approximately 0.001 to approximately 1000 mg / kg, more preferably approximately 0.01 to approximately 100 mg / kg, more preferably approximately 0.1 to approximately 50 mg / kg, and more preferably approximately 0.5 to approximately 10 mg / kg.

[0178] The term “CD3” or “differentiation cluster 3” refers to a multimeric protein complex composed of four distinct polypeptide chains, epsilon (ε), gamma (γ), and zeta (ζ), which assemble as three pairs (εγ, εδ, ζζ). The CD3 complex functions as a T cell coreceptor, non-covalently associating with the T cell receptor. It may refer to any form of CD3, as well as its variants, isoforms, and species homologs that retain at least some of the activity of CD3. Thus, the binding proteins defined and disclosed herein may also bind to CD3 from non-human species. In other cases, the binding proteins may be perfectly specific to human CD3 and may not exhibit species or other types of cross-reactivity. Unless otherwise indicated, such as by a specific reference to human CD3, CD3 includes the natural sequence CD3 of all mammalian species, e.g., humans, dogs, cats, horses, and cattle. The amino acid sequences of human CD3 gamma, delta, and zeta are shown in the NCBI (www.ncbi.nlm.nih.gov / ) reference sequences NP_000064.1, NP_000723.1, and NP_932170.1, respectively.

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

[0180] The term "CD33" refers to the myeloid cell surface antigen CD33, a sialic acid-binding immunoglobulin-like lectin (Siglec) that mediates intercellular interactions and maintains immune cells in a quiescent state. The amino acid sequence of human CD33 (hCD33) is shown in UniProt (www.uniprot.org) Reference No. P20138.

[0181] The term "tumor-specific activation of T cells" refers to the preferential activation of T cells in tumor tissue compared to non-tumor tissue.

[0182] Furthermore, the term “peptide” also encompasses peptides modified by glycosylation, for example, and proteins comprising two or more polypeptide chains, each with a length of 4 to 600 amino acids, such as insulin and immunoglobulins, which are crosslinked by disulfide bonds. The term “chemical or biochemical agent” is intended to include any naturally occurring or synthetic compound that 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, retinopathy (particularly proliferative retinopathy), neurodegenerative disorders, infections, metabolic disorders, and neoplastic disorders. Any of the recombinant binding proteins described herein can be used in the preparation of agents for the treatment of such disorders, particularly neoplastic disorders. A “medical condition” may be characterized by inappropriate cell proliferation. A medical condition may be a state of hyperproliferation. The present invention relates, in particular, to a method for treating a medical condition, comprising the step of administering to a patient in need of such treatment a therapeutically effective amount of the recombinant binding protein of the present invention or the pharmaceutical composition described above. In a preferred embodiment, the medical condition is a neoplastic disorder. The term “neoplastic disorder,” as used herein, refers to an abnormal state or condition of cells or tissue characterized by rapidly growing cell proliferation or tumors. In one embodiment, the medical condition is a malignant neoplastic disorder. In one embodiment, the medical condition is cancer, preferably leukemia, more preferably acute myeloid leukemia. The term "therapeutic dose" refers to the amount sufficient to produce the 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 immunogenic binding ability. Such fragments and variants are also well known in the art and are commonly used both in vitro and in vivo. Therefore, the term "antibody" encompasses intact immunoglobulin molecules, e.g., Fab, Fab', F(ab')2, and single-chain V-region fragments (scFv), bispecific antibodies, chimeric antibodies, antibody-fusion polypeptides, and unconventional antibodies.

[0185] The terms “cancer” and “malignant” are used herein to refer to or describe a physiological condition in mammals typically characterized by unregulated cell proliferation. Cancer encompasses solid tumors and humoral tumors, as well as primary and metastatic tumors. A “tumor” includes one or more cancerous cells. A solid tumor typically also includes the tumor stroma. Examples of cancer include, but are not limited to, primary and metastatic cancers, lymphomas, blastomas, sarcomas, and leukemias, as well as any other epithelial and lymphoid malignancies. Brain cancer, bladder cancer, breast cancer, ovarian cancer, clear cell kidney cancer, head and neck squamous cell carcinoma, lung adenocarcinoma, lung squamous cell carcinoma, malignant melanoma, non-small cell lung cancer (NSCLC), ovarian cancer, pancreatic cancer, prostate cancer, renal cell carcinoma, small cell lung cancer (SCLC), triple-negative breast cancer, acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML), diffuse large lung cancer Examples include B-cell lymphoma (DLBCL), follicular lymphoma, Hodgkin lymphoma (HL), mantle cell lymphoma (MCL), multiple myeloma (MM), myelodysplastic syndrome (MDS), non-Hodgkin lymphoma (NHL), squamous cell carcinoma of the head and neck (SCCHN), chronic myeloid leukemia (CML), small lymphocytic lymphoma (SLL), malignant mesothelioma, colorectal cancer, or gastric cancer. [Examples]

[0186] The starting materials and reagents disclosed below are known to those skilled in the art, are commercially available, and / or can be prepared using well-known techniques.

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

[0188] molecular biology Unless otherwise specified, the methods will be carried out according to known protocols (see, for example, Sambrook J., Fritsch EF., and Maniatis T., "Molecular Cloning: A Laboratory Manual," Cold Spring Harbor Laboratory (1989), New York).

[0189] Designed Ankyrin Repeat Protein Library Methods for generating a designed ankyrin repeat protein library are described, for example, in U.S. Patent No. 7,417,130; Binz et al., "J.Mol.Biol.", Vol. 332, pp. 489-503, 2003; Binz et al. (2004), cited above. By such methods, a designed ankyrin repeat protein library having randomized ankyrin repeat modules and / or randomized capping modules can be constructed. For example, such a library may be assembled based on an immobilized N-terminal capping module (e.g., the N-terminal capping module of SEQ ID NO: 69, 70, or 71), or a randomized N-terminal capping module (e.g., by SEQ ID NO: 72), and an immobilized C-terminal capping module (e.g., the C-terminal capping module of SEQ ID NO: 73, 74, or 75), or a randomized C-terminal capping module (e.g., by SEQ ID NO: 76). Preferably, such a library is assembled such that the randomized positions of the repeat or capping modules do not contain any amino acids C, G, M, N (before the G residue), and P.

[0190] Furthermore, such randomized modules within such libraries may include additional polypeptide loop insertions having randomized amino acid positions. An example of such polypeptide loop insertions is a complement-determining region (CDR) loop library of an antibody or de novo-generated peptide library. 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, an ankyrin repeat protein library may contain variable-length (e.g., 1 to 20 amino acids) randomized loops (having immobilized and randomized positions) inserted into one or more β-turns of an ankyrin repeat domain. Such an N-terminal capping module in any of the ankyrin repeat protein libraries preferably has a RILLAA motif, a RILLKA motif, or a RELLKA motif (e.g., located at positions 19-24 in SEQ ID NO: 1), and such a C-terminal capping module in any of the ankyrin repeat protein libraries preferably has a KLN motif, a KLA motif, or a KAA motif (e.g., located at the last three amino acids in SEQ ID NO: 1). SEQ ID NOs. 69-71 provide examples of N-terminal capping modules containing a RILLAA motif, a RILLKA motif, or a RELLKA motif, and SEQ ID NOs. 73-75 provide examples of C-terminal capping modules containing a KLN motif, a KLA motif, or a KAA motif.

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

[0192] Examples of designed ankyrin repeat protein libraries, such as N2C and N3C designed ankyrin repeat protein libraries, are described (U.S. Patent No. 7,417,130, Binz et al., 2003, cited above; Binz et al., 2004, cited above). The numbers N2C and N3C describe the number of randomized repeat modules present between the N-terminal and C-terminal capping modules.

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

[0194] Example 1: Selection of a binding protein containing an ankyrin repeat domain with binding specificity to CD33 Using a ribosome display (Hanes, J. and Pluckthun, A., "PNAS" Vol. 94, pp. 4937-42 (1997)), many ankyrin repeat proteins with binding specificity to human CD33 (hCD33) (UniProt ID P20138) were selected from the DARPin® library, similar to the description cited above by Binz et al. (2004). Binding of the selected clones to recombinant human CD33 targets (full-length extracellular domain (ECD) of CD33's ECD, and splice variants of CD33's ECD) was evaluated by homogeneous time-resolved fluorescence (HTRF) of crude extracts, demonstrating the successful selection of hundreds of hCD33-specific binding proteins. For example, the ankyrin repeat domains of SEQ ID NOs. 61, 3, 5, 7-9, and 14-16 constitute the amino acid sequences of the selected binding proteins, including ankyrin repeat domains with binding specificity to 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 a library of ankyrin repeat proteins as described above and an established protocol (e.g., Zahn, C., Amstutz, P., and Pluckthun, A., "Nat. Methods" Vol. 4, pp. 69-79, 2007), with the biotinylated extracellular domain (ECD) of human CD33 as the target protein. 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 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) CD33, which contains only the constant domain. A standard four-round ribosome selection process was employed, with the selection pressure from round 1 to round 4 increased by decreasing the target concentration and increasing the wash stringency (Binz et al., 2004, cited above). A deselection strategy was applied in each round by using streptavidin and neutraavidin beads in conjunction with biotinylated non-CD33 Fc domains. The number of reverse transcription (RT)-PCR cycles after each selection round was consistently reduced from 45 to 28, adjusted to the yield for enrichment of the binding agent.

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

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

[0198] The selected clones specifically bind to human CD33, as indicated by the crude extract HTRF. Individual selected ankyrin repeat proteins that specifically bind to hCD33 in solution were identified by homogeneous time-resolved fluorescence (HTRF) assay using crude extracts of ankyrin repeat protein-expressing E. coli cells, using a standard protocol. Ankyrin repeat protein clones selected by ribosome display were cloned into derivatives of the pQE30 (Qiagen) expression vector (pMPDV045) containing the C-terminal CD3-specific ankyrin repeat domain, followed by a Flag tag, transformed into E. coli XL1-Blue (Stratagene), seeded on LB-agar (containing 1% glucose and 50 μg / ml ampicillin), and incubated overnight at 37°C. Single colonies were seeded (each clone in a single well) into 96-well plates containing 160 μl of growth medium (TB containing 1% glucose and 50 μg / ml ampicillin), and incubated overnight at 37°C with shaking at 800 rpm. 8.5 μL of overnight cultures were seeded into 150 μL of fresh TB medium containing 50 μg / mL ampicillin in a new 96-well plate. After incubation at 37°C and 700 rpm for 120 minutes, expression was induced with IPTG (final concentration 0.5 mM) for 4 hours. Cells were collected, the pellet was frozen overnight at -20°C, and then resuspended in 8 μL of B-PERII (Thermo Scientific) and incubated at room temperature for 1 hour with shaking (900 rpm). Subsequently, 160 μL of PBS was added, and cell fragments were removed by centrifugation (3220 g for 15 minutes).

[0199] Extracts from each lysed clone were applied to the wells of a 384-well plate at a 1:1000 dilution (final concentration) 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 at room temperature for 60 minutes. HTRF was read using a Tecan M1000 with an excitation wavelength of 340 nm and an emission filter of 665 ± 10 nm. Screening of hundreds of clones with such 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 may optionally contain an additional G, S, or GS sequence at their N-terminus.

[0202] Manipulation of additional ankyrin repeat proteins with binding specificity to hCD33

[0203] [Table 3]

[0204] Sequence IDs 2, 4, 6, and 10-13 are ankyrin repeat proteins with binding specificity to hCD33, manipulated based on the sequences of DARPin® protein #1 (Sequence ID 1), DARPin® protein #3 (Sequence ID 3), DARPin® protein #5 (Sequence ID 5), and DARPin® protein #9 (Sequence ID 9), respectively.

[0205] For SEQ ID NOs: 1 and 3, the sequences were modified to reduce the number of aromatic residues and alter the surface charge. In both N-terminal capping modules, the RILLAA motif was replaced with RILLKA, and aspartic acid (position 18) was replaced with leucine. In both C-terminal capping modules, glutamic acid (position 18) was replaced with glutamic acid. For SEQ ID NOs: 3, an additional phenylalanine (position 14) in the N-terminal capping module was replaced with valine. For SEQ ID NOs: 1, an additional tryptophan (position 7) in the N-terminal capping module was replaced with valine, and the EDIA motif in the second internal repeat (positions 18-21) was replaced with LEIV. The manipulated mutants did not alter T cell killing (evaluated in combination with other TAA and CD3-binding ankyrin repeat domains) compared to the parental version, more than 2x as measured in a standard LDH killing assay after incubation for 48 hours using pan-T cells and MOLM-13 cells in a 5:1 ratio.

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

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

[0208] Expression of CD33-specific ankyrin repeat proteins For further analysis, selected clones exhibiting specific human CD33 binding in crude cell extract HTRF as described above were expressed in E. coli cells using a His tag (SEQ ID NO: 85) fused to their N-terminus for easy purification. The expressed proteins were purified according to a standard protocol. 0.11 ml of static overnight culture (TB, 1% glucose, 50 mg / l ampicillin; 37°C) was inoculated into 0.99 ml of culture (TB, 50 mg / l ampicillin, 37°C) in a 96-deep-well plate. After incubation at 37°C (700 rpm) for 2 hours, the cultures were induced with 0.5 mM IPTG and incubated at 37°C for 6 hours with shaking (900 rpm). Cells were collected, the pellet was frozen overnight at -20°C, and then resuspended in 50 μL of B-PERII (Thermo Scientific) supplemented with DNAase I (200 units / ml) and lysozyme (0.4 mg / ml). The cells were incubated at room temperature for 1 hour with shaking (900 rpm). Subsequently, 60 μL of low-salt sodium phosphate buffer was added, and cell debris was removed by centrifugation (3220 g for 15 minutes). After pooling eight individual expressions in total, cell debris was removed by centrifugation (3200 g for 60 minutes, 4°C). The supernatant was filtered using a MultiScreen filter plate (Millipore), purified using a 96-well Thermo HisPur cobalt spin plate, and the protein solution was rebuffered in PBS using a 96-well Thermo Zeba spin desalting plate. The purified proteins were 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 proteins, novel mutants with very high affinity and / or very low off-rates to the target protein were generated using affinity maturation. Thus, one of the initially identified CD33-specific binding proteins, DARPin® protein #2 ("parent" binding protein), was selected as a suitable starting point for affinity maturation. The affinity maturation procedure involved saturation mutagenesis at each randomized position of the ankyrin repeat domain used as the starting point. Sequences generated by the affinity maturation procedure were screened for lower off-rates by competitive HTRF. Briefly, single-amino acid point mutation mutants were generated on the parent plasmid (pMCHE1190) by standard QuikChange PCR using a primer with a single NNK degenerate codon to introduce all 20 amino acids to the potential binding site. Crude extracts (CE) of the ankyrin repeat protein containing the N-terminal His tag (SEQ ID NO: 85) were incubated with the biotinylated target, followed by the addition of excess unflag-tagged parent CD33-specific binding protein, and the HTRF signal was measured over time. Beneficial mutations identified based on higher HTRF signaling than the parent clone were combined in the binding protein using protein engineering. In this way, affinity mature protein #29 and DARPin® protein #30 were generated from DARPin® protein #2.

[0210] Next, this affinity-matured CD33-specific binding domain was subcloned into a derivative of the pQE30(Qiagen) expression vector to encode the N-terminal His tag (SEQ ID NO: 85), and subsequently, expression constructs encoding the CD33-specific binding domain (SEQ ID NO: 77 or 78), the peptide linker (SEQ ID NO: 65), and the C-terminal CD3-binding domain (SEQ ID NO: 57) were obtained. These constructs in a T cell engager format were expressed in E. coli cells and purified using their His tags according to a standard protocol. The proteins were tested in dose-dependent in vitro T cell activation and tumor cell killing assays using healthy donor PBMCs as effector cells (E) and primary T cells isolated from Molm-13-N1 tumor cells (5:1 E:T ratio) as target cells (T). Assay incubation of co-cultures for 48 hours and analysis by flow cytometry and LDH release were performed. All constructs were expressed in E. coli cells and purified using their His tags according to a standard protocol.

[0211] Example 2: Dissociation constant (K) of recombinant ankyrin repeat protein with binding specificity to human CD33 determined by surface plasmon resonance (SPR) analysis. D ) decision The binding affinity of purified ankyrin repeat proteins to recombinant human CD33 targets was analyzed using a ProteOn instrument (BioRad) and measured according to standard procedures known to those skilled in the art. For this purpose, DARPin® proteins #1, #3, #5, #7-#9, and #14-#16 of the present invention were subcloned and expressed in derivatives of the pQE30 (Qiagen) expression vector as described above to obtain 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] In short, SPR measurements were performed using a ProteOn XPR36 instrument (BioRad). The electrophoresis buffer was PBS pH 7.4 (PBST) containing 0.005% Tween20®. The full-length bio.hCD33 target (SEQ ID NO: 60) was immobilized to a level of 540 RU on an NLC chip (BioRad), and the spliced ​​variant (SEQ ID NO: 61) was immobilized to a level of 560 RU. The interaction of purified CD33-specific binding protein to the full-length and spliced ​​variants of CD33 ECD in a 96-well TCE format was measured by injecting 50 nM binding protein with association for 120 seconds and dissociation for 1200 seconds at a constant flow rate of 100 μL / min. The target was regenerated between individual measurements using 2 M MgCl2. The signal was double-referenced against a control lane treated with electrophoresis buffer (PBST).

[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 CD3-specific ankyrin repeat domains). D The value is shown. Dissociation constant (K D The K of the selected ankyrin repeat protein and the binding interaction with human cleavage and full-length CD33 was calculated from the estimated on-rate and off-rate using standard procedures known to those skilled in the art. D The value was determined to be within the range of 0.47 to 17 nM by single-trace SPR (see Table 2a). The values ​​in Table 2a are the average of multiple iterations.

[0214] [Table 4]

[0215] Furthermore, the binding affinity of two additional purified ankyrin repeat proteins to recombinant human CD33 targets was measured and analyzed for DARPin proteins #1, #3, #6, #7-#9, and #14-#16 using the same procedure as above. Briefly, bio.hCD33 was coated onto an 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 at a constant flow rate of 100 uL / min, with a 120-second association phase and a 1200-second dissociation phase. Targets were regenerated with 4 M MgCl2. Signals were double-referenced against control lanes treated with electrophoresis buffers 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 values ​​of the CD33-specific ankyrin repeat proteins of the present invention that bind to the full-length target of bio.hCD33. D Shows the value. K D The values ​​were calculated from estimated on-rates and off-rates using standard procedures known to those skilled in the art. The values ​​in Table 2b are the averages of multiple iterations.

[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 protein in female BALB / c mice To determine whether the CD33-specific ankyrin repeat domain of the present invention can have an appropriate serum half-life in vivo for use in the development of therapeutic agents, the pharmacokinetic profiles of DARPin® protein #2, DARPin® protein #29, and DARPin® protein #30 were analyzed in mice. For this purpose, the DARPin construct was subcloned and expressed as described above into derivatives of a pQE30 (Qiagen) expression vector encoding one of the following at its C-terminus: an N-terminal His tag (SEQ ID NO: 85), an HSA-binding ankyrin repeat domain for half-life extension (SEQ ID NO: 53), a peptide linker (SEQ ID NO: 65, etc.). For example, an expression vector encoding the following ankyrin repeat protein was constructed.

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

[0220] The mice were divided into two groups, each containing an equal number of animals. Four serum samples were collected from each mouse. Blood samples for pharmacokinetic studies were collected at 5 minutes, 4 hours, 24 hours, 48 ​​hours, 76 hours, 96 hours, and 168 hours after compound administration. The blood was allowed to coagulate at room temperature, then centrifuged to collect the serum.

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

[0222] Apply 100 μL of diluted serum samples (1:20 to 1:312500 in a 1:5 dilution step) or ankyrin repeat protein standard curve samples (0 and 50 to 0.0008 nmol / L in a 1:3 dilution step) at room temperature for 2 hours with shaking at 450 rpm. Wash the plate as described above.

[0223] Then incubate the wells with 100 μL of mouse anti-RGS-His-HRP IgG (Ab06, 1:2000 in PBST-C) and incubate for 1 hour at room temperature with shaking at 450 rpm. Wash the plate as described above. Develop the ELISA by using 100 μL / well of TMB substrate solution for 5 minutes and stopping by adding 100 μL of 1 mol / L H2SO4. Calculate the difference in absorbance at 450 nm and absorbance at 620 nm. Samples are measured in duplicate on two different plates.

[0224] Pharmacokinetic analysis Pharmacokinetic data analysis is performed at Molecular Partners using version 7.0 of the WinNonlin program as part of Phoenix 64, Pharsight, North Carolina. Calculation of pharmacokinetic parameters based on mean concentration-time data of animals administered via intravenous bolus injection is performed using non-compartmental 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] The maximum serum concentrations (Cmax) and their time to occurrence (Tmax) are obtained directly from the serum concentration-time profile. The area under the serum concentration-time curve (AUCinf) is determined by the linear trapezoidal rule up to the last sampling point (Tlast) and extrapolation to infinity assuming a single exponential decrease in the terminal phase. Extrapolation to infinity is performed using Clast / λz, where λz is the final rate constant estimated by log-linear regression, and Clast is the concentration estimated at Tlast by the final log-linear regression. The total serum clearance (Cl_pred) and apparent terminal phase 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 as follows: Vss = iv dose·AUMCinf / (AUCinf)². AUMCinf represents the total area under the first instant of the drug concentration-time curve extrapolated to infinity, using the same extrapolation procedure described for calculating AUCinf. To calculate the PK parameter based on a given concentration at a dose of nmol / L, the given dose values ​​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 the three ankyrin repeat proteins of the present 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 domain of the present invention, when combined with a half-life extension portion, such as a serum albumin-specific binding domain, can achieve an appropriate serum half-life in vivo, making it useful for the development of therapeutic agents.

[0228] Example 4: Determination of binding of the ankyrin repeat protein of the present invention to CD33-expressing tumor cells. The binding of the present invention's binding protein 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 in 96-well plates at a rate of 100,000 cells per well. Single-specificity formats of DARPin protein #2, DARPin protein #29, and DARPin protein #30 were titrated at a dilution ratio of 1:5, starting at 2000 nM. Tumor cells were resuspended in diluted DARPin protein and incubated at 4°C for 60 minutes. The assay was performed in PBS containing 2% fetal bovine serum but without 20 μM human serum albumin (HSA). After washing twice with phosphate-buffered saline (PBS), DARPin® protein-specific tumor cell binding was detected by adding 2 μg / mL of unlabeled primary anti-rabbit DARPin® antibody (anti-rabbit 1-1-1 antibody, CePower). Next, the cells were incubated at 4°C for at least 30 minutes. Afterward, the 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, the cells were washed twice and resuspended in Cytofix fixation buffer (BD Biosciences) at room temperature (RT) for 15 minutes. 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 (Figure 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 protein of the present invention binds to CD33 expressed on the cell surface with an EC50 of approximately 2 nM or less.

[0231] Example 5: Evaluation of the specificity and efficacy of the CD33-specific ankyrin repeat protein of the present invention by target-specific short-term T cell activation assay. The specificity and efficacy of the above-described exemplary CD33-specific ankyrin repeat proteins of the present invention were evaluated in an in vitro short-term T cell activation assay by FACS measurement of CD25 activation markers 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 a CD3-specific binding domain (SEQ ID NO: 57) in addition to the aforementioned CD33-specific ankyrin repeat domain, and are shown as DARPin protein #33, DARPin protein #31, and DARPin protein #32, respectively.

[0232] Therefore, 100,000 purified pan-T effector cells and 20,000 Molm-13 target cells per well were co-incubated in two sets with serial dilutions of selected DARPin protein in the presence of 600 μM human serum albumin at 37°C for 48 hours (E:T ratio 5:1). After 48 hours, the cells were washed and stained at 4°C for 30 minutes 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) antibody. After washing and fixation, the cells were analyzed using an Attune NxT (ThermoFisher) instrument. T cell activation was assessed by measuring CD25+ cells on Live / Dead-negative and CD8+ gated T cells. FACS data was analyzed using FlowJo software, and the data was plotted using GraphPad Prism8 (3-PL-fit). Figure 3 shows short-term T cell activation induced by DARPin protein #33, DARPin protein #31, and DARPin protein #32, as measured by the activation marker CD25. All of the CD33-specific binding proteins of the present invention tested were able to bind to CD733 expressed on tumor cells in T cell engager format and activate T cells.

[0233] Example 6: Evaluation of the specificity and efficacy of the CD33-specific ankyrin repeat protein of the present 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 present invention, DARPin protein #2, DARPin protein #29, and DARPin protein #30, in T cell engager formats (i.e., DARPin protein #33, DARPin protein #31, and DARPin protein #32, respectively) were also evaluated using an in vitro short-term cytotoxicity assay to measure LDH release. For this purpose, 100,000 purified pan-T effector cells and 20,000 Molm-13 target cells / well were co-incubated in double sets with serial dilutions of the indicated T cell engager proteins in the presence of 600 μM human serum albumin at 37°C for 48 hours (E:T ratio 5:1). After 48 hours 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, 30-minute incubation). Absorbance was measured from 492nm to 620nm using a TECAN infinite M1000Pro reader. OD values ​​were plotted using a GraphPad Prism8.

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

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

[0236] The competition for binding to human CD33 (purchased from Evitria) among monovalent CD33-specific binding proteins DARPin® protein #1, DARPin® protein #9, and DARPin® protein #14, as well as AMG330 analogs, was evaluated by HTRF and competitive ELISA.

[0237] First, crude extracts of three CD33-specific binding proteins, DARPin® protein #1, DARPin® protein #9, and DARPin® protein #14, were used for binding experiments to full-length (V / C2 domain) and cleaved CD33 (C2 domain) using HTRF. Binding to the CD33 target was detected using Strep-Tb and anti-His-D2HTRF reagents. Briefly, crude extracts of DARPin® protein #1, DARPin® protein #9, and DARPin® protein #14 were diluted 1:250 (finally 1:1000) with PBSTB and added to 384-well plates (PerkinElmer, 6008280). Next, the biotinylated targets hCD33-Fc(kih)-Avi (full-length CD33; purchased from Evitria) and hCD33-C2-Fc(kih)-Avi (cleaved CD33; purchased from Evitria) were each diluted to 8 nM (final 6 nM) and mixed with the HTRF reagents Mab Anti-6His-d2 and Strep-Tb, both diluted 1:200 (final 1:400). As shown in Figure 5, all three CD33-specific binding proteins, DARPin® protein #1, DARPin® protein #9, and DARPin® protein #14, showed binding to full-length CD33, but only DARPin® protein #9 bound to the cleaved 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, competitive ELISA experiments were performed using the CD33-binding benchmark molecule AMG330 in a similar manner. An AMG330 analog that binds to the apex of the V domain, as shown by Friedrich et al. (2014), was immobilized on microplates. Biotinylated CD33 targets, as well as 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 directly measured via streptavidin covalently bound to peroxidase. Briefly, Nunc MaxiSorp 96-well plates were coated overnight with 10 nM of DARPin® protein #1, DARPin® protein #9, and DARPin® protein #14, as well as 5 nM of an AMG330 analog. ELISA plates were washed three times and blocked with PBSTC (PBS containing 0.1% (v / v) Tween20® and 0.25% casein) at 450 rpm for 4 hours and 15 minutes. Meanwhile, 500 nM of the competing substances DARPin® protein #1, DARPin® protein #9, and DARPin® protein #14 were pre-incubated with 20 nM bio.hCD33 in a 1:1 ratio at room temperature for 2 hours. Bio.hCD33 without the competing substances was included as a positive control. The pre-incubated samples were then added to coated MaxiSorp plates and incubated at room temperature and 450 rpm for 30 minutes. After washing the plates three times with PBST, the target was detected using streptavidin-POD antibody (Roche, catalog number: 11 089 153 001). For detection, a newly prepared TMB buffer (30 mM citrate buffer, pH 4.1, 5% (v / v) TMB solution (Carl Roth GmbH) and 0.16% H2O2) was added, and the reaction was stopped with 1 M H2SO4. Absorbance was measured at OD450 and referenced to OD620 using a Sunrise microplate reader (Tecan). Data were analyzed by subtracting the buffer (PBS) value. A GraphPad Prism was used for analysis.

[0239] As expected, C2 domain-binding DARPin® protein #9 did not exhibit competitive binding similar to AMG330. Both V domain-binding molecules, DARPin® protein #1 and DARPin® protein #14, resulted in complete or partial inhibition of the ELISA signal due to competition. Therefore, DARPin® protein #1 binds to the V domain and exhibits partial competition with both AMG330 and DARPin® protein #14. In addition, DARPin® protein #14 also binds to the V domain and completely competes for binding to AMG330, while DARPin® protein #9 binds to the C2 domain, enabling co-binding of AMG330 to CD33 (Figure 6).

[0240] Example 8: In vivo efficacy evaluation of exemplary multi-domain TCE-binding proteins containing DARPin® protein #29 in PBMC humanized mice and MOLM-13 tumor models. DARPin® protein #29, formatted as a multi-domain T cell engager-binding protein, was tested in a humanized mouse model of peripheral blood mononuclear cells (PBMCs) containing the tumor cell line MOLM-13. The test included two ankyrin repeat domains with binding specificity to human serum albumin, two ankyrin repeat domains with binding specificity to tumor-associated antigen 1 (TAA1) and tumor-associated antigen 2 (TAA2), respectively, and one additional ankyrin repeat domain with binding specificity to CD3. 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 a standard rodent micro-isolator cage (room temperature 20±1°C, relative humidity 50±10%, and a 12-hour light-dark cycle). Mice were provided with irradiated food and bedding, and 0.22 μm filtered drinking water. All experiments were conducted with authorization from the cantonal and federal veterinary authorities in accordance with Swiss animal protection laws.

[0241] Mice were intraperitoneally injected with PBMC (5 × 10 6 PBMC prepared from buffy coats from two different donors) 2 days prior to xenografting of cancer cells. MOLM-13 cells were xenografted subcutaneously (s.c.) into the right flank of the mice. Two hPBMC donors were used. Treatments were intravenously (i.v.) injected 4 days after cancer cell transplantation. Treatments were performed as follows: - The DARPin® protein #29 in a multi-domain TCE format, or vehicle, was administered i.v. at 0.5 mg / kg three times a week for 2 weeks Tumor size was evaluated by caliper measurement. Tumor volume was calculated using the following formula: Tumor volume (mm 3 ) = 0.5 × length × width 2 .

[0242] As seen in Figure 7 (A - B), the DARPin® protein #29 in a multi-domain TCE format showed good efficacy with respect to inhibition of 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 multi-domain TCE-binding proteins comprising 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 multi-domain T cell engager-binding protein containing two ankyrin repeat domains with binding specificity to human serum albumin, two ankyrin repeat domains with binding specificity to tumor-associated antigen 1 (TAA1) and tumor-associated antigen 2 (TAA2), respectively, and one ankyrin repeat domain with binding specificity to 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, so that 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 CD33 (but not TAA1 and TAA2) expression was 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 serial dilutions of their respective multi-domain T cell engager-binding proteins in the presence of 20 μM human serum albumin at 37°C for 48 hours (E:T ratio 5:1). After 48 hours, the cells were washed and stained at 4°C for 30 minutes with 1:1,000 Live / Dead Green (Thermo Fisher), 1:400 mouse anti-human CD8 Pacific Blue (BD), and 1:100 mouse anti-human-CD25 PerCP-Cy5.5 (eBiosciences) antibody. After washing and fixation, the cells were analyzed using a FACS Canto II (BD) instrument. T cell activation was assessed by measuring CD25+ cells on Live / Dead-negative and CD8+ gated T cells. We will analyze the FACS data using FlowJo software and plot the data using GraphPad Prism8 (3-PL-fit).

[0245] The results demonstrate that both exemplary multi-domain T cell engager-binding proteins, including either DARPin® protein #29 (Figure 8A) or DARPin® protein #30 (Figure 8B), were able to potently activate T cells in the presence of wild-type expressing Molm-13 tumor cells (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 T cell activation was significantly reduced when CD33 expression was eliminated in Molm-13 tumor cells (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) are functional in relation to exemplary multi-domain T cell engager-binding proteins and significantly contribute to the overall potency of multispecific T cell engager proteins due to their ability to specifically bind to CD33 on target cells.

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

[0247] Those skilled in the art will recognize, or can confirm by routine experimentation, many equivalents to the particular aspects of the present invention described herein. Such equivalents are intended to be covered by the following claims.

[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, wherein the ankyrin repeat domain has binding specificity to human CD33, and the ankyrin repeat domain comprises an amino acid sequence having at least 90% amino acid sequence identity with any one of SEQ ID NOs: 1-16 and SEQ ID NOs: 77-78.

2. The aforementioned ankyrin repeat domains have a dissociation constant (K) of less than 100 nM in human CD33 in PBS. D ) and optionally select a K between 0.1 nM and 100 nM. D Joins with, and / or The ankyrin repeat domain has an EC range of 0.1 nM to 10 nM. 50 The recombinant binding protein according to claim 1, which binds to human CD33.

3. It further comprises a binding portion having binding specificity to a target expressed on immune cells, The recombinant binding protein according to claim 1 or 2, wherein the immune cell is a T cell and the target expressed on the immune cell is CD3.

4. The recombinant binding protein according to claim 3, wherein the binding portion having binding specificity to a target expressed on immune cells is an ankyrin repeat domain having binding specificity to human CD3.

5. The binding portion having binding specificity to a target expressed on immune cells is an ankyrin repeat domain having binding specificity to human CD3, and the ankyrin repeat domain having binding specificity to human CD3 contains an amino acid sequence that is at least 95% identical to any one of SEQ ID NOs. 55 to 59, or The recombinant binding protein according to claim 3, wherein the ankyrin repeat domain having binding specificity to human CD3 comprises one amino acid sequence from SEQ ID NOs. 55 to 59.

6. The ankyrin repeat domain having binding specificity to human CD33, and the binding portion having binding specificity to a target expressed on immune cells, are covalently bonded to a peptide linker. In the optional selection, the peptide linker is a peptide linker rich in proline-threonine. The recombinant binding protein according to claim 3, wherein, optionally, the amino acid sequence of the peptide linker has a length of 1 to 50 amino acids.

7. The binding protein further contains a half-life extension portion, The recombinant binding protein according to claim 1, wherein, optionally, the half-life extension portion is an ankyrin repeat domain having binding specificity to human serum albumin.

8. The ankyrin repeat domain having binding specificity to human serum albumin contains an amino acid sequence that is at least 95% identical to any one of the amino acid sequences of SEQ ID NOs. 52 to 54, or The recombinant binding protein according to claim 7, wherein the ankyrin repeat domain having binding specificity to human serum albumin comprises one amino acid sequence from SEQ ID NOs. 52 to 54.

9. The recombinant binding protein according to claim 1, wherein the binding protein further comprises at least one binding site having binding specificity to a target expressed in tumor cells, and the target expressed in tumor cells is different from human CD33.

10. A nucleic acid encoding the recombinant binding protein described in 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 pharmaceutically acceptable carrier and / or diluent.

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

13. The pharmaceutical composition according to claim 11, for use in the treatment of cancer, and optionally for use in the treatment of cancer characterized by humoral tumors.

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

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