CN117157327A

CN117157327A - Novel multispecific T cell conjugates based on DARPIN

Info

Publication number: CN117157327A
Application number: CN202280019430.0A
Authority: CN
Inventors: M·比安奇; N·雷什克; S·格利姆; C·瑞驰恩; B·施莱思; V·勒维斯基
Original assignee: Molecular Partners AG
Current assignee: Molecular Partners AG
Priority date: 2021-03-09
Filing date: 2022-03-09
Publication date: 2023-12-01

Abstract

The present application relates to multi-specific recombinant proteins comprising binding agents having binding specificity for different targets, such as CD3, CD33, CD123 and CD70. Furthermore, the present application relates to nucleic acids encoding such multispecific proteins, pharmaceutical compositions comprising such proteins or nucleic acids, and the use of such binding proteins, nucleic acids or pharmaceutical compositions in methods for treating or diagnosing diseases, such as cancer, e.g., acute Myelogenous Leukemia (AML), in mammals, including humans.

Description

Novel multispecific T cell conjugates based on DARPIN

Cross-reference to related patent applications

The present application claims the priority of US 63/158539 submitted at 3.9 in 2021, US 63/172,973 submitted at 4.9 in 2021 and US 63/265,184 submitted at 12.9 in 2021. The disclosures of these patent applications are incorporated by reference herein in their entirety for all purposes.

Technical Field

The present application relates to multi-specific recombinant proteins comprising binding agents having binding specificities for different targets (e.g., CD3, CD33, CD70 and CD 123). Furthermore, the present application relates to nucleic acids encoding such multispecific proteins, pharmaceutical compositions comprising such proteins or nucleic acids, and the use of such binding proteins, nucleic acids or pharmaceutical compositions in methods for treating or diagnosing diseases such as cancer (e.g., acute Myelogenous Leukemia (AML)) in mammals, including humans.

Background

Acute Myelogenous Leukemia (AML) is a heterogeneous and complex malignant disease characterized by rapid cell proliferation, invasive clinical processes, and generally high mortality. AML treatment resistance remains the leading cause of acute leukemia-related death (wine and Stone, ther Adv Hematol;2019; 10). While standard regimens employing chemotherapy remain the primary treatment for worldwide use, recent advances in immunotherapy have provided effective treatment options for chemotherapy-resistant AML. Such immunotherapeutic approaches include monoclonal antibodies, bispecific antibodies, and T cells expressing chimeric antigen receptors (CAR-T cells).

Monoclonal antibody-based therapies include mainly anti-CD 33 antibodies or anti-CD 123 antibodies, either as monotherapy or conjugated with cytotoxic agents (wine and Stone, ther Adv Hematol;2019; 10). However, these drugs have shown significant side effects or low efficacy. For example, treatment with gemtuzumab ozogamicin (gemtuzumab ozogamicin), a humanized anti-CD 33 monoclonal antibody conjugated to the antibiotic calicheamicin, has resulted in significant hematological and hepatotoxicity, while treatment with talcotuzumab (a humanized anti-CD 123 monoclonal antibody) has not provided effective therapeutic benefit. Currently, another human monoclonal antibody targeting CD70 is undergoing clinical trials (Gu Tuo bead mab), and despite the initial findings that appear promising, experts still express interest in their potential safety profile (see, e.g., www.clinicaltrialsarena.com/program/argexs-cusatuzumab-in-previous-untreated-aml-draw-variied-expert-formats).

CAR-T cell therapy is a method that strongly influences lymphoid malignancy management. Although there is great interest in applying this technology also to AML, in practice this has proven challenging. Regarding monoclonal antibodies, CD33 and CD123 have been considered as the most promising targets for CAR-T cell therapy in AML. However, preclinical models of these targets show extensive side effects on non-AML cells (on-target/off-tumor toxicity), and Cytokine Release Syndrome (CRS) is another recognized side effect.

Using two pairsT cell directed killing of tumor cells by specific antibodies is another recent therapeutic tool that has been used to treat various cancer types, including AML. These T cell conjugate (TCE) bispecific antibodies comprise two different variable regions: one binds to the T cell receptor complex subunit CD3 and the other binds to a tumor cell surface antigen. Binding of TCE to these two targets provides a functional link between cells, leading to T cell activation and cytotoxic activity against tumor cells, bypassing normal TCR-MHC interactions (Ellerman, methods;154:102-117 (2019)). AMG330 is a bispecific antibody against CD3 and CD33, which can cause T cell cytotoxicity against AML cells. Similarly, rituximab (flotatuzumab) is a dual affinity, re-targeting antibody Which uses two separate polypeptides, the heavy chain variable domain of one antibody is fused to the light chain variable domain of the other antibody to join CD3 on T cells and CD123 on AML cells.

However, such antibody-based TCE therapeutics suffer from a number of drawbacks such as high production costs and inability to target a variety of tumor surface markers, and they can cause serious side effects such as Cytokine Release Syndrome (CRS) (shimabakuro-Vornhagen et al, J Immunother Cancer;6 (1): 56 (2018); labrjin et al, nat Rev Drug Discov;18 (8): 585-608 (2019)) and/or targeted/extra-tumor toxicity (Weiner et al, cancer res.;55 (20); 4586-4593 (1995); weiner et al Cancer immunol 42 (3); 141-150 (1996)). Antibody-based T cell conjugates generally exhibit greater than 1000-fold higher affinity for CD3 when compared to native TCR-MHC interactions (Wu et al, pharmacol Ther;182:161-75 (2018); WO2014/167022; junnila et al, cancer Res.;19:5561-71 (2014); yang et al, J Immunol Aug.;15 (137): 1097-100 (1986)). This high affinity is associated with lower T cell activation and tumor cell killing efficiency (Bortoletto et al, eur. J. Immunol.,32;11:3102-3107 (2002); ellerman, methods;154:102-117 (2019); mandikian et al, mol. Cancer Ther.;17 (4); 776LP-785 (2018); vafa et al, frontiers in Oncology;10:446 (2020)). Furthermore, down-regulation of tumor surface markers targeted by TCE can lead to tumor resistance to TCE therapy.

Acute Myelogenous Leukemia (AML) is one type of cancer that exemplifies in many ways the challenges of cancer therapies and the shortcomings of currently available cancer therapies, as discussed above. For AML, the medical need for high mortality remains high and the treatment of recurrent or refractory AML continues to be therapeutically challenging. Currently, excessive Antibody Drug Conjugate (ADC) and targeted T cell conjugate therapies have entered clinical development of AML, but those therapies are often accompanied by dose-limiting toxicity. The greatest challenges appear to be limited target specificity and over-stimulation of the immune system, leading to, for example, bone marrow toxicity and Cytokine Release Syndrome (CRS), respectively. Furthermore, resistance to targeted cancer therapies may evolve due to down-regulation of individual targets in tumor cells. Thus, new methods or drug molecules are needed to address these challenges of cancer therapies and the shortcomings of currently available cancer therapies, as exemplified in AML.

Thus, there remains a need for new molecules, such as immune cell conjugates, e.g., TCE proteins, that have beneficial properties that address one or more of the shortcomings of the therapeutic proteins previously described. Such novel molecules may be useful in therapeutic methods for treating neoplastic diseases (e.g., acute myelogenous leukemia).

Disclosure of Invention

The present invention provides a recombinant protein comprising a first binding agent that specifically binds to a protein expressed on the surface of an immune cell and at least two binding agents that specifically bind to tumor-associated antigens, wherein the two binding agents specifically bind to different tumor-associated antigens. Furthermore, the invention provides nucleic acids encoding such binding proteins and pharmaceutical compositions comprising such binding proteins or nucleic acids. The invention also provides the use of such binding proteins, nucleic acids or pharmaceutical compositions in methods for the localized activation of immune cells, such as T cells, in a tumor environment, and for the treatment of diseases such as acute myeloid leukemia in mammals, including humans.

The recombinant proteins of the invention target at least two different tumor-associated antigens (TAAs). When the at least two TAAs are present in tumor cells simultaneously, the recombinant proteins of the invention may exhibit significantly increased tumor specificity by increased avidity. Also due to such increased avidity, the multispecific proteins of the present invention may have a greater window of potency than the respective single target-specific controls. Furthermore, the proteins of the invention can induce significantly less cytokine release than current therapeutic molecules, indicating an improved therapeutic window. Moreover, by targeting the at least two different TAAs, the proteins of the invention may be more resistant to tumor resistance developing as a result of down-regulation of the target.

In one aspect, the invention provides a recombinant protein, wherein the recombinant protein comprises a first binding agent that specifically binds to a protein expressed on the surface of an immune cell and at least two binding agents that specifically bind to tumor-associated antigens, wherein the two binding agents specifically bind to different tumor-associated antigens. In one aspect, the invention provides such a recombinant protein, wherein the recombinant protein is capable of simultaneously binding the protein expressed on the surface of an immune cell and the two different tumor-associated antigens.

In one aspect, the invention provides such recombinant proteins wherein the tumor-associated antigen specifically bound by the two binding agents is co-expressed in tumor cells.

In one aspect, the invention provides a recombinant protein, wherein the recombinant protein comprises a first binding agent that specifically binds to a protein expressed on the surface of an immune cell and at least three binding agents that specifically bind to tumor-associated antigens, wherein the three binding agents specifically bind to different tumor-associated antigens. In one aspect, the invention provides such a recombinant protein, wherein the recombinant protein is capable of simultaneously binding the protein expressed on the surface of an immune cell and the three different tumor-associated antigens.

In one aspect, the invention provides such recombinant proteins wherein the tumor-associated antigens specifically bound by the three binding agents are co-expressed in tumor cells.

In one aspect, the invention provides such recombinant proteins, wherein the tumor cell is a tumor cell from a liquid tumor, preferably wherein the liquid tumor is leukemia, more preferably wherein the leukemia is Acute Myelogenous Leukemia (AML).

In one aspect, the invention provides a recombinant protein wherein said recombinant protein is capable of having a lower dissociation constant (K _D ) Binding to a surface displaying the tumor-associated antigen.

In one aspect, the invention provides such a recombinant protein, wherein the surface displaying the tumor-associated antigen is the surface of the tumor cell.

In one aspect, the invention provides such a recombinant protein, wherein the immune cell is a T cell.

In one aspect, the invention provides such a recombinant protein wherein the protein expressed on the surface of an immune cell is a protein that is part of a T cell receptor complex, preferably the part of the T cell receptor complex is CD3.

In one aspect, the invention provides such a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides such a recombinant protein wherein the first binding agent is a designed ankyrin repeat domain having binding specificity for the protein expressed on the surface of an immune cell.

In one aspect, the invention provides such a recombinant protein wherein the first binding agent is a designed ankyrin repeat domain having binding specificity for CD 3.

In one aspect, the invention provides a recombinant protein wherein said ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID NOs 1 to 5 (preferably SEQ ID NO: 2). The invention also provides such recombinant proteins wherein said ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID No. 3.

In one aspect, the invention provides such a recombinant protein, wherein the binding agent that specifically binds to a tumor-associated antigen is selected from the group consisting of (i) a second binding agent that specifically binds to a first tumor-associated antigen (TAA 1), (ii) a third binding agent that specifically binds to a second tumor-associated antigen (TAA 2), and (iii) a fourth binding agent that specifically binds to a third tumor-associated antigen (TAA 3).

In one aspect, the invention provides such a recombinant protein, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides such a recombinant protein, wherein the second binding agent is a designed ankyrin repeat domain having binding specificity for the TAA1, preferably wherein TAA1 is CD33.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID NOs 15 and 67 to 70. The present invention provides such recombinant proteins wherein said ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID NOs 15, 67 to 70 and 111 to 112 (preferably SEQ ID NO: 111).

In one aspect, the invention provides such a recombinant protein, wherein the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides such a recombinant protein, wherein the third binding agent is a designed ankyrin repeat domain having binding specificity for the TAA2, preferably wherein TAA2 is CD123.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID NOs 6 and 65 to 66. The present invention provides such recombinant proteins wherein said ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID NOs 6, 65 to 66 and 102 to 106 (preferably SEQ ID NO: 105).

In one aspect, the invention provides such a recombinant protein, wherein the fourth binding agent is a designed ankyrin repeat domain having binding specificity for said TAA3, preferably wherein TAA3 is CD70.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD70 comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID No. 64. The present invention provides such recombinant proteins wherein said ankyrin repeat domain having binding specificity for CD70 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID nos. 64 and 107 to 110 (preferably SEQ ID No. 109).

In one aspect, the invention provides a recombinant protein wherein said ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID NOs 1 to 5 (preferably SEQ ID NO: 2); wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID NOs 15, 67 to 70; and wherein said ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID NOs 6, 65 to 66. The present invention provides such recombinant proteins wherein said ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID NOs 1 to 5 (preferably SEQ ID NO: 3); wherein the ankyrin repeat domain with binding specificity for CD33 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID NOs 15, 67 to 70 and 111 to 112 (preferably SEQ ID NO: 111); and wherein said ankyrin repeat domain with binding specificity for CD123 comprises an amino acid sequence having at least 85% identity with any of the amino acid sequences of SEQ ID NOs 6, 65 to 66 and 102 to 106 (preferably SEQ ID NO 105).

In one aspect, the invention provides a recombinant protein wherein said ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID NOs 1 to 5 (preferably SEQ ID NO: 2); wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID NOs 15, 67 to 70; and wherein said ankyrin repeat domain having binding specificity for CD70 comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID No. 64. The invention also provides such recombinant proteins wherein said ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID NOs 1 to 5 (preferably SEQ ID NO: 3); wherein the ankyrin repeat domain with binding specificity for CD33 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID NOs 15, 67 to 70 and 111 to 112 (preferably SEQ ID NO: 111); and wherein said ankyrin repeat domain having binding specificity for CD70 comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID nos. 64 and 107 to 110 (preferably SEQ ID No. 109).

In one aspect, the invention provides a recombinant protein wherein said ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID NOs 1 to 5 (preferably SEQ ID NO: 2); wherein the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID NOs 6, 65 to 66; and wherein said ankyrin repeat domain having binding specificity for CD70 comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID No. 64. The invention also provides such recombinant proteins wherein said ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID NOs 1 to 5 (preferably SEQ ID NO: 3); wherein the ankyrin repeat domain with binding specificity for CD123 comprises a sequence corresponding to SEQ ID NOs 6, 65 to 66; and 102 to 106 (preferably SEQ ID NO: 105) having at least 85% identity; and wherein said ankyrin repeat domain having binding specificity for CD70 comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID nos. 64 and 107 to 110 (preferably SEQ ID No. 109).

In one aspect, the invention provides a recombinant protein wherein said ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID NOs 1 to 5 (preferably SEQ ID NO: 2); wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID NOs 15, 67 to 70; wherein the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID NOs 6, 65 to 66; and wherein said ankyrin repeat domain having binding specificity for CD70 comprises an amino acid sequence having at least 85% identity to the amino acid sequence of SEQ ID No. 64. The invention also provides such recombinant proteins wherein said ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID NOs 1 to 5 (preferably SEQ ID NO: 3); wherein the ankyrin repeat domain with binding specificity for CD33 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID NOs 15, 67 to 70 and 111 to 112 (preferably SEQ ID NO: 111); wherein the ankyrin repeat domain with binding specificity for CD123 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID NOs 6, 65 to 66 and 102 to 106 (preferably SEQ ID NO 105); and wherein said ankyrin repeat domain having binding specificity for CD70 comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID nos. 64 and 107 to 110 (preferably SEQ ID No. 109).

In one aspect, the invention provides such a recombinant protein wherein the first binding agent, the second binding agent, and/or the third binding agent are covalently linked to a peptide linker.

In one aspect, the invention provides a recombinant protein wherein the protein comprises a polypeptide having an amino acid sequence with at least 80% identity to any one of the amino acid sequences of SEQ ID NOs 7 to 10 and 58 to 62, preferably wherein the protein comprises a polypeptide having the amino acid sequence of any one of SEQ ID NOs 7 to 10 and 58 to 62. The present invention also provides a recombinant protein wherein the protein comprises a polypeptide having an amino acid sequence with at least 80% identity to any of the amino acid sequences of SEQ ID NOs 11 to 14, 78 to 86 and 95 to 101, preferably wherein the protein comprises a polypeptide having an amino acid sequence of any of the amino acid sequences of SEQ ID NOs 11 to 14, 78 to 86 and 95 to 101. The invention also provides such recombinant proteins, wherein the proteins comprise polypeptides having an amino acid sequence with at least 80% identity to any of the amino acid sequences of SEQ ID NOS 95 to 101, preferably SEQ ID NO 95 or SEQ ID NO 96. The invention also provides such recombinant proteins, wherein the proteins comprise a polypeptide having the amino acid sequence of any one of the amino acid sequences of SEQ ID NO:95 to 101, preferably SEQ ID NO:95 or SEQ ID NO: 96.

In one aspect, the invention provides such recombinant proteins wherein the proteins are present at less than 10 in PBS ^-6 Dissociation constant of M (K _D ) Binds human CD3.

In one aspect, the invention provides such recombinant proteins wherein the proteins are present at less than 10 in PBS ^-7 Dissociation constant of M (K _D ) Binds human CD33.

In one aspect, the invention provides such recombinant proteins wherein the proteins are present at less than 10 in PBS ^-7 Dissociation constant of M (K _D ) Binds human CD123.

In one aspect, the invention provides such recombinant proteins wherein the proteins are present at less than 10 in PBS ^-7 Dissociation constant of M (K _D ) Binds human CD70.

In one aspect, the invention provides such recombinant proteins, wherein the binding protein is expressed as an EC in the range of 1nM to 400nM ₅₀ Binds human CD3.

In one aspect, the present invention provides such recombinant proteins, wherein the protein further comprises a half-life extending moiety, preferably wherein the half-life extending moiety is a binding agent that specifically binds human serum albumin.

In one aspect, the invention provides such recombinant proteins wherein the half-life extending moiety is a designed ankyrin repeat domain having binding specificity for human serum albumin.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for human serum albumin comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NOs 34 to 36.

In one aspect, the present invention provides such a recombinant protein, wherein the recombinant protein is present at less than 10 in PBS ^-7 Dissociation constant of M (K _D ) Binding human serum albumin.

In one aspect, the invention also provides nucleic acids encoding such recombinant proteins.

In one aspect, the invention also provides a pharmaceutical composition comprising such a recombinant protein or a nucleic acid encoding such a recombinant protein, and a pharmaceutically acceptable carrier and/or diluent.

In one aspect, the present invention also provides a method of treating a medical condition, the method comprising the step of administering to a patient in need thereof a therapeutically effective amount of such a recombinant protein or a therapeutically effective amount of a pharmaceutical composition comprising said recombinant protein.

In one aspect, the present invention provides a method of treating a medical condition, wherein the medical condition is a cancer, preferably a liquid tumor, more preferably a leukemia, even more preferably an acute myelogenous leukemia.

In one aspect, the present invention provides a recombinant protein as defined herein or a pharmaceutical composition comprising said recombinant protein, for use in therapy.

In one aspect, the present invention provides a recombinant protein as defined herein or a pharmaceutical composition comprising said recombinant protein for use in the treatment of cancer, preferably in the treatment of liquid tumors.

In one aspect, the present invention provides a recombinant protein as defined herein or a pharmaceutical composition comprising said recombinant protein for use in the treatment of cancer, wherein said cancer is leukemia, preferably wherein said cancer is acute myelogenous leukemia.

Drawings

FIG. 1FIG. 1A. Pharmacokinetic analysis of exemplary CD3 specific engineered ankyrin repeat proteins in female BALB/c mice. The figure shows that after a single intravenous bolus administration of 1mg/kg,protein #16,Protein #17,/>Protein #18 and->Group mean serum concentration-time curve for protein #19 in female BALB/c mice (mean +/-max/min, n=3/group). FIG. 1B. Pharmacokinetic analysis of exemplary specific engineered ankyrin repeat proteins in female BALB/c mice. The figure shows the ++1 mg/kg after a single intravenous bolus administration >Protein #51, < >>Protein #50, < >>Protein #54, < >>Protein #54 and->Group mean serum concentration-time curve for protein #55 in female BALB/c mice (mean +/-max/min, n=3/group).

FIG. 2 (A-C).Surface Plasmon Resonance (SPR) analysis of ankyrin repeat proteins binding to human CD3, byProtein #53 (FIG. 2A), a->Protein #54 (FIG. 2B) and +.>Protein #9 (fig. 2C) is exemplified. Various concentrations of purified ankyrin repeat protein were applied to GLC chips with immobilized human CD3 for binding rate and dissociation rate measurements. The SPR trace analysis obtained was used to determine ankyrin repeat CD3 interactions. RU, resonance unit; s, time in seconds.

FIG. 3 (A-C).Short term T cell activation as measured by activation marker CD 25. Pan-T cells and MOLM-13 cells were incubated at a 1:1 E:T ratio and T cell activation was assessed by FACS after 24 hours of co-culture in the presence of serial dilutions of the indicated molecules. Activated T cells were gated as viable cd8+/cd25+ cells. A known reference T cell conjugate is shown: AMG330 with binding specificity for CD33 and Fu Tuozhu mab with binding specificity for CD123, selected recombinant protein that binds to MOLM-13 cells (without half-life extension); (FIG. 3A) Protein #27 (two tumor antigen specific binding domains with binding specificity for CD123 and CD 33) and a protein having only one tumor antigen specific binding domain (/ a)>Protein 40 and->Protein 41). (FIG. 3B)Protein #28 and->Protein #29 (two tumor antigen specific binding domains with binding specificity for CD70 and CD 33) and a protein having only one tumor antigen specific binding domain (/ a)>ProteinsMatter 42 and->Protein 43). (FIG. 3C)/(S)>Protein #30 (three tumor antigen specific binding domains with binding specificity for CD70, CD123 and CD 33) and a protein with only one tumor antigen specific binding domain (/ a->Protein 44, < >>Protein 45 and->Protein 46).

FIG. 4 (A-C)Short term T cell activation as measured by activation marker CD 25. Pan-T cells and MOLM-13 cells were incubated at a 1:1 E:T ratio and T cell activation was assessed by FACS after 24 hours of co-culture in the presence of serial dilutions of the indicated molecules. Activated T cells were gated as viable cd8+/cd25+ cells. A known reference T cell conjugate is shown: AMG330 with binding specificity for CD33 and Fu Tuozhu mab with binding specificity for CD123, and selected recombinant proteins that bind to MOLM-13 cells; (FIG. 4A) Protein #27 is similar to a protein having two tumor antigen specific binding domains and one half-life extending domain at the N-terminus (+.>Protein # 47), a similar protein with two half-life extending domains located at the N-terminus (++>Protein # 48) and the like having one half-life extending domain at the C-terminus (/ -amino acid sequence)>Protein # 49); (FIG. 4B)/(S)>Protein #29 is similar to a protein having two tumor antigen specific binding domains and one half-life extending domain at the N-terminus (+.>Protein # 50), a similar protein with two half-life extending domains located at the N-terminus (++>Protein # 51) and the like having one half-life extending domain at the C-terminus (/ -amino acid sequence)>Protein # 52); (FIG. 4C)/(S)>Protein #31 is similar to a protein having three tumor antigen specific binding domains and one half-life extending domain at the N-terminus (+.>Protein # 53) compared to a similar protein with two half-life extending domains located at the N-terminus (++>Protein # 54) and a similar protein (++f) with one half-life extending domain at the C-terminus>Protein # 55).

FIG. 5 (A-C).Short term target cell killing (LDH). Pan-T cells and Molm-13 cells were incubated at an E:T ratio of 5:1 and tumor cell killing was assessed by LDH release in the supernatant after co-cultivation in the presence of serial dilutions of the indicated molecules for 48 hours. Reference control molecules (known reference T cell conjugates: AMG330 with binding specificity for CD33 and Fu Tuozhu mab with binding specificity for CD 123) and multispecific recombinant proteins with two or three tumor-specific binding domains are shown; (FIG. 5A)Protein #27 (two tumor antigen specific binding domains with binding specificity for CD123 and CD 33) and protein having only one tumor antigen specific binding domainProtein 40 and->Protein 41). (FIG. 5B)/(S)>Protein #28Protein #29 (two tumor antigen specific binding domains with binding specificity for CD70 and CD 33) and a protein having only one tumor antigen specific binding domain (/ a)>Protein 42 and->Protein 43). (FIG. 5C)/(S)>Protein #30 (three tumor antigen specific binding domains with binding specificity for CD70, CD123 and CD 33) and protein having only one tumor antigen specific binding domain Protein 44, < >>Protein 45 and->Protein 46).

FIG. 6 (A-C)Short term target cell killing (LDH). Pan-T cells and Molm-13 cells were incubated at an E:T ratio of 5:1 and tumor cell killing was assessed by LDH release in the supernatant after co-cultivation in the presence of serial dilutions of the indicated molecules for 48 hours. Reference control molecules (known reference T cell conjugates: AMG330 with binding specificity for CD33 and Fu Tuozhu mab with binding specificity for CD 123) and multispecific recombinant proteins with two or three tumor-specific binding domains are shown; (FIG. 6A)Protein #27 is similar to a protein having two tumor antigen specific binding domains and one half-life extending domain at the N-terminus (+.>Protein # 47), a similar protein with two half-life extending domains located at the N-terminus (++>Protein # 48) and the like having one half-life extending domain at the C-terminus (/ -amino acid sequence)>Protein # 49); (FIG. 6B)/(S)>Protein #29 is similar to a protein having two tumor antigen specific binding domains and one half-life extending domain at the N-terminus (+.>Protein # 50), a similar protein with two half-life extending domains located at the N-terminus (++ >Protein # 51) and similar proteins having a half-life extending domain at the C-terminusProtein # 52); (FIG. 6C)/(S)>Protein #31 is similar to a protein having three tumor antigen specific binding domains and one half-life extending domain at the N-terminus (+.>Protein # 63) compared to a similar protein with two half-life extending domains located at the N-terminus (++>Protein # 54) and a similar protein (++f) with one half-life extending domain at the C-terminus>Protein # 55).

FIG. 7Killing target cells. Shows a reference control molecule (known reference T cell conjugate: AMG330 with binding specificity for CD33 and Fu Tuozhu mab with binding specificity for CD 123) andprotein #31. The data show that in tumor cell killing assays, when compared to clinical baseline, the +.>Protein #5 exerts similar potency and efficacy.

FIG. 8Killing target cells. Curve 7 represents the Molm13 parental cells expressing all three targets (CD 70, CD123 and CD 33).Protein #31 showed full potency. Curves 4-6 represent a single KO cell, meaning that only two targets are still expressed. Here->Protein #5 was still active, indicating the potential to combat tumor heterogeneity. Curves 1-3 represent double KO cells, meaning that only one target is still expressed to mimic a healthy tissue compartment. Here, a- >Protein #31 had significantly lower activity, meaning increased selectivity for healthy tissue.

Fig. 9 (a-D).Ifnγ levels. (average of three donors). All three donors (D1-D3) had a calculated mean concentration value of ifnγ in the blood circuit system. Plasma samples were collected from the blood circuit system at 0 (zero) hours, 2 hours, 4 hours, 8 hours and 24 hours. The calculated LLOQ is indicated by a dashed line. Each point is the mean and error bars SD of three donors. Fu Tuozhu mab is presented in figure 9A,protein #27 is presented in FIG. 9B, < >>Protein #29 is presented in FIG. 9C, and +.>Protein #31 is presented in figure 9D. Data and value>ULOQ。

FIG. 10 (A-D)IL-2 levels (average of three donors). The calculated mean concentration values of IL-2 in the blood circuit system for all three donors (D1-D3). Plasma samples were collected from the blood circuit system at 0 (zero) hours, 2 hours, 4 hours, 8 hours and 24 hours. The calculated LLOQ is indicated by a dashed line. Each point is the mean and error bars SD of three donors. Fu Tuozhu mab is presented in figure 10A,protein #27 is presented in FIG. 10B, < >>Protein #29 is presented in FIG. 10C, and +.>Protein #31 is presented in figure 10D.

FIG. 11 (A-D).IL-6 levels (average of three donors). The calculated mean concentration values of IL-6 in the blood circuit system for all three donors (D1-D3). Plasma samples were collected from the blood circuit system at 0 (zero) hours, 2 hours, 4 hours, 8 hours and 24 hours. The calculated LLOQ is indicated by a dashed line. Each point is the mean and error bars SD of three donors. Fu Tuozhu mab is presented in figure 11A,protein #27 is presented in FIG. 11B, < >>Protein #29 is presented in FIG. 11C, and +.>Protein #31 is presented in fig. 11D. Data and value>ULOQ。

FIG. 12 (A-D).IL-8 levels (average of three donors). The calculated mean concentration values of IL-8 in the blood circuit system for all three donors (D1-D3). Plasma samples were collected from the blood circuit system at 0 (zero) hours, 2 hours, 4 hours, 8 hours and 24 hours. The calculated LLOQ is indicated by a dashed line. Each point is the mean and error bars SD of three donors. Fu Tuozhu mab is presented in figure 11A,protein #27 is presented in FIG. 11B, < >>Protein #29 is presented in FIG. 11C, and +.>Protein #31 is presented in fig. 11D. Data and value>ULOQ。

Fig. 13 (a-D).Tnfα levels (average of three donors). The calculated mean concentration values of TNFα in the blood circuit system for all three donors (D1-D3). Plasma samples were collected from the blood circuit system at 0 (zero) hours, 2 hours, 4 hours, 8 hours and 24 hours. The calculated LLOQ is indicated by a dashed line. Each point is the mean and error bars SD of three donors. Fu Tuozhu mab is presented in figure 13A, Protein #27 is presented in FIG. 13B, < >>Protein #29 is presented in FIG. 13C, and +.>Protein #31 is presented in fig. 13D. Data and value>ULOQ。

FIG. 14 (A-D).Platelet count. Blood was withdrawn from the circuit at time 0 (zero sample), 2 hours, 4 hours, 8 hours and 24 hours using Sysmex bloodThe liquid analyzer automatically counts PLT counts. Each dot represents the average of three donors (and error bar SD): fig. 14A shows trastuzumab and fig. 14B shows trastuzumabProtein #27 +.in FIG. 14C>Protein #29, and +.in FIG. 14D>Protein #31.

FIG. 15 (A-B).Against the selected recombinant proteinProtein 27, < >>Protein 40Protein 41 measured, binding rate/dissociation rate constant of CD33 target.

FIG. 16 (A-B).Against the selected recombinant proteinProtein 27, < >>Protein 40Binding rate/dissociation rate constant of CD123 target measured by protein 41.

Fig. 17.K of recombinant protein-human CD33 interaction _D Values.

Fig. 18.K of recombinant protein-human CD123 interaction _D Values.

Fig. 19:to human CD3,Protein #1 and->Protein #2, < >>Protein #3 and->Protein #4 purified SDS-PAGE gel analysis of four selected ankyrin repeat proteins with binding specificity. M corresponds to protein size markers (reducing SDS-PAGE, nuPAGE 4% -12%, bis Tris (invitrogen) gel; 5. Mu.g/lane; MES buffer; immediate blue staining). Molecular weight (kDa) of the marker protein is indicated. Lane 1: protein size markers; lane 2: purified +. >Protein #1; lane 3: purified +.>Protein #2; lane 4: purified +.>Protein #3; lane 5: purified +.>Protein #4.

FIG. 20 (A-C).FIGS. 20A and 20B illustrate binding of ankyrin repeat proteins to CD3 of T cells. Binding to CD3 on Pan-T cells was assessed by Mirrorball. Reference control molecules (known reference T cell conjugates: AMG330 with binding specificity for CD33 and Fu Tuozhu mab with binding specificity for CD 123) and selected ankyrin repeat proteins: does not have half-life periodExtension (HLE)Protein 7, (-)>Protein 8,Protein 9 and->Protein 10 (FIG. 20A) or +.>Protein 11,Protein 12, (-) ->Protein 13 and->Protein 14 (FIG. 20B). The half-life extended protein (B) showed similar binding compared to the corresponding non-HLE molecule shown in (a). Pan-T cells from 5 different donors were tested, one representative donor being shown here. Negative control designed ankyrin repeat proteins with binding specificity only for CD33 and CD123, with or without half-life extension, respectively. FIG. 20C shows the selected multispecific binding protein +.>Protein #56, < > >Protein #57, < >>Protein #58, < >>Protein #59, < >>Protein #60, < >>Protein #61 and->Tumor cell binding of protein #62 on Molm-13N1 cells.

FIG. 21Short term T cell activation as measured by activation marker CD 25. Pan-T cells and MOLM-13 cells were incubated at a 1:1 E:T ratio and T cell activation was assessed by FACS after 24 hours of co-culture in the presence of serial dilutions of the indicated molecules. Activated T cells were gated as viable cd8+/cd25+ cells. A known reference T cell conjugate is shown: AMG330 with binding specificity to CD33 and Fu Tuozhu monoclonal antibody with binding specificity to CD123 and binding domain with two tumor specificity to MOLM-13 cellsProtein 8) or only one tumor specific binding domain (/ ->Protein 23 and->Protein 24).Protein 8 induced T cell activation corresponds to the reference molecule, however +.>Protein 23Protein 24 showed lower potency. Representative data are shown using Pan-T cells from one donor.

FIG. 22 (A-B)Short term target cell killing (LDH). Pan-T cells and Molm-13 cells were incubated at an E:T ratio of 5:1 and tumor cell killing was assessed by LDH release in the supernatant after co-cultivation in the presence of serial dilutions of the indicated molecules for 48 hours. Reference control molecules (known reference T cell conjugates: AMG330 with binding specificity for CD33 and Fu Tuozhu mab with binding specificity for CD 123) and selected ankyrin repeat proteins: without half-life extension (HLE) Protein 7, (-)>Protein 8>Protein 9Protein 10 (FIG. 22A) or +.>Protein 11, (-)>Protein 12,Protein 13 and->Protein 14 (FIG. 22B). (FIG. 22A) for proteins without half-life extension, by +.>Protein 8 and->Protein 9 induced tumor cell killing is comparable to the reference molecule, however +.>Protein 10 and->Protein 7 showed lower cytotoxic potency. (FIG. 22B) half-life extending proteins showed reduced efficacy to 1/4-1/70 compared to the corresponding non-HLE molecules shown in (A). Pan-T cells from 5 different donors were tested, one representative donor being shown here. Negative control designed ankyrin repeat proteins with binding specificity for CD33 and CD123, with or without half-life extension, respectively.

FIG. 23 (A-B)Short term T cell activation as measured by activation marker CD 25. Pan-T cells and Molm-13 cells were incubated at a ratio of E:T of 1:1, and T cell activation was assessed by FACS after co-culture in the presence of serial dilutions of the indicated molecules for 24 hours. Activated T cells were gated as viable cd8+/cd25+ cells. Reference control molecules (known reference T cell conjugates: AMG330 with binding specificity for CD 33) and selected ankyrin repeat proteins are shown: without half-life extension (HLE) Protein 7, (-)>Protein 8>Protein 9 and->Protein 10 (FIG. 23A) or +.>Protein 11, (-)>Protein 12,Protein 13 and->Protein 14 (FIG. 23B). (FIG. 23A) in the absence of half-life extension, the method consists of +.>Protein 8 and->Protein 9 induced T cell activation was comparable to baseline, howeverProtein 10 and->Protein 7 showed lower potency. (FIG. 23B) half-life extending proteins showed reduced efficacy to 1/4-1/100 compared to the corresponding non-HLE molecules shown in (A). Pan-T cells from 7 different donors were tested, one representative donor being shown here. Negative control: designed ankyrin repeat protein with binding specificity only for CD33 and CD123, with or without half-life extension, respectively.

FIGS. 24A to 24BShort term T cell activation as measured by ifnγ secretion. Pan-T cells and Molm-13 cells were incubated at a ratio of E:T of 1:1. After 24 hours of co-cultivation in the presence of serial dilutions of the indicated molecules, the culture supernatants were analyzed by ELISAIfnγ secretion in the fluid. Reference control molecules (known reference T cell conjugates: AMG330 with binding specificity for CD 33) and selected ankyrin repeat proteins are shown: without half-life extension (HLE) Protein 7, (-)>Protein 8>Protein 9 and->Protein 10 (FIG. 24A) or +.>Protein 11, (-)>Protein 12, (-) ->Protein 13Protein 14 (FIG. 24B). (FIG. 24A) in the absence of half-life extension, the method consists of +.>Protein 8 and->Protein 9 induced T-cell activation corresponds to the reference molecule, however +.>Protein 10Protein 7 showed lower potency. (FIG. 24B) half-life extending proteins showed reduced efficacy to 1/3-1/20 compared to the corresponding non-HLE molecules shown in (A). Pan-T cells from 4 different donors were tested, one representative donor being shown here. Negative control designed ankyrin repeat proteins with binding specificity only for CD33 and CD123, with or without half-life extension, respectively.

Fig. 25.Long-term tumor cell killing. Pan-T cells and Molm-13 cells were incubated at an E:T ratio of 5:1, and tumor cell killing was assessed by co-culturing for 6 days with IntuCyte in the presence of serial dilutions of the indicated molecules. Tumor cell killing was calculated as the ratio between area under annexin V staining curve and cell proliferation. Two reference control molecules (known reference T cell conjugates: AMG330 with binding specificity for CD33 and Fu Tuozhu mab with binding specificity for CD 123) and selected ankyrin repeat proteins are shown: without half-life extension (HLE) Protein 7, (-)>Protein 8>Protein 9 and->Protein 10 or with HLEProtein 11, (-)>Protein 12, (-) ->Protein 13 and->Protein 14. The data show effective and specific tumor cell killing of most of the test proteins, comparable to the reference molecule, independent of half-life extension. Only low affinity +.>Protein 7 showed a significant decrease in killing efficacy. The concentrations were from left to right 2nM diluted 1/10 for the baseline and test proteins and 20nM diluted 1/10 for the half-life extending molecules. Pan-T cells from 5 different donors were used, one representative donor being shown here.

FIG. 26 (A-B)Long term T cell activation. Pan-T cells and Molm-13 cells were incubated at a ratio of E:T of 1:1, and T cell activation was assessed by FACS after co-culturing for 5 days in the presence of serial dilutions of the indicated molecules. Activated T cells were gated as viable cd8+/cd25+ cells. Reference control molecules (known reference T cell conjugates: AMG330 with binding specificity for CD33 and Fu Tuozhu mab with binding specificity for CD 123) and selected ankyrin repeat proteins: without half-life extension (HLE)Protein 7, (-) >Protein 8,Protein 9 and->Protein 10 or +.>Protein 11, (-)>Protein 12, (-) ->Protein 13 and->Protein 14. (FIG. 26A) in the absence of half-life extension, the method consists of +.>Protein 8 and->Protein 9 induced T cell activation is comparable to the reference molecule, howeverProtein 7 and->Protein 10 showed a decrease in potency to less than 1/100. (FIG. 26B) proteins with prolonged half-lives showed a decrease in potency below 1/10 compared to the corresponding non-HLE molecules shown in (A). Pan-T cells from 2 different donors were used, one representative donor being shown here.

FIG. 27 (A-B)Long-term T cell proliferation. Pan-T cells and Molm-13 cells were incubated at a ratio of E:T of 1:1, and T cell proliferation was assessed by FACS after co-culturing for 5 days in the presence of serial dilutions of the indicated molecules. The proliferating T cells were gated as CellTrace Violet positive cells that showed at least one cell division. Two reference control molecules (known reference T cell conjugates: AMG330 with binding specificity for CD33 and Fu Tuozhu mab with binding specificity for CD 123) and selected ankyrin repeat proteins are shown: without half-life extension (HLE) Protein 7, (-)>Protein 8>Protein 9 and->Protein 10 or +.>Protein 11, (-)>Protein 12, (-) ->Protein 13 and->Protein 14. (FIG. 27A) in the absence of half-life extension, the method consists of +.>Protein 8 and->Protein 9 induced T cell proliferation is comparable to the reference molecule, however +.>Protein 7 and->Protein 10 showed a decrease in potency to less than 1/100. (FIG. 27B) DARPin with an extended half-life showed a decrease in potency below 1/30 compared to the corresponding non-HLE molecule shown in (A). Pan-T cells from 2 different donors were used, one representative donor being shown here.

FIG. 28 (A-F).Ifnγ levels. Plasma samples were collected from the whole blood circuit system at 0 (zero) hours, 4 hours, 8 hours, 24 hours, and 48 hours. Vehicle was the negative control. For AMG330 analog (fig. 28A), votuzumab-mab analog (fig. 28B),Protein 7 (FIG. 28C), ->Protein 8 (FIG. 28D), ->Protein 9 (FIG. 28E) and +.>Protein 10 (fig. 28F), presents the average of two donors over time. No time zero value is presented. The dashed line represents the lower limit of quantitation (LLOQ). All values are below the upper limit of quantification<ULOQ)。

FIG. 29 (A-F)Tnfα levels. Plasma samples were collected from the whole blood circuit system at 0 (zero) hours, 4 hours, 8 hours, 24 hours, and 48 hours. Vehicle was the negative control. For AMG330 analog (fig. 29A), votuzumab-mab analog (fig. 29B), Protein 7 (FIG. 29C), ->Protein 8 (FIG. 29D), ->Protein 9 (FIG. 29E) and +.>Protein 10 (fig. 29F), presents the average of two donors over time. No time zero value is presented. The dashed line represents LLOQ. All values are<ULOQ。

FIG. 30 (A-F).T cell activation (CD 25 positive cells%). Blood cells were collected from the whole blood circuit system at 0 (zero) hours, 4 hours, 8 hours, 24 hours, 48 hours, stained with fluorophore-labeled antibodies and analyzed by flow cytometry. For AMG330 analog (fig. 30A), votuzumab-mab analog (fig. 30B),Protein 7 (FIG. 30C),Protein 8 (FIG. 30D), ->Protein 9 (FIG. 30E) and +.>Protein 10 (fig. 30F), CD25 positive T cell% over time was reported as the average of two donors. Vehicle was the negative control.

FIG. 31 (A-F)T cell activation (CD 69 positive cell%). Blood cells were collected from the whole blood circuit system at 0 (zero) hours, 4 hours, 8 hours, 24 hours, 48 hours, stained with fluorophore-labeled antibodies and analyzed by flow cytometry. For AMG330 analog (fig. 31A), votuzumab-mab analog (fig. 31B),Protein 7 (FIG. 31C),Protein 8 (FIG. 31D), ->Protein 9 (FIG. 31E) and +.>Protein 10 (FIG. 31F), reported CD69 positive T cell% over time The average of two donors is reported. Vehicle was the negative control.

FIG. 32 (A-F)T cell viability (% dead cells of all cd3+). Blood cells were collected from the whole blood circuit system at 0 (zero) hours, 4 hours, 8 hours, 24 hours, 48 hours, stained with fluorophore-labeled antibodies and analyzed by flow cytometry. For AMG330 analog (fig. 32A), votuzumab-mab analog (fig. 32B),Protein 7 (FIG. 32C),Protein 8 (FIG. 32D), ->Protein 9 (FIG. 32E) and +.>Protein 10 (fig. 32F), the viability of dye-positive T cells over time (i.e., dead cells%) was reported as the average of two donors. Vehicle was the negative control.

FIG. 33 (A-F)Cd33+ cell viability (all cd33+ dead cells%). Blood cells were collected from the whole blood circuit system at 0 (zero) hours, 4 hours, 8 hours, 24 hours, 48 hours, stained with fluorophore-labeled antibodies and analyzed by flow cytometry. For AMG330 analog (fig. 33A), votuzumab-mab analog (fig. 33B), and,Protein 7 (FIG. 33C), ->Protein 8 (FIG. 33D), ->Protein 9 (FIG. 33E) and +.>Protein 10 (fig. 33F), the viability of dye-positive cd33+ cells over time (i.e., dead cells%) was reported as the average of two donors. Vehicle was the negative control.

FIG. 34 (A-F)Cd123+ cell viability (all cd123+ dead cells%). Blood cells were collected from the whole blood circuit system at 0 (zero) hours, 4 hours, 8 hours, 24 hours, 48 hours, stained with fluorophore-labeled antibodies and analyzed by flow cytometry. For AMG330 analog (fig. 34A), votuzumab-mab analog (fig. 34B),Protein 7 (FIG. 34C), ->Protein 8 (FIG. 34D), ->Protein 9 (FIG. 34E) and +.>Protein 10 (fig. 34F), the viability of dye-positive cd123+ cells over time (i.e., dead cells%) was reported as the average of two donors. Vehicle was the negative control.

FIG. 35 (A-F)Cell count of T cells (cells per 10000 beads). Blood cells were collected from the whole blood circuit system at 0 (zero) hours, 4 hours, 8 hours, 24 hours, 48 hours, stained with fluorophore-labeled antibodies and analyzed by flow cytometry. For AMG330 analog (fig. 35A), votuzumab-mab analog (fig. 35B),Protein 7 (FIG. 35C), ->Protein 8 (FIG. 35D), ->Protein 9 (FIG. 35E) and +.>Protein 10 (fig. 35F), cell counts per 10000 beads over time were reported as the average of two donors. Vehicle was the negative control.

FIG. 36 (A-F) Cell count of cd33+ cells (cells per 10000 beads). Blood cells were collected from the whole blood circuit system at 0 (zero) hours, 4 hours, 8 hours, 24 hours, 48 hours, stained with fluorophore-labeled antibodies and analyzed by flow cytometry. For AMG330 analog (fig. 36A), votuzumab-mab analog (fig. 36B), and,Protein 7 (FIG. 36C), ->Protein 8 (FIG. 36D), ->Protein 9 (FIG. 36E) and +.>Protein 10 (fig. 36F), cell counts per 10000 beads over time were reported as the average of two donors. Vehicle was the negative control.

FIG. 37 (A-F)Cell count of cd123+ cells (cells per 10000 beads). Blood cells were collected from the whole blood circuit system at 0 (zero) hours, 4 hours, 8 hours, 24 hours, 48 hours, stained with fluorophore-labeled antibodies and analyzed by flow cytometry. For AMG330 analog (fig. 37A), votuzumab-mab analog (fig. 37B),Protein 7 (FIG. 37C), ->Protein 8 (FIG. 37D), ->Protein 9 (FIG. 37E) and +.>Protein 10 (fig. 37F), cell counts per 10000 beads over time were reported as the average of two donors. Vehicle was the negative control.

FIG. 38 (A-B).Against the selected recombinant protein Protein 20, < >>Protein 21The binding rate (fig. 38A) and dissociation rate (fig. 38B) constants of the CD33 targets measured by protein 22.

FIG. 39 (A-B).Against the selected recombinant proteinProtein 20, < >>Protein 21The binding rate (fig. 39A) and dissociation rate (fig. 39B) constants of CD123 targets measured by protein 22.

Fig. 40.K of recombinant protein-human CD33 interaction _D Values.

Fig. 41.K of recombinant protein-human CD123 interaction _D Values.

FIG. 42 (A-C).Mean tumor volume and SD.

FIG. 43 (A-L).Single tumor growth curve.

FIG. 44 (A-H): ifnγ levels. All 3 donors in the blood circuit system of IFN gamma calculation average concentration value. Plasma samples were collected from the blood circuit system at 0 (zero) hours, 2 hours, 4 hours, 8 hours and 24 hours. Each dot represents the average of three donors (and error bar SD): fu Tuozhu monoclonal antibody FIG. 44A,Protein #58 FIG. 44B,Protein #59 FIG. 44C, < >>Protein #57 FIG. 44D, < > protein->Protein #60 FIG. 44E,Protein #56 FIG. 44F, < >>Protein #61 FIG. 44G, < > protein->Protein #74 figure 44H. The calculated LLOQ is indicated by a dashed line.

Fig. 45 (a-H):IL-2 levels. Calculated mean concentration values of IL-2 in the blood circuit system for all 3 donors. Plasma samples were collected from the blood circuit system at 0 (zero) hours, 2 hours, 4 hours, 8 hours and 24 hours. Each dot represents the average of three donors (and error bar SD): fu Tuozhu monoclonal antibody FIG. 45A, Protein #58 FIG. 45B, < > protein->Protein #59 FIG. 45C,>protein #57 FIG. 45D, < > protein->Protein #60 FIG. 45E, < > protein->Protein #56 FIG. 45F,>protein #61 FIG. 45G, < > protein->Protein #74 FIG. 45H. The calculated LLOQ is indicated by a dashed line.

FIG. 46 (A-H): IL-6 levels. The calculated mean concentration of IL-6 in the blood circuit system was calculated for all 3 donors. Plasma samples were collected from the blood circuit system at 0 (zero) hours, 2 hours, 4 hours, 8 hours and 24 hours. Each dot represents the average of three donors (and error bar SD): fu Tuozhu monoclonal antibody FIG. 46A,Protein #58 FIG. 46B, < > protein->Protein #59 FIG. 46C,>protein #57 FIG. 46D, < > protein->Protein #60 FIG. 46E, < > protein #60>Protein #56 FIG. 46F,>protein #61 FIG. 46G,>protein #74 fig. 46H. The calculated LLOQ is indicated by a dashed line.

Fig. 47 (a-H):IL-8 levels. Calculated mean concentration values of IL-8 in the blood circuit system for all 3 donors. Plasma samples were collected from the blood circuit system at 0 (zero) hours, 2 hours, 4 hours, 8 hours and 24 hours. Each dot represents the average of three donors (and error bar SD): fu Tuozhu monoclonal antibody FIG. 47A,Protein #58 FIG. 47B, < > protein->Protein #59 FIG. 47C, < > protein->Protein #57 FIG. 47D, < > protein->Protein #60 FIG. 47E, < > protein- >Protein #56 FIG. 47F,>protein #61 FIG. 47G, < > protein->Protein #74 fig. 47H. The calculated LLOQ is indicated by a dashed line.

FIG. 48 (A-H).Tnfα levels. The calculated mean concentration values of tnfα in the blood circuit system for all 3 donors. From blood at 0 (zero) hours, 2 hours, 4 hours, 8 hours and 24 hoursThe circuit system collects plasma samples. Each dot represents the average of three donors (and error bar SD): fu Tuozhu monoclonal antibody FIG. 48A,Protein #58 FIG. 48B,>protein #59 FIG. 48C,>protein #57 FIG. 48D, < > protein->Protein #60 FIG. 48E, < > protein->Protein #56 FIG. 48F,>protein #61 FIG. 48G,>protein #74 figure 44H. The calculated LLOQ is indicated by a dashed line.

FIG. 49 (A-H).Platelet count. Blood samples were taken from the blood circuit system at time 0 (zero) and at 2, 4, 8 and 24 hours and platelets were counted automatically using a Sysmex XN-L350 blood (H) analyzer. Each dot represents the average of three donors (and error bar SD): fu Tuozhu monoclonal antibody FIG. 49A,Protein #58 FIG. 49B,Protein #59 FIG. 49C, < >>Proteins#57 FIG. 49D, #57>Protein #60 FIG. 49E,Protein #56 FIG. 49F,>protein #61 FIG. 49G, < > protein>Protein #74 fig. 49H. The calculated LLOQ is indicated by a dashed line. / >

FIG. 50 (A-H)White blood cell count. Blood samples were taken from the blood circuit system at time 0 (zero) and at 2, 4, 8 and 24 hours and leukocytes were counted automatically using a Sysmex XN-L350 hematology analyzer. Each dot represents the average of three donors (and error bar SD): fu Tuozhu monoclonal antibody FIG. 50A,Protein #58 FIG. 50B,Protein #59 FIG. 50C, < > protein->Protein #57 FIG. 50D, < > protein->Protein #60 FIG. 50E,Protein #56 FIG. 50F, < > protein->Protein #61 FIG. 50G, < > protein->Protein #74 fig. 50H. The calculated LLOQ is indicated by a dashed line.

FIG. 51 (A-E)FIG. 51A. Targeting CD123-CD33-CD70Protein #56, < >>Protein #57, < >>Protein #58, < >>Protein #59, < >>Protein #60 and->Efficacy titration curve (T cell activation) of protein #61 on Molm13 tumor cells co-cultured with human PanT cells. EC50 values are shown in pM. FIG. 51B. CD123-CD33-CD70 targeting +.>Protein #56, < >>Protein #57 and corresponding negative control +.>Protein #74, < >>Protein #75, < >>Protein #76,Efficacy titration curve (T cell activation) of protein #77 on Molm13 tumor cells co-cultured with human PanT cells. EC50 values are shown in pM. FIG. 51C +.The CD123-CD33-CD70 targeting +. >Efficacy titration curves (T cell activation) of protein #56 and the known reference control molecules AMG330 analog and votuzumab analog on Molm13 tumor cells co-cultured with human PanT cells. EC50 values are shown in pM. FIG. 51D. Targeting CD123-CD33-CD70Efficacy titration curve (T cell activation) of protein #56 on Molm13 wild type or various TAA knockout tumor cells. EC50 values are shown in pM. FIG. 51E +.The CD123-CD33-CD70 targeting +.>Efficacy titration curve (T cell activation) of protein #57 on Molm13 wild type or various TAA knockout tumor cells. EC50 values are shown in pM.

FIG. 52 (A-E)FIG. 52A. Targeting CD123-CD33-CD70Protein #56, < >>Protein #57, < >>Protein #58, < >>Protein #59, < >>Protein #60 and->Efficacy titration curve (cell killing) of protein #61 on Molm13 tumor cells co-cultured with human PanT cells. EC50 values are shown in pM. FIG. 52B. CD123-CD33-CD70 targeting +.>Protein #56, < >>Protein #57 and corresponding negative control +.>Protein #74, < >>Protein #75, < >>Protein #76,Efficacy titration curve (cell killing) of protein #77 on Molm13 tumor cells co-cultured with human PanT cells. EC50 values are shown in pM. FIG. 52℃ Targeting CD123-CD33-CD70 +. >Efficacy titration curves (cell killing) of protein #56 and the known reference control molecules AMG330 analog and votuzumab analog on Molm13 tumor cells co-cultured with human PanT cells. EC50 values are shown in pM. FIG. 52D targeting CD123-CD33-CD70Efficacy titration curve (cell killing) of protein #56 on Molm13 wild type or various TAA knockout tumor cells. EC50 values are shown in pM.

FIG. 53 (A-B).FIG. 53A. TargetTo CD123-CD33-CD70Efficacy titration curves (allogeneic and autologous T cell activation) of protein #56 on BMMC tumor cells in AML patients. EC50 values are shown in pM. FIG. 53B +.>Efficacy titration curve (cell killing) of protein #56 on BMMC tumor cells of AML patients.

Fig. 54 (a-B):FIG. 5A selected multispecific for targeting CD123-CD33-CD70 compared to Fu Tuozhu mabEfficacy titration curve of protein on BMMC tumor cells of AML patients (T cell activation). FIG. 54B selected multispecific +.A.The targeting CD123-CD33-CD70 compared to Fu Tuozhu mab>Efficacy titration curves of proteins on BMMC tumor cells in AML patients (tumor cell killing).

FIG. 55Median expression in different LSC and HSC samples, expressed as Δmfi.

FIG. 56ACompared with HSC (open bars)Protein #56 and->Protein #57 preferentially kills LSCs (solid bars), confirming the window of opportunity exists between LSCs and HSCs

FIG. 57 (A-F)Killing of Molm-13 cells and release of ifnγ. The killing of Molm-13 cells is shown by the black squares/curves and the concentration of ifnγ is shown by the black circles/curves. Drawing of the figureEffects of protein #56 on cell killing and cytokine release. Figure->Effects of protein #57 on cell killing and cytokine release. Drawing of the figureEffects of protein #59 on cell killing and cytokine release. Figure->Effects of protein #62 on cell killing and cytokine release. Fig. 57E Fu Tuozhu effect of mab on cell killing and cytokine release. Fig. 57f. Effect of amg330 on cell killing and cytokine release.

Fig. 58 (a-B).FIG. 58A shows the intraperitoneal injection of hBMC, subcutaneous xenograft of MOLM-13 tumor cells two days after hBMC injection and PBS1X (black circles), 0.5mg/kgTumor growth over time in protein #56 (black squares) or 0.5mg/kg AMG330 (black triangles) treated mice (n=5 mice/donor/2 hPBMC donors used). Treatment was initiated on day 4 after tumor cell xenograft. Data are expressed as mean + SEM. Fig. 58B. Evaluation of tumor volume at day 17 post tumor cell xenograft in the mice depicted in fig. 58A.

FIG. 59 (A-L).After 2 days of incubation, autologous killing of patient-derived AML cells and release of cytokines (IFN-g, TNFa, IL-2). Cell killing is shown as% living cells by black squares/curves and cytokine concentration is shown by black circles/curves. Fig. 59A Fu Tuozhu effect of mab analogs on cell killing and ifnγ release. Fig. 59B Fu Tuozhu effect of mab analogs on cell killing and tnfα release. FIG. 59C Fu Tuozhu effect of monoclonal antibody analogs on cell killing and IL-2 release. Fig. 59D.Effect of AMG330 analogs on cell killing and ifnγ release. Fig. 59e. Effects of amg330 analogs on cell killing and tnfα release. FIG. 59F. Influence of AMG330 analogues on cell killing and IL-2 release. Drawing of the figureEffects of protein #56 on cell killing and ifnγ release. Figure->Effects of protein #56 on cell killing and tnfα release. Figure->Effects of protein #56 on cell killing and IL-2 release. Figure->Effects of protein #57 on cell killing and ifnγ release. Figure->Effects of protein #57 on cell killing and tnfα release. Figure->Effects of protein #57 on cell killing and IL-2 release.

FIG. 60 (A-L).After 5 days of incubation, autologous killing of patient-derived AML cells and release of cytokines (IFN-g, TNFa, IL-2). Cell killing is shown as% living cells by black squares/curves and cytokine concentration is shown by black circles/curves. Fig. 60A Fu Tuozhu effect of mab analogs on cell killing and ifnγ release. FIG. 60B Fu Tuozhu effect of monoclonal antibody analogs on cell killing and TNFα release. FIG. 60C Fu Tuozhu effect of monoclonal antibody analogs on cell killing and IL-2 release. Figure 60d. Effect of amg330 analogues on cell killing and ifnγ release. Figure 60e. Effect of amg330 analogues on cell killing and tnfα release. FIG. 60F. Influence of AMG330 analogues on cell killing and IL-2 release. Drawing of the figure Effects of protein #56 on cell killing and ifnγ release. Figure->Effects of protein #56 on cell killing and tnfα release. Figure->Effects of protein #56 on cell killing and IL-2 release. Figure->Effects of protein #57 on cell killing and ifnγ release. Figure->Effects of protein #57 on cell killing and tnfα release. Figure->Effects of protein #57 on cell killing and IL-2 release.

FIG. 61 (A-L).Patient-derived AML cells after 2 days incubation in the presence of human Pan-T cells (E: T ratio 4:1) were allokilled and cytokine (IFN-g, TNFa, IL-2) released. Cell killing is shown as% living cells by black squares/curves and cytokine concentration is shown by black circles/curves. Fig. 61A Fu Tuozhu effect of mab analogs on cell killing and ifnγ release. Fig. 61B Fu Tuozhu effect of mab analogs on cell killing and tnfα release. FIG. 61C Fu Tuozhu effect of monoclonal antibody analogs on cell killing and IL-2 release. The effect of amg330 analogues on cell killing and ifnγ release. Fig. 61e, effect of amg330 analogues on cell killing and tnfα release. FIG. 61F. Influence of AMG330 analogues on cell killing and IL-2 release. Drawing of the figure Effects of protein #56 on cell killing and ifnγ release.Figure->Effects of protein #56 on cell killing and tnfα release. Figure->Effects of protein #56 on cell killing and IL-2 release. Figure->Effects of protein #57 on cell killing and ifnγ release. Figure->Effects of protein #57 on cell killing and tnfα release. Figure->Effects of protein #57 on cell killing and IL-2 release.

FIG. 62 (A-H): cytokine release in whole blood from 2 healthy donors spiked with Molm13 cells. FIG. 62A. IFN release in donor 1 blood; FIG. 62B. IFN release in donor 2 blood; FIG. 62C-IL-2 release in donor 1 blood; FIG. 62D. IL-2 release from donor 2 blood; FIG. 62E. IL-6 release in donor 1 blood; FIG. 62F. IL-6 release in donor 2 blood; FIG. 62G TNFa release in donor 1 blood; fig. 62H, TNFa release in donor 2 blood.

Fig. 63. SPR trace of simultaneous binding of protein #56 to serum albumin, CD70, CD123, CD33 and CD 3. (a) And (2)>Binding of protein #56 to immobilized HSA. (b) hCD70 and->Protein #56 complexCompound->Or combination of PBST injections. (c) hCD123 and +.>Protein #56/hCD70 complex +.>Or and->Binding of protein #56 complex (■) or PBST injection. (d) hCD33 and- >Protein #56/hCD70/hCD123 complex +.>Or withBinding of protein #56 complex (×t) or PBST injection. (e) scCD3Protein #56/hCD70/hCD123/hCD33 Complex +.>) Is combined with or is associated with->Protein #56 complex (-, or PBST injection) followed by a 180s dissociation period. The injection protocol is described in table 11.

FIG. 64.Protein #56 is "measured" and "calculated" simultaneously with all targetsComparison of bound SPR traces. "measurement" binding trace, taken from FIG. 63->By combination->Separate single control injection SPR traces of protein #56 binding to serum albumin, CD70, CD123, CD33 or CD3, respectively, produced "calculated" traces.

FIG. 65: intraperitoneal injection of hBMC (n=5 mice/donor/2 to 6 hBMC donors depending on treatment), subcutaneous xenograft MOLM-13 cells and eight days after tumor cell injection with PBS1X (vehicle), 0.5mg/kgProtein #74, 0.5mg/kg +.>Protein #75, 0.5mg/kg +.>Protein #56, 0.5mg/kg +.>Tumor growth over time in mice treated with protein #57, 0.5mg/kg AMG330 analog, and 0.5mg/kg Fu Tuozhu mab analog.

FIG. 66: tumor growth over time in mice injected intraperitoneally with hPBMC (n=5 mice/donor/2 to 6 hPBMC donors used depending on treatment), subcutaneously xenografted MOLM-13 cells and treated with PBS1X (vehicle). 2mg/kg eight days after tumor cell injection Protein #56, 0.5mg/kg +.>Protein #56, 0.2mg/kg +.>Protein #56 and 0.02mg/kg +.>Protein #56.

FIG. 67 (A-C): the frequency of CD45 expressing human immune cells (i.e., cd45+) (fig. 67A) and activated human T cells (CD 4 expressing, fig. 67B; or CD8 expressing, fig. 67C) in dissociated MOLM-13 tumors after treatment (measured by T cell activation markers CD25 and CD 69). Activated T cells were gated as living cd4+/cd25+/cd69+ cells (fig. 67B) and living cd8+/cd25+/cd69+ cells (fig. 67C).

FIG. 68 (A-D)Cytokine and chemokine release in mouse serum after treatment. IFNγ levels, 68B.IL-6 levels, 68C.IL-2 levels, and 68D.TNFa levels.

FIG. 69 (A-B)Cytokine and chemokine release in the tumor environment of mice after treatment. Levels of ifnγ; graph 69b. Levels of il-6.

Detailed Description

Disclosed herein are recombinant proteins comprising a first binding agent that specifically binds to a protein expressed on the surface of an immune cell and at least two binding agents that specifically bind to tumor-associated antigens, wherein the two binding agents specifically bind to different tumor-associated antigens.

More specifically, disclosed herein are recombinant proteins comprising engineered ankyrin repeat domains with binding specificities for different targets such as CD3, CD33, CD123 and CD 70. Also disclosed herein are nucleic acids encoding the binding proteins, pharmaceutical compositions comprising the binding proteins or nucleic acids, and methods of using the binding proteins, nucleic acids, or pharmaceutical compositions. A library of engineered ankyrin repeat proteins (WO 2002/020565; binz et al, nat. Biotechnol.22,575-582,2004; stumpp et al, drug discovery. Today 13,695-701,2008) can be used to select target-specifically engineered ankyrin repeat domains that bind their targets with high affinity. Such target-specifically designed ankyrin repeat domains can then be used as valuable components of recombinant binding proteins for the treatment of diseases. Designed ankyrin repeat proteins are a class of binding molecules that have the potential to overcome the limitations of monoclonal antibodies, allowing novel therapeutic approaches. Such ankyrin repeat proteins may comprise a single designed ankyrin repeat domain, or may comprise a combination of two or more designed ankyrin repeat domains with the same or different target specificities (Stumpp et al Drug discovery.today 13,695-701,2008; U.S. Pat. No. 9,458,211). An ankyrin repeat protein comprising only a single engineered ankyrin repeat domain is a small protein (14 kDa) that can be selected to bind with high affinity and specificity to a given target protein. These features, as well as the possibility of combining two or more engineered ankyrin repeat domains in one protein, make engineered ankyrin repeat proteins ideal agonist, antagonist and/or inhibitor drug candidates. In addition, such ankyrin repeat proteins can be engineered to carry various effector functions, such as cytotoxic agents or half-life extenders, to achieve entirely new pharmaceutical forms. In summary, engineered ankyrin repeat proteins are examples of next generation protein therapeutics with potential beyond existing antibody drugs.

Is Molecular Partners AG, switzerland.

Molecules expressed on the surface of tumor cells (e.g., CD33, CD123, and CD 70) are potential targets for anti-cancer therapies, particularly if they are expressed on tumor cells, but not or at much lower levels on healthy cells. By way of example, CD33, CD123 and CD70 are expressed on the surface of AML blast cells and leukemia stem cells, but they are not simultaneously expressed or are simultaneously expressed at much lower levels on the surface of healthy cells (including hematopoietic stem cells).

In one aspect, the invention provides a recombinant protein comprising (1) a first binding agent that specifically binds to a protein expressed on the surface of an immune cell and (2) at least two binding agents that specifically bind to tumor-associated antigens, wherein the two binding agents specifically bind to different tumor-associated antigens.

In one aspect, the invention provides a recombinant protein comprising (1) a first binding agent that specifically binds to a protein expressed on the surface of an immune cell, and (2) at least two binding agents that specifically bind to tumor-associated antigens, wherein the two binding agents specifically bind to different tumor-associated antigens, and wherein the recombinant protein is capable of simultaneously binding the protein expressed on the surface of an immune cell and the two different tumor-associated antigens.

In one aspect, the invention provides a recombinant protein comprising (1) a first binding agent that specifically binds to a protein expressed on the surface of an immune cell, and (2) at least three binding agents that specifically bind to tumor-associated antigens, wherein the three binding agents specifically bind to different tumor-associated antigens.

In one aspect, the invention provides a recombinant protein comprising (1) a first binding agent that specifically binds to a protein expressed on the surface of an immune cell, and (2) at least three binding agents that specifically bind to tumor-associated antigens, wherein the three binding agents specifically bind to different tumor-associated antigens, and wherein the recombinant protein is capable of simultaneously binding the protein expressed on the surface of an immune cell and the three different tumor-associated antigens.

In one aspect, the tumor-associated antigen is co-expressed in tumor cells. In one aspect, the tumor cell is a tumor cell from a liquid tumor. In another aspect, the tumor cell is a liquid tumor cell from leukemia. In one aspect, the leukemia is Acute Myelogenous Leukemia (AML).

In one aspect, the invention provides a recombinant protein wherein said recombinant protein comprises only one of said binding agents that specifically binds a tumor associated antigen when compared to a recombinant protein comprising said binding agent that specifically binds a tumor associated antigen The recombinant protein can be isolated with a low dissociation constant (K _D ) Binding to a surface displaying the tumor-associated antigen.

In one aspect, the invention provides a recombinant protein wherein the surface displaying the tumor-associated antigen is the surface of the tumor cell.

In one aspect, the invention provides recombinant proteins wherein the lower dissociation constant (K _D ) Is a corresponding dissociation constant of the recombinant protein comprising only one of the binding agents that specifically binds a tumor-associated antigen of at least about 1/2, at least about 1/4, at least about 1/10, at least about 1/20, at least about 1/40, or at least about 1/100.

In one aspect, the invention provides a recombinant protein, wherein the immune cell is a T cell.

In one aspect, the invention provides a recombinant protein, wherein the T cell is a cd8+ cytotoxic T cell.

In one aspect, the invention provides a recombinant protein wherein the protein expressed on the surface of an immune cell is a protein that is part of a T cell receptor complex.

In one aspect, the invention provides a recombinant protein wherein the protein that is part of a T cell receptor complex is CD3.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein wherein the first binding agent is a designed ankyrin repeat domain having binding specificity for the protein expressed on the surface of an immune cell.

In one aspect, the invention provides a recombinant protein wherein the first binding agent is a designed ankyrin repeat domain having binding specificity for CD 3.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to any one of the amino acid sequences of SEQ ID NOs 1 to 5 (preferably SEQ ID NOs 2 or 3), and wherein a at the penultimate position of SEQ ID NOs 1 to 4 is optionally substituted with L and/or a at the last position of SEQ ID NOs 1 to 4 is optionally substituted with N; or wherein L at the penultimate position of SEQ ID NO. 5 is optionally substituted with A and/or N at the last position of SEQ ID NO. 5 is optionally substituted with A.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD3 comprises the amino acid sequence of SEQ ID NOs 1 to 5, preferably SEQ ID NOs 2 or 3, and wherein a at the penultimate position of SEQ ID NOs 1 to 4 is optionally substituted with L and/or a at the last position of SEQ ID NOs 1 to 4 is optionally substituted with N; or wherein L at the penultimate position of SEQ ID NO. 5 is optionally substituted with A and/or N at the last position of SEQ ID NO. 5 is optionally substituted with A.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to the amino acid sequence of SEQ ID No. 2, and wherein a at the penultimate position of SEQ ID No. 2 is optionally substituted with L, and/or a at the last position of SEQ ID No. 2 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD3 comprises the amino acid sequence of SEQ ID No. 2, and wherein a at the penultimate position of SEQ ID No. 2 is optionally substituted with L and/or a at the last position of SEQ ID No. 2 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to the amino acid sequence of SEQ ID NO:3, and wherein a at the penultimate position of SEQ ID NO:3 is optionally substituted with L, and/or a at the last position of SEQ ID NO:3 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD3 comprises the amino acid sequence of SEQ ID No. 3, and wherein a at the penultimate position of SEQ ID No. 3 is optionally substituted with L and/or a at the last position of SEQ ID No. 3 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein wherein the binding agent that specifically binds to a tumor-associated antigen is selected from the group consisting of (i) a second binding agent that specifically binds to a first tumor-associated antigen (TAA 1), (ii) a third binding agent that specifically binds to a second tumor-associated antigen (TAA 2), and (iii) a fourth binding agent that specifically binds to a third tumor-associated antigen (TAA 3).

In one aspect, the invention provides a recombinant protein wherein the binding agent that specifically binds to a tumor-associated antigen is selected from the group consisting of (i) a second binding agent that specifically binds to a first tumor-associated antigen (TAA 1), (ii) a third binding agent that specifically binds to a second tumor-associated antigen (TAA 2), and (iii) a fourth binding agent that specifically binds to a third tumor-associated antigen (TAA 3), and wherein the selected binding agents are capable of simultaneously binding to their respective tumor-associated antigen targets.

In one aspect, the invention provides a recombinant protein, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain. In one aspect, the invention provides a recombinant protein wherein the second binding agent is a designed ankyrin repeat domain having binding specificity for the TAA 1.

In one aspect, the invention provides a recombinant protein wherein the TAA1 is CD33.

In one aspect, the invention provides a recombinant protein wherein the second binding agent is a designed ankyrin repeat domain having binding specificity for CD33.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to any one of the amino acid sequences of SEQ ID NOs 15, 67 to 70 and 111 to 112, and wherein a at the penultimate position of SEQ ID NOs 15, 67 to 70 and 111 to 112 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 15, 67 to 70 and 111 to 112 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD33 comprises any of the amino acid sequences of SEQ ID NOs 15, 67 to 70 and 111 to 112, and wherein a at the penultimate position of SEQ ID NOs 15, 67 to 70 and 111 to 112 is optionally substituted with L and/or a at the last position of SEQ ID NOs 15, 67 to 70 and 111 to 112 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to the amino acid sequence of SEQ ID NO:15, and wherein a at the penultimate position of SEQ ID NO:15 is optionally substituted with L, and/or a at the last position of SEQ ID NO:15 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD33 comprises the amino acid sequence of SEQ ID No. 15, and wherein a at the penultimate position of SEQ ID No. 15 is optionally substituted with L and/or a at the last position of SEQ ID No. 15 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to the amino acid sequence of SEQ ID No. 67, and wherein a at the penultimate position of SEQ ID No. 67 is optionally substituted with L, and/or a at the last position of SEQ ID No. 67 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD33 comprises the amino acid sequence of SEQ ID No. 67, and wherein a at the penultimate position of SEQ ID No. 67 is optionally substituted with L and/or a at the last position of SEQ ID No. 67 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to the amino acid sequence of SEQ ID No. 68, and wherein a at the penultimate position of SEQ ID No. 68 is optionally substituted with L, and/or a at the last position of SEQ ID No. 68 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD33 comprises the amino acid sequence of SEQ ID No. 68, and wherein a at the penultimate position of SEQ ID No. 68 is optionally substituted with L and/or a at the last position of SEQ ID No. 68 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to the amino acid sequence of SEQ ID No. 69, and wherein a at the penultimate position of SEQ ID No. 69 is optionally substituted with L, and/or a at the last position of SEQ ID No. 69 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD33 comprises the amino acid sequence of SEQ ID No. 69, and wherein a at the penultimate position of SEQ ID No. 69 is optionally substituted with L and/or a at the last position of SEQ ID No. 70 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to the amino acid sequence of SEQ ID No. 13, and wherein a at the penultimate position of SEQ ID No. 70 is optionally substituted with L and/or a at the last position of SEQ ID No. 70 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD33 comprises the amino acid sequence of SEQ ID No. 70, and wherein a at the penultimate position of SEQ ID No. 70 is optionally substituted with L and/or a at the last position of SEQ ID No. 70 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to the amino acid sequence of SEQ ID No. 111, and wherein a at the penultimate position of SEQ ID No. 111 is optionally substituted with L, and/or a at the last position of SEQ ID No. 111 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD33 comprises the amino acid sequence of SEQ ID No. 111, and wherein a at the penultimate position of SEQ ID No. 111 is optionally substituted with L and/or a at the last position of SEQ ID No. 111 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to the amino acid sequence of SEQ ID No. 112, and wherein a at the penultimate position of SEQ ID No. 112 is optionally substituted with L, and/or a at the last position of SEQ ID No. 112 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD33 comprises the amino acid sequence of SEQ ID No. 112, and wherein a at the penultimate position of SEQ ID No. 112 is optionally substituted with L and/or a at the last position of SEQ ID No. 112 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is a designed ankyrin repeat domain having binding specificity for the protein expressed on the surface of an immune cell, and wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is a designed ankyrin repeat domain having binding specificity for CD3, and wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to any of the amino acid sequences of SEQ ID nos. 1 to 5, or wherein a at the penultimate position of SEQ ID nos. 1 to 4 is optionally substituted with L, and/or wherein L at the penultimate position of SEQ ID No. 5 is optionally substituted with a, and/or N at the last position of SEQ ID No. 5 is optionally substituted with a; and wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD3 comprises the amino acid sequence of SEQ ID NOs 1 to 5, and wherein a at the penultimate position of SEQ ID NOs 1 to 4 is optionally substituted with L and/or a at the last position of SEQ ID NOs 1 to 4 is optionally substituted with N, or wherein L at the penultimate position of SEQ ID NO 5 is optionally substituted with a and/or N at the last position of SEQ ID NO 5 is optionally substituted with a; and wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the second binding agent is a designed ankyrin repeat domain having binding specificity for CD 33.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any of the amino acid sequences of SEQ ID NOs 15, 67 to 70, and 111 to 112.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises any one of the amino acid sequences of SEQ ID NOs 15, 67 to 70, and 111 to 112.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is a designed ankyrin repeat domain having binding specificity for the protein expressed on the surface of an immune cell, and wherein the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is a designed ankyrin repeat domain having binding specificity for CD3, and wherein the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to any of the amino acid sequences of SEQ ID nos. 1 to 5, or wherein a at the penultimate position of SEQ ID nos. 1 to 4 is optionally substituted with L, and/or wherein L at the penultimate position of SEQ ID No. 5 is optionally substituted with a, and/or N at the last position of SEQ ID No. 5 is optionally substituted with a; and wherein the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD3 comprises the amino acid sequence of SEQ ID NOs 1 to 5, and wherein a at the penultimate position of SEQ ID NOs 1 to 4 is optionally substituted with L and/or a at the last position of SEQ ID NOs 1 to 4 is optionally substituted with N, or wherein L at the penultimate position of SEQ ID NO 5 is optionally substituted with a and/or N at the last position of SEQ ID NO 5 is optionally substituted with a; and wherein the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the third binding agent is a designed ankyrin repeat domain having binding specificity for CD 123.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or engineered repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any of the amino acid sequences of SEQ ID NOs 6, 65 to 66, and 102 to 106, and/or a at the last position of SEQ ID NOs 6, 65 to 66, and 102 to 106 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises any of the amino acid sequences of SEQ ID NOs 6, 65 to 66, and 102 to 106, and wherein a at the penultimate position of SEQ ID NOs 6, 65 to 66, and 102 to 106 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 6, 65 to 66, and 102 to 106 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or engineered repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO:6, and wherein a at the penultimate position of SEQ ID NO:6 is optionally substituted with L, and/or a at the last position of SEQ ID NO:6 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises the amino acid sequence of SEQ ID No. 6, and wherein a at the penultimate position of SEQ ID No. 6 is optionally substituted with L, and/or a at the last position of SEQ ID No. 6 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or engineered repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO:65, and wherein a at the penultimate position of SEQ ID NO:65 is optionally substituted with L, and/or a at the last position of SEQ ID NO:65 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises the amino acid sequence of SEQ ID NO:65, and wherein a at the penultimate position of SEQ ID NO:65 is optionally substituted with L, and/or a at the last position of SEQ ID NO:65 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or engineered repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO:66, and wherein a at the penultimate position of SEQ ID NO:66 is optionally substituted with L, and/or a at the last position of SEQ ID NO:66 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises the amino acid sequence of SEQ ID NO:66, and wherein a at the penultimate position of SEQ ID NO:66 is optionally substituted with L, and/or a at the last position of SEQ ID NO:66 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or engineered repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO:102, and wherein a at the penultimate position of SEQ ID NO:102 is optionally substituted with L, and/or a at the last position of SEQ ID NO:102 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises the amino acid sequence of SEQ ID NO:102, and wherein a at the penultimate position of SEQ ID NO:102 is optionally substituted with L, and/or a at the last position of SEQ ID NO:102 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or engineered repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO:103, and wherein a at the penultimate position of SEQ ID NO:103 is optionally substituted with L, and/or a at the last position of SEQ ID NO:103 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises the amino acid sequence of SEQ ID NO:103, and wherein a at the penultimate position of SEQ ID NO:103 is optionally substituted with L, and/or a at the last position of SEQ ID NO:103 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or engineered repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO 104, and wherein a at the penultimate position of SEQ ID NO 104 is optionally substituted with L, and/or a at the last position of SEQ ID NO 104 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises the amino acid sequence of SEQ ID No. 104, and wherein a at the penultimate position of SEQ ID No. 104 is optionally substituted with L, and/or a at the last position of SEQ ID No. 104 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or engineered repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO:105, and wherein a at the penultimate position of SEQ ID NO:105 is optionally substituted with L, and/or a at the last position of SEQ ID NO:105 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises the amino acid sequence of SEQ ID No. 105, and wherein a at the penultimate position of SEQ ID No. 105 is optionally substituted with L, and/or a at the last position of SEQ ID No. 105 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or engineered repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO:106, and wherein a at the penultimate position of SEQ ID NO:106 is optionally substituted with L, and/or a at the last position of SEQ ID NO:106 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises the amino acid sequence of SEQ ID No. 106, and wherein a at the penultimate position of SEQ ID No. 106 is optionally substituted with L, and/or a at the last position of SEQ ID No. 106 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is a designed ankyrin repeat domain having binding specificity for the protein expressed on the surface of an immune cell, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is a designed ankyrin repeat domain having binding specificity for CD3, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to any of the amino acid sequences of SEQ ID nos. 1 to 5, or wherein a at the penultimate position of SEQ ID nos. 1 to 4 is optionally substituted with L, and/or wherein L at the penultimate position of SEQ ID No. 5 is optionally substituted with a, and/or N at the last position of SEQ ID No. 5 is optionally substituted with a; and wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain; and wherein the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD3 comprises the amino acid sequence of SEQ ID NOs 1 to 5, and wherein a at the penultimate position of SEQ ID NOs 1 to 4 is optionally substituted with L and/or a at the last position of SEQ ID NO 6 is optionally substituted with N, or wherein L at the penultimate position of SEQ ID NO 5 is optionally substituted with a and/or N at the last position of SEQ ID NO 5 is optionally substituted with a; and wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain; and wherein the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is a designed ankyrin repeat domain having binding specificity for CD33, and wherein the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any of the amino acid sequences of SEQ ID NOs 15, 67 to 70, and 111 to 112; and wherein the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain; and wherein the ankyrin repeat domain having binding specificity for CD33 comprises any one of the amino acid sequences of SEQ ID NOs 15, 67 to 70 and 111 to 112; and wherein the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain; and wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to the amino acid sequence of SEQ ID No. 15; and wherein the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain; and wherein said ankyrin repeat domain having binding specificity for CD33 comprises the amino acid sequence of SEQ ID No. 15; and wherein the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID No. 67; wherein the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises the amino acid sequence of SEQ ID NO:67, wherein the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or engineered repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID No. 68, wherein the third binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or engineered repeat domain.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises the amino acid sequence of SEQ ID NO:68, wherein the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or engineered repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID No. 69, wherein the third binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or engineered repeat domain.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises the amino acid sequence of SEQ ID NO:69, wherein the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or engineered repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID No. 70, wherein the third binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or engineered repeat domain.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises the amino acid sequence of SEQ ID No. 70, wherein the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain; and wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to the amino acid sequence of SEQ ID No. 111; and wherein the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain; and wherein said ankyrin repeat domain with binding specificity for CD33 comprises the amino acid sequence of SEQ ID No. 111; and wherein the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain; and wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to the amino acid sequence of SEQ ID No. 112; and wherein the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain; and wherein said ankyrin repeat domain with binding specificity for CD33 comprises the amino acid sequence of SEQ ID No. 112; and wherein the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the third binding agent is a designed ankyrin repeat domain having binding specificity for CD 123.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any of the amino acid sequences of SEQ ID NOs 6, 65-66, and 102-106.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises any one of the amino acid sequences of SEQ ID NOs 6, 65 to 66, and 102 to 106.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 6.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises the amino acid sequence of SEQ ID No. 6.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 65.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises the amino acid sequence of SEQ ID No. 65.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 66.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises the amino acid sequence of SEQ ID No. 66.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID No. 102.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises the amino acid sequence of SEQ ID No. 102.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 103.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises the amino acid sequence of SEQ ID No. 103.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO 104.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises the amino acid sequence of SEQ ID No. 104.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID No. 105.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises the amino acid sequence of SEQ ID No. 105.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 106.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises the amino acid sequence of SEQ ID No. 106.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the fourth binding agent is a designed ankyrin repeat domain having binding specificity for CD 70.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD70 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any of the amino acid sequences of SEQ ID nos. 64 and 107 to 110.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD70 comprises any one of the amino acid sequences of SEQ ID NOs 64 and 107 to 110.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD70 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID No. 64.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD70 comprises the amino acid sequence of SEQ ID No. 64.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD70 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 107.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD70 comprises the amino acid sequence of SEQ ID No. 107.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD70 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID No. 108.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD70 comprises the amino acid sequence of SEQ ID No. 108.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD70 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID No. 109.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD70 comprises the amino acid sequence of SEQ ID No. 109.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD70 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of SEQ ID NO: 110.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, wherein the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD70 comprises the amino acid sequence of SEQ ID NO: 110.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or engineered repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any of the amino acid sequences of SEQ ID nos. 6, 65 to 66, and 102 to 106, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 96%, at least about 98%, or at least about 99% amino acid sequence identity to any of SEQ ID nos. 6, 65 to 66 and 102 to 106.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the anchor protein repeat domain having binding specificity for CD33 comprises any of the amino acid sequences of SEQ ID NOs 15, 67 to 70, and 111 to 112, and wherein the anchor protein repeat domain having binding specificity for CD123 comprises any of the amino acid sequences of SEQ ID NOs 6, 65 to 66, and 102 to 106.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is a engineered ankyrin repeat domain having binding specificity for the protein expressed on the surface of an immune cell, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to any of the amino acid sequences of SEQ ID nos. 6, 65 to 66 and 102 to 106, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 98%, or at least about 99% amino acid sequence identity to any of the amino acid sequences of SEQ ID nos. 6, 65 to 66 and 102 to 106.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is a engineered ankyrin repeat domain having binding specificity for CD3, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any of the amino acid sequences of SEQ ID nos. 6, 65 to 66, and 102 to 106, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 98%, or at least about 99% amino acid sequence identity to any of SEQ ID nos. 6, 65 to 66 and 102 to 106.

In one aspect, the invention provides a recombinant protein wherein said ankyrin repeat domain having binding specificity for CD3 comprises a sequence identical to SEQ ID NO:1 to 5, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical, and wherein the amino acid sequence of SEQ ID NO:1 to 4, and/or a at the last position of SEQ ID NO:1 to 4 is optionally substituted with L, or wherein L at the last position of SEQ ID NO:5 is optionally substituted with a, and/or N at the last position of SEQ ID NO:5 is optionally substituted with a, wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to any of the amino acid sequences of SEQ ID NOs 15, 67 to 70 and 111 to 112, such as at least about 86%, such as at least about 85%, for CD123, such as at least about 98% or at least about 99% identity to any of the amino acid sequences of SEQ ID NOs 6, 65 to 66 and 106 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%, or at least about 99% identical.

In one aspect, the invention provides a recombinant protein, wherein the ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence of SEQ ID NO:1 to 5, and wherein a at the penultimate position of SEQ ID NO:1 to 4 is optionally substituted with L, and/or a at the last position of SEQ ID NO:1 to 4 is optionally substituted with N, or wherein L at the penultimate position of SEQ ID NO:5 is optionally substituted with a, and/or N at the last position of SEQ ID NO:5 is optionally substituted with a, wherein the ankyrin repeat domain having binding specificity for CD33 comprises at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identical, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises at least about 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 91%, at least about 92%, at least about 94%, at least about 95%, at least about 96% and at least about amino acid sequence having at least about 98% or at least about 95% of any of the amino acid sequences of SEQ ID NO:15, 67 to 70 and 111 to 112.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is a designed ankyrin repeat domain having binding specificity for the protein expressed on the surface of an immune cell, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises any one of the amino acid sequences of SEQ ID NOs 15, 67 to 70 and 111 to 112, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises any one of the amino acid sequences of SEQ ID NOs 6, 65 to 66 and 102 to 106.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is a engineered ankyrin repeat domain having binding specificity for CD3, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises any one of the amino acid sequences of SEQ ID NOs 15, 67 to 70 and 111 to 112, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises any one of the amino acid sequences of SEQ ID NOs 6, 65 to 66 and 102 to 106.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to any of the amino acid sequences of SEQ ID NOs 1 to 4, or wherein a at the penultimate position of SEQ ID NOs 1 to 4 is optionally substituted with N, and/or wherein L at the penultimate position of SEQ ID NOs 5 is optionally substituted with a, and/or N at the last position of SEQ ID NOs 5 is optionally substituted with a, wherein the ankyrin repeat domain having binding specificity for CD33 comprises any of SEQ ID NOs 15, 67 to 70 and 111, or at least about 99% identity to any of the amino acid sequences of SEQ ID NOs 112, and wherein the ankyrin repeat domain having binding specificity for any of SEQ ID NOs 112 to any of SEQ ID NOs 6 to 112 comprises amino acid sequences of SEQ ID NOs 106 to 102.

In one aspect, the invention provides a recombinant protein, wherein the ankyrin repeat domain having binding specificity for CD3 comprises the amino acid sequence of SEQ ID NOs 1 to 5, and wherein a at the penultimate position of SEQ ID NOs 1 to 4 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 1 to 4 is optionally substituted with N, or wherein L at the penultimate position of SEQ ID NO 5 is optionally substituted with a, and/or N at the last position of SEQ ID NO 5 is optionally substituted with a, wherein the ankyrin repeat domain having binding specificity for CD33 comprises any of the amino acid sequences of SEQ ID NOs 15, 67 to 70 and 111 to 112, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises any of the amino acid sequences of SEQ ID NOs 6, 65 to 66 and 102 to 106.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD3 comprises the amino acid sequence of SEQ ID NOs 1 to 5, and wherein a at the penultimate position of SEQ ID NOs 1 to 4 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 1 to 4 is optionally substituted with N, or wherein L at the penultimate position of SEQ ID NOs 5 is optionally substituted with a, and/or N at the last position of SEQ ID NOs 5 is optionally substituted with a, wherein the ankyrin repeat domain having binding specificity for CD33 comprises any of the amino acid sequences of SEQ ID NOs 15, 67 to 70, and 111 to 112, and wherein a at the penultimate position of SEQ ID NOs 15, 67 to 70, and 111 to 112 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 15, 67 to 70, and 111 to 112 is optionally substituted with N, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises any of SEQ ID NOs 15, 67 to 70, and 111 to 112, and wherein a at the penultimate position of SEQ ID NOs 6 to 106, 67 to 70, and 111 to 112 is optionally substituted with L, and/or amino acid sequence of SEQ ID NOs 6 to 102, 66 to 102 and 102 to 102.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or engineered repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any of the amino acid sequences of SEQ ID nos. 64 and 107 to 110, and wherein the ankyrin repeat domain having binding specificity for CD70 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 98%, or at least about 99% identity to any of the amino acid sequences of SEQ ID nos. 64 and 107 to 110.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the anchor protein repeat domain having binding specificity for CD33 comprises any of the amino acid sequences of SEQ ID NOs 15, 67 to 70, and 111 to 112, and wherein the anchor protein repeat domain having binding specificity for CD70 comprises any of the amino acid sequences of SEQ ID NOs 64 and 107 to 110.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is a engineered ankyrin repeat domain having binding specificity for the protein expressed on the surface of an immune cell, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, 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% or at least about 99% identity to any of the amino acid sequences of SEQ ID nos. 64 and 107 to 110, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 96%, at least about 98% or at least about 99% amino acid sequence identity to any of SEQ ID nos. 64 and 107 to 110.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is a engineered ankyrin repeat domain having binding specificity for CD3, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any of the amino acid sequences of SEQ ID nos. 64 and 107 to 110, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 98%, or at least about 99% amino acid sequence identity to any of SEQ ID nos. 64 and 107 to any of the amino acid sequences of SEQ ID nos. 110.

In one aspect, the invention provides a recombinant protein wherein said ankyrin repeat domain having binding specificity for CD3 comprises a sequence identical to SEQ ID NO:1 to 5, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical, and wherein the amino acid sequence of SEQ ID NO: the a at the penultimate position of SEQ ID NOs 1 to 4 is optionally substituted with L and/or the a at the last position of SEQ ID NOs 1 to 4 is optionally substituted with N, or wherein the L at the penultimate position of SEQ ID NO 5 is optionally substituted with a and/or the N at the last position of SEQ ID NO 5 is optionally substituted with a, wherein said ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to any of the amino acid sequences of SEQ ID NOs 15, 67 to 70 and 111 to 112, and wherein said ankyrin repeat domain having binding specificity for CD70 comprises an amino acid sequence having at least 85%, such as at least about 87%, such as at least about 86%, at least about 110 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%, or at least about 99% identical.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD3 comprises the amino acid sequence of SEQ ID NO:1 to 5, and wherein a at the penultimate position of SEQ ID NO:1 to 4 is optionally substituted with L, and/or a at the last position of SEQ ID NO:1 to 4 is optionally substituted with N, or wherein L at the penultimate position of SEQ ID NO:5 is optionally substituted with a, and/or N at the last position of SEQ ID NO:5 is optionally substituted with a, wherein said ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to any of the amino acid sequences of SEQ ID NO:15, 67 to 70 and 111 to 112, and wherein said ankyrin repeat domain having binding specificity for CD70 comprises an amino acid sequence having at least about 85%, such as at least about 86%, at least about 87%, at least about 90%, at least about 91%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to any of the amino acid sequences of SEQ ID NO:64 and 107 to any of the amino acid sequences of at least about 86 to at least about 70.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is a designed ankyrin repeat domain having binding specificity for the protein expressed on the surface of an immune cell, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises any one of the amino acid sequences of SEQ ID NOs 15, 67 to 70 and 111 to 112, and wherein the ankyrin repeat domain having binding specificity for CD70 comprises any one of the amino acid sequences of SEQ ID NOs 64 and 107 to 110.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is a engineered ankyrin repeat domain having binding specificity for CD3, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises any one of the amino acid sequences of SEQ ID NOs 15, 67 to 70 and 111 to 112, and wherein the ankyrin repeat domain having binding specificity for CD70 comprises any one of the amino acid sequences of SEQ ID NOs 64 and 107 to 110.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to any of the amino acid sequences of SEQ ID nos. 1 to 4, or wherein a at the penultimate position of SEQ ID nos. 1 to 4 is optionally substituted with N, and/or wherein L at the penultimate position of SEQ ID No. 5 is optionally substituted with a, and/or N at the last position of SEQ ID No. 5 is optionally substituted with a, wherein the ankyrin repeat domain having binding specificity for CD33 comprises any of the amino acid sequences of SEQ ID nos. 15, 67 to 70 and 111, and any of the amino acid sequences of SEQ ID nos. 112 and 110 to any of the amino acid sequences of SEQ ID nos. 112.

In one aspect, the invention provides a recombinant protein, wherein the ankyrin repeat domain having binding specificity for CD3 comprises the amino acid sequence of SEQ ID NOs 1 to 5, and wherein a at the penultimate position of SEQ ID NOs 1 to 4 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 1 to 4 is optionally substituted with N, or wherein L at the penultimate position of SEQ ID NO 5 is optionally substituted with a, and/or N at the last position of SEQ ID NO 5 is optionally substituted with a, wherein the ankyrin repeat domain having binding specificity for CD33 comprises any of the amino acid sequences of SEQ ID NOs 15, 67 to 70 and 111 to 112, and wherein the ankyrin repeat domain having binding specificity for CD70 comprises any of the amino acid sequences of SEQ ID NOs 64 and 107 to 110.

In one aspect, the invention provides a recombinant protein, wherein the ankyrin repeat domain having binding specificity for CD3 comprises the amino acid sequence of SEQ ID NOs 1 to 5, and wherein a at the penultimate position of SEQ ID NOs 1 to 4 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 1 to 4 is optionally substituted with N, or wherein L at the penultimate position of SEQ ID NO 5 is optionally substituted with a, and/or N at the last position of SEQ ID NO 5 is optionally substituted with a, wherein the ankyrin repeat domain having binding specificity for CD33 comprises the amino acid sequence of SEQ ID NOs 15, 67 to 70 and 111 to 112, and wherein a at the penultimate position of SEQ ID NOs 15, 67 to 70 and 111 to 112 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 15, 65 to 70 and 111 to 112 is optionally substituted with N, and wherein the ankyrin repeat domain having binding specificity for CD70 comprises the amino acid sequence of SEQ ID NOs 15, 67 to 70 and 111 to 112, and wherein a at the penultimate position of SEQ ID NOs 110 and 107 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises a sequence that hybridizes to SEQ ID NO:15, 67 to 70, and 111 to 112, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any of the amino acid sequences of SEQ ID NOs 6, 65 to 66, and 102 to 106, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 98%, at least about 99% Amino acid sequence that is at least about 98% or at least about 99% identical.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and wherein the anchor protein repeat domain having binding specificity for CD33 comprises any of the amino acid sequences of SEQ ID NOs 15, 67 to 70, and 111 to 112, and wherein the anchor protein repeat domain having binding specificity for CD123 comprises any of the amino acid sequences of SEQ ID NOs 6, 65 to 66, and 102 to 106, and wherein the anchor protein repeat domain having binding specificity for CD70 comprises any of the amino acid sequences of SEQ ID NOs 64 and 107 to 110.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is a designed ankyrin repeat domain having binding specificity for the protein expressed on the surface of an immune cell, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises a sequence that hybridizes to SEQ ID NO:15, 67 to 70, and 111 to 112, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to any of the amino acid sequences of SEQ ID NOs 6, 65 to 66, and 102 to 106, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96% Amino acid sequence that is at least about 98% or at least about 99% identical.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is a engineered ankyrin repeat domain having binding specificity for CD3, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to any of the amino acid sequences of SEQ ID NOs 15, 67 to 70 and 111 to 112, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 98% or at least about 99% identity to any of the amino acid sequences of SEQ ID NOs 6, 65 to 66 and 102 to 106, wherein the ankyrin repeat domain having at least about 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 92%, at least about 93%, at least about 96%, at least about 95%, at least about 98% or at least about 98% identity to any of the amino acid sequences of SEQ ID NOs.

In one aspect, the invention provides a recombinant protein wherein said ankyrin repeat domain having binding specificity for CD3 comprises a sequence identical to SEQ ID NO:1 to 5, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical, and wherein the amino acid sequence of SEQ ID NO:1 to 4, and/or a at the last position of SEQ ID NO:1 to 4 is optionally substituted with L, or wherein L at the last position of SEQ ID NO:5 is optionally substituted with a, and/or N at the last position of SEQ ID NO:5 is optionally substituted with a, wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to any of the amino acid sequences of SEQ ID NOs 15, 67 to 70 and 111 to 112, such as at least about 86%, such as at least about 85%, for CD123, such as at least about 98% or at least about 99% identity to any of the amino acid sequences of SEQ ID NOs 6, 65 to 66 and 106 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%, or at least about 99% identical, and wherein the ankyrin repeat domain having binding specificity for CD70 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to any of the amino acid sequences of SEQ ID NOs 64 and 107 to 110.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD3 comprises the amino acid sequence of SEQ ID NO:1 to 5, and wherein a at the penultimate position of SEQ ID NO:1 to 4 is optionally substituted with L, and/or a at the last position of SEQ ID NO:1 to 4 is optionally substituted with N, or wherein L at the penultimate position of SEQ ID NO:5 is optionally substituted with a, and/or N at the last position of SEQ ID NO:5 is optionally substituted with a, wherein said ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity, and wherein said ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence having at least about 85%, such as at least about 86%, at least about 87%, at least about 90%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 94%, at least about 98% or at least about 95% identity to any of the amino acid sequences of SEQ ID NO:15, 67 to 70 and 111 to 112, and wherein the ankyrin repeat domain having binding specificity for CD70 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to any of the amino acid sequences of SEQ ID nos. 64 and 107 to 110.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is a designed ankyrin repeat domain having binding specificity for the protein expressed on the surface of immune cells, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises any of the amino acid sequences of SEQ ID NOs 15, 67 to 70 and 111 to 112, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises any of the amino acid sequences of SEQ ID NOs 6, 65 to 66 and 102 to 106, and wherein the ankyrin repeat domain having binding specificity for CD70 comprises any of the amino acid sequences of SEQ ID NOs 64 and 107 to 110.

In one aspect, the invention provides a recombinant protein, wherein the first binding agent is a engineered ankyrin repeat domain having binding specificity for CD3, and wherein the ankyrin repeat domain having binding specificity for CD33 comprises any one of the amino acid sequences of SEQ ID NOs 15, 67 to 70 and 111 to 112, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises any one of the amino acid sequences of SEQ ID NOs 6, 65 to 66 and 102 to 106, and wherein the ankyrin repeat domain having binding specificity for CD70 comprises any one of the amino acid sequences of SEQ ID NOs 64 and 107 to 110.

In one aspect, the invention provides a recombinant protein wherein the anchor protein repeat domain having binding specificity for CD3 comprises an amino acid sequence having at least 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98% or at least about 99% identity to any of the amino acid sequences of SEQ ID NOs 1 to 4, or wherein a at the penultimate position of SEQ ID NOs 1 to 4 is optionally substituted with N, and/or wherein L at the penultimate position of SEQ ID NOs 5 is optionally substituted with a, and/or N at the last position of SEQ ID NOs 5 is optionally substituted with a, wherein the anchor protein repeat domain having binding specificity for CD33 comprises an amino acid sequence of any of SEQ ID NOs 15, 67 to 70 and 111 to amino acid sequence of SEQ ID NOs 112 and wherein the anchor domain having binding specificity for any of SEQ ID NOs 6 to 112 comprises amino acid sequence of any of SEQ ID NOs 110 to 102 and any of the amino acid sequences of SEQ ID NOs 102 to 102.

In one aspect, the invention provides a recombinant protein, wherein the ankyrin repeat domain having binding specificity for CD3 comprises the amino acid sequence of SEQ ID NOs 1 to 5, and wherein a at the penultimate position of SEQ ID NOs 1 to 4 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 1 to 4 is optionally substituted with N, or wherein L at the penultimate position of SEQ ID NO 5 is optionally substituted with a, and/or N at the last position of SEQ ID NO 5 is optionally substituted with a, wherein the ankyrin repeat domain having binding specificity for CD33 comprises any of the amino acid sequences of SEQ ID NOs 15, 67 to 70 and 111 to 112, and wherein the ankyrin repeat domain having binding specificity for CD123 comprises any of the amino acid sequences of SEQ ID NOs 6, 65 to 66 and 102 to 106, and wherein the ankyrin repeat domain having binding specificity for CD70 comprises any of the amino acid sequences of SEQ ID NOs 64 to 107.

In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD3 comprises the amino acid sequence of SEQ ID NO:1 to 5, and wherein a at the penultimate position of SEQ ID NO:1 to 4 is optionally substituted with L, and/or a at the last position of SEQ ID NO:1 to 4 is optionally substituted with N, or wherein L at the penultimate position of SEQ ID NO:5 is optionally substituted with a, and/or N at the last position of SEQ ID NO:5 is optionally substituted with a, wherein the anchor protein repeat domain having binding specificity for CD33 comprises any of the amino acid sequences of SEQ ID NO:15, 67 to 70 and 111 to 112, and wherein a at the penultimate position of SEQ ID NO:15, 67 to 70 and 111 to 112 is optionally substituted with L, and/or a at the last position of SEQ ID NO:15, 67 to 70 and 111 to 112 is optionally substituted with N, and wherein the anchor protein repeat domain having binding specificity for CD123 comprises any of the amino acid sequences of SEQ ID NO:6, 65 to 66 and 102 to 106, and wherein any of the penultimate positions of SEQ ID NO:6, 67 to 70 and 111 to 112 is optionally substituted with L at any of the penultimate positions of 6, 67 to 70 and 110 to 102, and wherein the anchor protein repeat domain having binding specificity for CD123 comprises any of 6 to 66 to 106, and amino acid at the last position of 110 to 110 and/or the last position of 111 to 112 is optionally substituted with N, and/or A at the last position of SEQ ID NOS: 64 and 107 to 110 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein, wherein the recombinant protein comprises the first binding agent, the second binding agent, and the third binding agent, and wherein the recombinant protein is capable of binding the targets of each of the first binding agent, the second binding agent, and the third binding agent simultaneously.

In one aspect, the invention provides a recombinant protein, wherein the recombinant protein comprises the first binding agent, the second binding agent, and the third binding agent, and wherein the first binding agent, the second binding agent, and the third binding agent are arranged from N-terminus to C-terminus according to the following formula: (third binder) - (second binder) - (first binder).

In one aspect, the invention provides a recombinant protein wherein the first binding agent, the second binding agent, and the third binding agent are arranged from N-terminus to C-terminus according to the formula: (second binding agent) - (third binding agent) - (first binding agent).

In one aspect, the invention provides a recombinant protein, and wherein the first binding agent, the second binding agent, and the third binding agent are arranged from N-terminus to C-terminus according to the formula: (first binding agent) - (second binding agent) - (third binding agent).

In one aspect, the invention provides a recombinant protein wherein the first binding agent, the second binding agent, and the third binding agent are arranged from N-terminus to C-terminus according to the formula: (first binding agent) - (third binding agent) - (second binding agent).

In one aspect, the invention provides a recombinant protein, wherein the recombinant protein comprises the first binding agent, the second binding agent, and the fourth binding agent, and wherein the recombinant protein is capable of binding the targets of each of the first binding agent, the second binding agent, and the fourth binding agent simultaneously.

In one aspect, the invention provides a recombinant protein, wherein the recombinant protein comprises the first binding agent, the second binding agent, and the fourth binding agent, and wherein the first binding agent, the second binding agent, and the fourth binding agent are arranged from N-terminus to C-terminus according to the following formula: (fourth binder) - (second binder) - (first binder).

In one aspect, the invention provides a recombinant protein wherein the first binding agent, the second binding agent, and the fourth binding agent are arranged from N-terminus to C-terminus according to the formula: (second binding agent) - (fourth binding agent) - (first binding agent).

In one aspect, the invention provides a recombinant protein wherein the first binding agent, the second binding agent, and the fourth binding agent are arranged from N-terminus to C-terminus according to the formula: (first binding agent) - (second binding agent) - (fourth binding agent).

In one aspect, the invention provides a recombinant protein wherein the first binding agent, the second binding agent, and the fourth binding agent are arranged from N-terminus to C-terminus according to the formula: (first binding agent) - (fourth binding agent) - (second binding agent).

In one aspect, the invention provides a recombinant protein, wherein the recombinant protein comprises the first binding agent, the third binding agent, and the fourth binding agent, and wherein the recombinant protein is capable of binding the targets of each of the first binding agent, the third binding agent, and the fourth binding agent simultaneously.

In one aspect, the invention provides a recombinant protein, wherein the recombinant protein comprises the first binding agent, the third binding agent, and the fourth binding agent, and wherein the first binding agent, the third binding agent, and the fourth binding agent are arranged from N-terminus to C-terminus according to the following formula: (fourth binder) - (third binder) - (first binder).

In one aspect, the invention provides a recombinant protein wherein the first binding agent, the third binding agent, and the fourth binding agent are arranged from N-terminus to C-terminus according to the formula: (third binder) - (fourth binder) - (first binder).

In one aspect, the invention provides a recombinant protein wherein the first binding agent, the third binding agent, and the fourth binding agent are arranged from N-terminus to C-terminus according to the formula: (first binding agent) - (third binding agent) - (fourth binding agent).

In one aspect, the invention provides a recombinant protein wherein the first binding agent, the third binding agent, and the fourth binding agent are arranged from N-terminus to C-terminus according to the formula: (first binding agent) - (fourth binding agent) - (third binding agent).

In one aspect, the invention provides a recombinant protein, wherein the recombinant protein comprises the first binding agent, the second binding agent, the third binding agent, and the fourth binding agent, and wherein the recombinant protein is capable of binding the targets of the first binding agent, the second binding agent, the third binding agent, and the fourth binding agent, respectively, simultaneously.

In one aspect, the invention provides a recombinant protein, wherein the recombinant protein comprises the first binding agent, the second binding agent, the third binding agent, and the fourth binding agent, and wherein the first binding agent, the second binding agent, the third binding agent, and the fourth binding agent are arranged from N-terminus to C-terminus according to the following formula: (fourth binder) - (third binder) - (second binder) - (first binder).

In one aspect, the invention provides a recombinant protein, wherein the recombinant protein comprises the first binding agent, the second binding agent, the third binding agent, and the fourth binding agent, and wherein the first binding agent, the second binding agent, the third binding agent, and the fourth binding agent are arranged from N-terminus to C-terminus according to the following formula: (second binding agent) - (third binding agent) - (fourth binding agent) - (first binding agent).

In one aspect, the invention provides a recombinant protein wherein the first binding agent, the second binding agent, the third binding agent, and/or the fourth binding agent are covalently linked to a peptide linker.

In one aspect, the invention provides a recombinant protein comprising a peptide linker, wherein the peptide linker is a proline-threonine rich peptide linker.

In one aspect, the invention provides a recombinant protein comprising a peptide linker, wherein the amino acid sequence of the peptide linker has a length of 1 to 50 amino acids. In one aspect, the invention provides a recombinant protein comprising a peptide linker, wherein the amino acid sequence of the peptide linker has a length of 1 to 30 amino acids.

In one aspect, the invention provides a recombinant protein wherein a at the penultimate position of any one of SEQ ID NOs 1, 2, 3, 4, 6, 15, 64 to 70 and 102 to 112 is optionally substituted with L and/or a at the last position of any one of SEQ ID NOs 1, 2, 3, 4, 6, 15, 64 to 70 and 102 to 112 is optionally substituted with N; or wherein L at the penultimate position of SEQ ID NO. 5 is optionally substituted with A and/or N at the last position of SEQ ID NO. 5 is optionally substituted with A.

In one aspect, the invention provides a recombinant protein wherein any of the first, second, or third ankyrin repeat domains additionally comprises G, S or GS at the N-terminus.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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% or at least about 99% identical to any of the amino acid sequences of SEQ ID NOs 7 to 10 and 58 to 62, preferably wherein the protein comprises a polypeptide having an amino acid sequence of any of SEQ ID NOs 7 to 10 and 58 to 62.

In one aspect, the invention provides recombinant proteins comprising a polypeptide consisting of an amino acid sequence having at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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% or at least about 99% identity to any of the amino acid sequences of SEQ ID NOS.7-10 and 58-62. In one aspect, the invention provides a recombinant protein comprising a polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs 7 to 10 and 58 to 62.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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% or at least about 99% identical to the amino acid sequence of SEQ ID NO. 7, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO. 7.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO. 8, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO. 8.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO 9, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO 9.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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% or at least about 99% identical to the amino acid sequence of SEQ ID NO. 10, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO. 10.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO. 58, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO. 58.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO 59, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO 59.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO. 60, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO. 60.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO. 61, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO. 61.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO. 62, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO. 62.

In one aspect, the invention provides recombinant proteins wherein the proteins are present at or below about 10 in PBS ^-6 M, or equal to or less than about 5X 10 ^-7 Dissociation constant of M (K _D ) Binds human CD3.

In one aspect, the present invention provides a recombinant protein comprising a first binding agent that specifically binds to a protein expressed on the surface of an immune cell, a second binding agent that specifically binds to a first tumor-associated antigen, and a third binding agent that specifically binds to a second tumor-associated antigen, wherein the first binding protein is present in PBS at a level of about 10 or less ^-6 M, or equal to or less than about 5X 10 ^-7 Dissociation constant of M (K _D ) Binds human CD3. Thus, in one aspect, the binding protein is present at or below about 10 in PBS ^-6 Dissociation constant of M (K _D ) Binds human CD3. In another aspect, the binding protein is present at or below about 5×10 in PBS ^-7 Dissociation constant of M (K _D ) Binds human CD3.

In one aspectThe recombinant protein comprises a first binding agent that specifically binds to a protein expressed on the surface of an immune cell, wherein the binding protein is at or below about 10 in PBS ^-6 Dissociation constant of M (K _D ) Binding human CD3, and wherein the ankyrin repeat domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 1 to 5, wherein a at the penultimate position of SEQ ID NOs 1 to 4 is optionally substituted with L and/or a at the last position of SEQ ID NOs 1 to 4 is optionally substituted with N; or wherein L at the penultimate position of SEQ ID NO. 5 is optionally substituted with A and/or N at the last position of SEQ ID NO. 5 is optionally substituted with A. In another aspect, the recombinant protein comprises an ankyrin repeat domain having binding specificity for CD3, wherein the binding protein is at or below about 5 x 10 in PBS ^-7 M binds human CD3, and wherein the ankyrin repeat domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 1 to 5, wherein a at the penultimate position of SEQ ID NOs 1 to 5 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 1 to 4 is optionally substituted with N; or wherein L at the penultimate position of SEQ ID NO. 5 is optionally substituted with A and/or N at the last position of SEQ ID NO. 5 is optionally substituted with A. Thus, in one aspect, the protein is present at less than about 5X 10 in PBS ^-7 Dissociation constant of M (K _D ) Binds human CD3 and the ankyrin repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs 1 to 5. In one aspect, the binding protein is present at less than about 5×10 in PBS ^-7 Dissociation constant of M (K _D ) Binds human CD3 and the ankyrin is heavyThe multiple domain comprises an amino acid sequence having at least 90% amino acid sequence identity to any one of SEQ ID NOs 1 to 5. In another aspect, the protein is present in less than about 5×10 in PBS ^-7 Dissociation constant of M (K _D ) Binds human CD3 and the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity to any one of SEQ ID NOs 1 to 5; and in another aspect, the binding protein is present at less than about 5X 10 in PBS ^-7 Dissociation constant of M (K _D ) Binds human CD3 and the ankyrin repeat domain comprises an amino acid sequence having at least 95% amino acid sequence identity to any one of SEQ ID NOs 1 to 5. In one aspect, the binding protein is present at less than about 5×10 in PBS ^-7 Dissociation constant of M (K _D ) Binds human CD3 and the ankyrin repeat domain comprises an amino acid sequence having at least 98% amino acid sequence identity to any one of SEQ ID NOs 1 to 5; and in one aspect, the binding protein is present at less than about 5X 10 in PBS ^-7 Dissociation constant of M (K _D ) Binds human CD3 and the ankyrin repeat domain comprises the amino acid sequence of any one of SEQ ID NOs 1 to 5. Thus, in one aspect, the recombinant protein comprises a first binding agent that specifically binds to a protein expressed on the surface of an immune cell, wherein the binding protein is at or below about 5 x 10 in PBS ^-7 Dissociation constant of M (K _D ) Binds human CD3, and wherein the ankyrin repeat domain comprises the amino acid sequence of any one of SEQ ID NOs 1 to 5, wherein a at the penultimate position of SEQ ID NOs 1 to 4 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 1 to 4 is optionally substituted with N; or wherein L at the penultimate position of SEQ ID NO. 5 is optionally substituted with A and/or N at the last position of SEQ ID NO. 5 is optionally substituted with A.

In one aspect, the invention provides a recombinant protein comprising a first binding agent that specifically binds to a protein expressed on the surface of an immune cell, a second binding agent that specifically binds to a first tumor-associated antigen, a second tumor-associated antigenWherein the binding protein binds specifically to a third tumor-associated antigen in PBS at a level of about 10 or less ^-6 M, or equal to or less than about 5X 10 ^-7 Dissociation constant of M (K _D ) Binds human CD3. Thus, in one aspect, the binding protein is present at or below about 10 in PBS ^-6 Dissociation constant of M (K _D ) Binds human CD3. In another aspect, the binding protein is present at or below about 5×10 in PBS ^-7 Dissociation constant of M (K _D ) Binds human CD3.

In one aspect, the recombinant protein comprises a first binding agent that specifically binds to a protein expressed on the surface of an immune cell, wherein the binding protein is at or below about 10 in PBS ^-6 M binds human CD3, and wherein the ankyrin repeat domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 1 to 5, wherein a at the penultimate position of SEQ ID NOs 1 to 4 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 1 to 4 is optionally substituted with N; or wherein L at the penultimate position of SEQ ID NO. 5 is optionally substituted with A and/or N at the last position of SEQ ID NO. 5 is optionally substituted with A. In another aspect, the recombinant protein comprises an ankyrin repeat domain having binding specificity for CD3, wherein the binding protein is at or below about 5 x 10 in PBS ^-7 M binds human CD3, and wherein the ankyrin repeat domain comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 1 to 5, wherein a at the penultimate position of SEQ ID NOs 1 to 4 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 1 to 4 is optionally substituted with N; or it is L in the penultimate position of SEQ ID No. 5 is optionally substituted with A and/or N in the last position of SEQ ID No. 5 is optionally substituted with A. Thus, in one aspect, the protein is present at less than about 5X 10 in PBS ^-7 The dissociation constant (KD) of M binds human CD3 and the ankyrin repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs 1 to 5. In one aspect, the binding protein is present at less than about 5×10 in PBS ^-7 Dissociation constant of M (K _D ) Binds human CD3 and the ankyrin repeat domain comprises an amino acid sequence having at least 90% amino acid sequence identity to any one of SEQ ID NOs 1 to 5. In another aspect, the protein is present in less than about 5×10 in PBS ^-7 Dissociation constant of M (K _D ) Binds human CD3 and the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity to any one of SEQ ID NOs 1 to 5; and in another aspect, the binding protein is present at less than about 5X 10 in PBS ^-7 Dissociation constant of M (K _D ) Binds human CD3 and the ankyrin repeat domain comprises an amino acid sequence having at least 95% amino acid sequence identity to any one of SEQ ID NOs 1 to 5. In one aspect, the binding protein is present at less than about 5×10 in PBS ^-7 Dissociation constant of M (K _D ) Binds human CD3 and the ankyrin repeat domain comprises an amino acid sequence having at least 98% amino acid sequence identity to any one of SEQ ID NOs 1 to 5; and in one aspect, the binding protein is present at less than about 5X 10 in PBS ^-7 Dissociation constant of M (K _D ) Binds human CD3 and the ankyrin repeat domain comprises the amino acid sequence of any one of SEQ ID NOs 1 to 5. Thus, in one aspect, the recombinant protein comprises a first binding agent that specifically binds to a protein expressed on the surface of an immune cell, wherein the binding protein is at or below about 5 x 10 in PBS ^-7 Dissociation constant of M (K _D ) Binds human CD3, and wherein the ankyrin repeat domain comprises the amino acid sequence of any one of SEQ ID NOS: 1 to 5, wherein at the penultimate position of SEQ ID NOS: 1 to 4Optionally substituted with L and/or A at the last position of SEQ ID NOS 1 to 4 is optionally substituted with N; or wherein L at the penultimate position of SEQ ID NO. 5 is optionally substituted with A and/or N at the last position of SEQ ID NO. 5 is optionally substituted with A. In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, and a third binding agent having binding specificity for CD123, wherein the protein is present in PBS at a level of about 10 or less ^-6 M, or equal to or less than about 10 ^-7 M, or equal to or less than about 10 ^-8 M, or equal to or less than about 10X 10 ^-9 The dissociation constant (KD) of M binds to human CD33.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, and a third binding agent having binding specificity for CD123, wherein the protein is present in PBS at a level of about 10 or less ^-6 Dissociation constant of M (K _D ) Binds human CD33, and wherein the second binding agent comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 15, 67 to 70 and 111 to 112, wherein a at the penultimate position of SEQ ID NOs 15, 67 to 70 and 111 to 112 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 15, 67 to 70 and 111 to 112 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, and a third binding agent having binding specificity for CD123, wherein the protein is present in PBS at a level of about 10 or less ^-7 Dissociation constant of M (K _D ) Binds human CD33, and wherein the second binding agent comprises a polypeptide having at least 80% amino acid sequence that binds to any one of SEQ ID NOs 15, 67 to 70, and 111 to 112,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity, wherein a at the penultimate position of SEQ ID NOs 15, 67 to 70 and 111 to 112 is optionally substituted by L and/or a at the last position of SEQ ID NOs 15, 67 to 70 and 111 and 112 is optionally substituted by N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, and a third binding agent having binding specificity for CD123, wherein the protein is present in PBS at a level of about 10 or less ^-8 Dissociation constant of M (K _D ) Binds human CD33, and wherein the second binding agent comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 15, 67 to 70 and 111 to 112, wherein a at the penultimate position of SEQ ID NOs 15, 67 to 70 and 111 and 112 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 15, 67 to 70 and 111 and 112 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, and a third binding agent having binding specificity for CD123, wherein the protein is present in PBS at a level of about 10 or less ^-9 Dissociation constant of M (K _D ) Binds human CD33, and wherein the second binding agent comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 15, 67 to 70 and 111 to 112, wherein a at the penultimate position of SEQ ID NOs 15, 67 to 70 and 111 to 112 is optionally substituted with L, and/orThe A at the last position of SEQ ID NOS 15, 67 to 70 and 111 to 112 is optionally substituted by N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, and a third binding agent having binding specificity for CD123, wherein the protein is present in PBS at a level of about 10 or less ^-10 Dissociation constant of M (K _D ) Binds human CD33, and wherein the second binding agent comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 15, 67 to 70 and 111 to 112, wherein a at the penultimate position of SEQ ID NOs 15, 67 to 70 and 111 to 112 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 15, 67 to 70 and 111 to 112 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, and a third binding agent having binding specificity for CD123, wherein the protein is present in PBS at a level of about 10 or less ^-6 M, or equal to or less than about 10 ^-7 M, or equal to or less than about 10 ^- ⁸ M, or equal to or less than about 10 ^-9 M, or equal to or less than about 10 ^-10 Dissociation constant of M (K _D ) Binds human CD123.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, and a third binding agent having binding specificity for CD123, wherein the protein is present in PBS at a level of about 10 or less ^-6 Dissociation constant of M (K _D ) Binds human CD123 and wherein the third binding agent comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89% to any of SEQ ID NOs 6, 65 to 66 and 102 to 106An amino acid sequence of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity, wherein a at the penultimate position of SEQ ID NOs 6, 65 to 66 and 102 to 106 is optionally substituted by L and/or a at the last position of SEQ ID NOs 6, 65 to 66 and 102 to 106 is optionally substituted by N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, and a third binding agent having binding specificity for CD123, wherein the protein is present in PBS at a level of about 10 or less ^-7 Dissociation constant of M (K _D ) Binds human CD123 and wherein the third binding agent comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 6, 65 to 66 and 102 to 106, wherein a at the penultimate position of SEQ ID NOs 6, 65 to 66 and 102 to 106 is optionally substituted with L and/or a at the last position of SEQ ID NOs 6, 65 to 66 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, and a third binding agent having binding specificity for CD123, wherein the protein is present in PBS at a level of about 10 or less ^-8 Dissociation constant of M (K _D ) Binds human CD123 and wherein the third binding agent comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 6, 65 to 66 and 102 to 106, wherein a at the penultimate position of SEQ ID NOs 6, 65 to 66 and 102 to 106 is optionally substituted with L and/or a at the last position of SEQ ID NOs 6, 65 to 66 and 102 to 106 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, and a third binding agent having binding specificity for CD123, wherein the protein is present in PBS at a level of about 10 or less ^-9 Dissociation constant of M (K _D ) Binds human CD123 and wherein the third binding agent comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 6, 65 to 66 and 102 to 106, wherein a at the penultimate position of SEQ ID NOs 6, 65 to 66 and 102 to 106 is optionally substituted with L and/or a at the last position of SEQ ID NOs 6, 65 to 66 and 102 to 106 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, and a third binding agent having binding specificity for CD123, wherein the protein is present in PBS at a level of about 10 or less ^-10 Dissociation constant of M (K _D ) Binds human CD123 and wherein the third binding agent comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 6, 65 to 66 and 102 to 106, wherein a at the penultimate position of SEQ ID NOs 6, 65 to 66 and 102 to 106 is optionally substituted with L and/or a at the last position of SEQ ID NOs 6, 65 to 66 and 102 to 106 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, and a fourth binding agent having binding specificity for CD70, wherein the protein is present in PBS at a level of about 10 or less ^-6 M, or equal to or less than about 10 ^-7 M, or equal to or less than about 10 ^- ⁸ M, or equal to or less than about 10 ^-9 M, or equal to or less than about 10 ^-10 Dissociation constant of M (K _D ) Binds human CD70.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, and a fourth binding agent having binding specificity for CD70, wherein the protein is present in PBS at a level of about 10 or less ^-6 Dissociation constant of M (K _D ) Binds human CD70, and wherein the fourth binding agent comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 64 and 107 to 110, wherein a at the penultimate position of SEQ ID NOs 64 and 107 to 110 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 64 and 107 to 110 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, and a fourth binding agent having binding specificity for CD70, wherein the protein is present in PBS at a level of about 10 or less ^-7 Dissociation constant of M (K _D ) Binds human CD70, and wherein the fourth binding agent comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 64 and 107 to 110, wherein a at the penultimate position of SEQ ID NOs 64 and 107 to 110 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 64 and 107 to 110 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having binding specificity for CD3Wherein the protein is present in PBS at a level of about 10 or less, a second binding agent having binding specificity for CD33, and a fourth binding agent having binding specificity for CD70 ^-8 Dissociation constant of M (K _D ) Binds human CD70, and wherein the fourth binding agent comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 64 and 107 to 110, wherein a at the penultimate position of SEQ ID NOs 64 and 107 to 110 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 64 and 107 to 110 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, and a fourth binding agent having binding specificity for CD70, wherein the protein is present in PBS at a level of about 10 or less ^-9 Dissociation constant of M (K _D ) Binds human CD70, and wherein the fourth binding agent comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 64 and 107 to 110, wherein a at the penultimate position of SEQ ID NOs 64 and 107 to 110 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 64 and 107 to 110 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, and a fourth binding agent having binding specificity for CD70, wherein the protein is present in PBS at a level of about 10 or less ^-10 Dissociation constant of M (K _D ) Binds human CD70 and wherein the second binding agent comprises a polypeptide having at least 80%, 81%, 82%, 83%, 84%, amino acid sequence that is identical to any one of SEQ ID nos. 64 and 107 to 110,An amino acid sequence of 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity, wherein a at the penultimate position of SEQ ID nos. 64 and 107 to 110 is optionally substituted with L and/or a at the last position of SEQ ID nos. 64 and 107 to 110 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, a third binding agent having binding specificity for CD123, and a fourth binding agent having binding specificity for CD70, wherein the protein is present in PBS at a level of about 10 or less ^-6 M, or equal to or less than about 10 ^-7 M, or equal to or less than about 10 ^-8 M, or equal to or less than about 10X 10 ^-9 Dissociation constant of M (K _D ) Binds human CD33.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, a third binding agent having binding specificity for CD123, and a fourth binding agent having binding specificity for CD70, wherein the protein is present in PBS at a level of about 10 or less ^-6 Dissociation constant of M (K _D ) Binds human CD33, and wherein the second binding agent comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 15, 67 to 70 and 111 to 112, wherein a at the penultimate position of SEQ ID NOs 15, 67 to 70 and 111 to 112 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 16, 67 to 70 and 111 to 112 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, a third binding agent having binding specificity for CD123 A cocktail and a fourth binding agent having binding specificity for CD70, wherein the protein is present in PBS at a level of about 10 or less ^-7 Dissociation constant of M (K _D ) Binds human CD33, and wherein the second binding agent comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 15, 67 to 70 and 111 to 112, wherein a at the penultimate position of SEQ ID NOs 15, 67 to 70 and 111 to 112 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 15, 67 to 70 and 111 to 112 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, a third binding agent having binding specificity for CD123, and a fourth binding agent having binding specificity for CD70, wherein the protein is present in PBS at a level of about 10 or less ^-8 Dissociation constant of M (K _D ) Binds human CD33, and wherein the second binding agent comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 15, 67 to 70 and 111 to 112, wherein a at the penultimate position of SEQ ID NOs 15, 67 to 70 and 111 to 112 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 15, 67 to 70 and 111 to 112 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, a third binding agent having binding specificity for CD123, and a fourth binding agent having binding specificity for CD70, wherein the protein is present in PBS at a ratio of equal to or less than about 10X 10 ^-9 Dissociation constant of M (K _D ) Binds human CD33, and wherein the second binding agentComprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 15, 67 to 70 and 111 to 112, wherein a at the penultimate position of SEQ ID NOs 15, 67 to 70 and 111 to 112 is optionally substituted by L and/or a at the last position of SEQ ID NOs 15, 67 to 70 and 111 to 112 is optionally substituted by N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, a third binding agent having binding specificity for CD123, and a fourth binding agent having binding specificity for CD70, wherein the protein is present in PBS at a ratio of equal to or less than about 10X 10 ^-9 Dissociation constant of M (K _D ) Binds human CD33, and wherein the second binding agent comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 15, 67 to 70 and 111 to 112, wherein a at the penultimate position of SEQ ID NOs 15, 67 to 70 and 111 to 112 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 15, 67 to 70 and 111 to 112 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, a third binding agent having binding specificity for CD123, and a fourth binding agent having binding specificity for CD70, wherein the protein is present in PBS at a level of about 10 or less ^-6 M, or equal to or less than about 10 ^-7 M, or equal to or less than about 10 ^-8 M, or equal to or less than about 10X 10 ^-9 Dissociation constant of M (K _D ) Binds human CD123.

In one aspect, the invention provides a recombinant protein comprising a polypeptide of the invention A first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, a third binding agent having binding specificity for CD123, and a fourth binding agent having binding specificity for CD70, wherein the protein is present in PBS at a level of about 10 or less ^-6 Dissociation constant of M (K _D ) Binds human CD123 and wherein the third binding agent comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 6, 65 to 66 and 102 to 106, wherein a at the penultimate position of SEQ ID NOs 6, 65 to 66 and 102 to 106 is optionally substituted with L and/or a at the last position of SEQ ID NOs 6, 65 to 66 and 102 to 106 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, a third binding agent having binding specificity for CD123, and a fourth binding agent having binding specificity for CD70, wherein the protein is present in PBS at a level of about 10 or less ^-7 Dissociation constant of M (K _D ) Binds human CD123 and wherein the third binding agent comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 6, 65 to 66 and 102 to 106, wherein a at the penultimate position of SEQ ID NOs 6, 65 to 66 and 102 to 106 is optionally substituted with L and/or a at the last position of SEQ ID NOs 6, 65 to 66 and 102 to 106 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, a third binding agent having binding specificity for CD123, and a fourth binding agent having binding specificity for CD70, wherein the proteinThe mass is equal to or less than about 10 in PBS ^-8 Dissociation constant of M (K _D ) Binds human CD123 and wherein the third binding agent comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 6, 65 to 66 and 102 to 106, wherein a at the penultimate position of SEQ ID NOs 6, 65 to 66 and 102 to 106 is optionally substituted with L and/or a at the last position of SEQ ID NOs 6, 65 to 66 and 102 to 106 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, a third binding agent having binding specificity for CD123, and a fourth binding agent having binding specificity for CD70, wherein the protein is present in PBS at a ratio of equal to or less than about 10X 10 ^-9 Dissociation constant of M (K _D ) Binds human CD123 and wherein the third binding agent comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 6, 65 to 66 and 102 to 106, wherein a at the penultimate position of SEQ ID NOs 6, 65 to 66 and 102 to 106 is optionally substituted with L and/or a at the last position of SEQ ID NOs 6, 65 to 66 and 102 to 106 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, a third binding agent having binding specificity for CD123, and a fourth binding agent having binding specificity for CD70, wherein the protein is present in PBS at a level of about 10 or less ^-6 M, or equal to or less than about 10 ^-7 M, or equal to or less than about 10 ^-8 M, or equal to or less than about 10 ^-9 M, or equal to or less than about 10 ^-10 Dissociation constant of M (K _D ) Binds human CD70.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, a third binding agent having binding specificity for CD123, and a fourth binding agent having binding specificity for CD70, wherein the protein is present in PBS at a level of about 10 or less ^-6 Dissociation constant of M (K _D ) Binds human CD70, and wherein the fourth binding agent comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 64 and 107 to 110, wherein a at the penultimate position of SEQ ID NOs 64 and 107 to 110 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 64 and 107 to 110 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, a third binding agent having binding specificity for CD123, and a fourth binding agent having binding specificity for CD70, wherein the protein is present in PBS at a level of about 10 or less ^-7 Dissociation constant of M (K _D ) Binds human CD70, and wherein the fourth binding agent comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 64 and 107 to 110, wherein a at the penultimate position of SEQ ID NOs 64 and 107 to 110 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 64 and 107 to 110 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, a first binding agent having binding specificity for CD123, a second binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD123, a third binding agent having binding specificity for CD123, a fourth binding agent having binding specificity for CDA third binding agent having binding specificity and a fourth binding agent having binding specificity for CD70, wherein the protein is present in PBS at a level of about 10 or less ^-8 Dissociation constant of M (K _D ) Binds human CD70, and wherein the fourth binding agent comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 64 and 107 to 110, wherein a at the penultimate position of SEQ ID NOs 64 and 107 to 110 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 64 and 107 to 110 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, a third binding agent having binding specificity for CD123, and a fourth binding agent having binding specificity for CD70, wherein the protein is present in PBS at a level of about 10 or less ^-9 Dissociation constant of M (K _D ) Binds human CD70, and wherein the fourth binding agent comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100% amino acid sequence identity to any one of SEQ ID NOs 64 and 107 to 110, wherein a at the penultimate position of SEQ ID NOs 64 and 107 to 110 is optionally substituted with L, and/or a at the last position of SEQ ID NOs 64 and 107 to 110 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein comprising a first binding agent having binding specificity for CD3, a second binding agent having binding specificity for CD33, a third binding agent having binding specificity for CD123, and a fourth binding agent having binding specificity for CD70, wherein the protein is present in PBS at a level of about 10 or less ^-10 Dissociation constant of M (K _D ) Binds human CD70 and wherein the fourth binding agent comprises a binding agent to SEQ ID NO 64 and 107 to 110, wherein a at the penultimate position of SEQ ID nos. 64 and 107 to 110 is optionally substituted with L and/or a at the last position of SEQ ID nos. 64 and 107 to 110 is optionally substituted with N.

Dissociation constant (K) for recombinant binding proteins of the invention by Surface Plasmon Resonance (SPR) analysis _D ) Is described in example 4. Thus, in one aspect, the binding specificity of a recombinant binding protein of the invention for CD3, CD33, CD123 or CD70 is determined by Surface Plasmon Resonance (SPR) in PBS. In one aspect, the binding specificity of the recombinant binding proteins of the invention is determined by Surface Plasmon Resonance (SPR) in PBS as described in example 3.

In one aspect, the invention provides a recombinant protein comprising a first binding agent that specifically binds to a protein expressed on the surface of an immune cell, a second binding agent that specifically binds to a first tumor-associated antigen, a third binding agent that specifically binds to a second tumor-associated antigen, and/or a fourth binding antigen that specifically binds to a third tumor-associated antigen, wherein the protein has an EC of less than 10nM ₅₀ Binds human CD3.

In one aspect, the invention provides a recombinant protein comprising a first binding agent that specifically binds to a protein expressed on the surface of an immune cell, a second binding agent that specifically binds to a first tumor-associated antigen (TAA 1), a third binding agent that specifically binds to a second tumor-associated antigen (TAA 2), and/or a fourth binding agent that specifically binds to a third tumor-associated antigen (TAA 3), wherein the protein binds to human CD3 with an EC50 in the range of about 1nM to about 400nM, preferably wherein the protein binds to human CD3 with an EC50 in the range of about 1nM to about 10 nM.

Typical and preferred determinations of CD3 binding (EC 50) of the recombinant binding proteins of the invention on T cells with binding specificity for CD3 by imaging cytometry using Mirrorball laser scanning are described in example 4 (with primary human T cells). Thus, in one aspect, the CD3 binding (EC 50) of the recombinant binding proteins of the invention is determined on primary human T cells by Mirrorball laser scanning imaging cytometry, as described in example 4.

In one aspect, the recombinant protein comprises an ankyrin repeat domain having binding specificity for CD3, wherein the binding protein has an EC in the range of about 1nM to about 10nM ₅₀ Binding to human CD3 on a T cell, and wherein the binding protein comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% amino acid sequence identity to any one of SEQ ID NOs 1 to 5, wherein a at the penultimate position of SEQ ID NOs 1 to 4 is optionally substituted with L and/or a at the last position of SEQ ID NOs 1 to 4 is optionally substituted with N; or wherein L at the penultimate position of SEQ ID NO. 5 is optionally substituted with A and/or N at the last position of SEQ ID NO. 5 is optionally substituted with A. Thus, in one aspect, the protein is present in an EC in the range of about 1nM to about 10nM ₅₀ Binds human CD3 on T cells and the ankyrin repeat domain comprises an amino acid sequence having at least 80% amino acid sequence identity to any one of SEQ ID NOs 1 to 5. In one aspect, the protein binds human CD3 on T cells with an EC50 in the range of about 1nM to about 10nM, and the ankyrin repeat domain comprises an amino acid sequence having at least 90% amino acid sequence identity to any one of SEQ ID NOs 1 to 5. In another aspect, the protein binds human CD3 on T cells with an EC50 in the range of about 1nM to about 10nM, and the ankyrin repeat domain comprises an amino acid sequence having at least 93% amino acid sequence identity to any one of SEQ ID NOs 1 to 5; and in another aspect, the protein has an EC in the range of about 1nM to about 10nM ₅₀ Binds human CD3 on T cells and the ankyrin repeat domain comprises a sequence having at least 95% amino groups with any one of SEQ ID NOs 1 to 5Amino acid sequence of acid sequence identity. In one aspect, the protein is present in an EC in the range of about 1nM to about 10nM ₅₀ Binds human CD3 on T cells and the ankyrin repeat domain comprises an amino acid sequence having at least 98% amino acid sequence identity to any one of SEQ ID NOs 1 to 5; and in one aspect, the protein is present in an EC in the range of about 1nM to about 10nM ₅₀ Binds to human CD3 on T cells and the ankyrin repeat domain comprises the amino acid sequence of any one of SEQ ID NOs 1 to 5. Thus, in one aspect, the recombinant protein comprises an ankyrin repeat domain having binding specificity for CD3, wherein the protein has an EC in the range of about 1nM to about 10nM ₅₀ Binds human CD3 on T cells, and wherein the ankyrin repeat domain comprises the amino acid sequence of any one of SEQ ID NOs 1 to 5, wherein a at the penultimate position of SEQ ID NOs 1 to 5 is optionally substituted with L and/or a at the last position of SEQ ID NOs 1 to 5 is optionally substituted with N.

In one aspect, the invention provides a recombinant protein comprising (i) a first binding agent that specifically binds to a protein expressed on the surface of an immune cell, and (ii) a second binding agent that specifically binds to a first tumor-associated antigen (TAA 1), a third binding agent that specifically binds to a second tumor-associated antigen (TAA 2), and/or a fourth binding agent that specifically binds to a third tumor-associated antigen (TAA 3), and wherein the recombinant protein further comprises a half-life extending moiety. Thus, in one aspect, the protein further comprises a half-life extending moiety, wherein the half-life extending moiety is a binding agent that specifically binds human serum albumin. In one aspect, the binding agent having binding specificity for human serum albumin is a designed ankyrin repeat domain having binding specificity for human serum albumin. In one aspect, the engineered ankyrin repeat domain having binding specificity for human serum albumin comprises an amino acid sequence having at least about 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of any one of SEQ ID NOs 34 to 36. In one aspect, the engineered ankyrin repeat domain having binding specificity for human serum albumin comprises an amino acid sequence that is identical to any one of SEQ ID NOs 34 to 36.

In one aspect, the invention provides a recombinant protein comprising (i) a first binding agent that specifically binds to a protein expressed on the surface of an immune cell, wherein the first binding agent is a designed ankyrin repeat domain having binding specificity for the protein expressed on the surface of an immune cell, preferably a designed ankyrin repeat domain having binding specificity for CD3, and (ii) a second binding agent that specifically binds to a first tumor-associated antigen (TAA 1), wherein the second binding agent is a designed ankyrin repeat domain having binding specificity for the TAA1, preferably TAA1 is CD33, specifically binds to a second tumor-associated antigen (TAA 2), wherein the third binding agent is a designed ankyrin repeat domain having binding specificity for the TAA2, preferably TAA2 is CD123, and/or a fourth binding agent that specifically binds to a third tumor-associated antigen (TAA 3), wherein the fourth binding agent is a designed ankyrin repeat domain having binding specificity for the TAA3, preferably TAA3, and wherein the half-life is further comprises a recombinant protein 70. Thus, in one aspect, the recombinant protein further comprises a half-life extending moiety, wherein the half-life extending moiety is a binding agent that specifically binds human serum albumin. In one aspect, the binding agent having binding specificity for human serum albumin is a designed ankyrin repeat domain having binding specificity for human serum albumin. In one aspect, the engineered ankyrin repeat domain having binding specificity for human serum albumin comprises an amino acid sequence having at least about 85%, such as at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identity to the amino acid sequence of any one of SEQ ID NOs 34 to 36. In one aspect, the engineered ankyrin repeat domain having binding specificity for human serum albumin comprises an amino acid sequence that is identical to any one of SEQ ID NOs 34 to 36.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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% or at least about 99% identical to any of the amino acid sequences of SEQ ID NOs 11 to 14, 78 to 86 and 95 to 101, preferably wherein the protein comprises a polypeptide having an amino acid sequence of any of the SEQ ID NOs 11 to 14, 78 to 86 and 95 to 101.

In one aspect, the invention provides recombinant proteins comprising a polypeptide consisting of an amino acid sequence having at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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% or at least about 99% identity to any of the amino acid sequences of SEQ ID NOs 11 to 14, 78 to 86 and 95 to 101. In one aspect, the invention provides a recombinant protein comprising a polypeptide consisting of the amino acid sequence of any one of SEQ ID NOs 11 to 14, 78 to 86 and 95 to 101.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO. 11, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO. 11.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO. 12, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO. 12.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO. 13, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO. 13.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO. 14, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO. 14.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO:78, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO: 78.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO. 79, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO. 79.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO. 80, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO. 80.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO. 81, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO. 81.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO. 82, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO. 82.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO 83, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO 83.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO 84, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO 84.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO. 85, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO. 85.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO. 86, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO. 86.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO. 95, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO. 95.

In one aspect, the present invention provides a recombinant protein comprising a first ankyrin repeat domain, a second ankyrin repeat domain, a third ankyrin repeat domain, a fourth ankyrin repeat domain, a fifth ankyrin repeat domain and a sixth ankyrin repeat domain, wherein said first ankyrin repeat domain is present in PBS at or below 10 ^- ⁷ Dissociation constant of M (K _D ) Specifically binds human serum albumin, wherein the second ankyrin repeat domain is present in PBS at or below 10 ^-7 Dissociation constant of M (K _D ) Specifically binds human serum albumin, wherein the third ankyrin repeat domain is present in PBS at or below 10 ^-7 Dissociation constant of M (K _D ) Specifically binds to human CD33, wherein the fourth ankyrin repeat domain is present in PBS at or below 10 ^-7 Dissociation constant of M (K _D ) Specifically binds to human CD123, wherein the fifth ankyrin repeat domain is at or below 10 in PBS ^-7 Dissociation constant of M (K _D ) Specifically binds human CD70, wherein the sixth ankyrin repeat domain is at or below 10 in PBS ^-6 Dissociation constant of M (K _D ) Specifically binds human CD3, wherein the first ankyrin repeat domain, the second ankyrin repeat domain, the third ankyrin repeat domain, the fourth ankyrin repeat domain, the fifth ankyrin repeat domain and the sixth ankyrin repeat domain are arranged from N-terminus to C-terminus according to the formula: (first ankyrin repeat domain) - (second ankyrin repeat domain) - (third ankyrin repeat domain) - (fourth ankyrin repeat domain) - (fifth ankyrin repeat domain) - (sixth ankyrin repeat domain). In one embodiment, the first ankyrin repeat domain, the second ankyrin repeat domain, the third ankyrin repeat domain, the fourth ankyrin repeat domain, the fifth ankyrin repeat domain and the sixth ankyrin repeat domain are each at 10 in PBS ^-6 M and 10 ^-12 Dissociation constant between M (K _D ) Specifically bind to its target (as described above).In one embodiment, the binding protein is present in an EC in the range of 1nM to 400nM ₅₀ Binds human CD3. In one embodiment, the recombinant protein is expressed in an EC of 1nM or less ₅₀ Binding to Molm-13 tumor cells. In one embodiment, the recombinant protein is capable of binding CD3, CD33, CD123 and CD70 simultaneously. In one embodiment, the recombinant protein comprises a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO 95. In one embodiment, the recombinant protein comprises the amino acid sequence of SEQ ID NO. 95. In one embodiment, the recombinant protein further comprises G, S or GS, preferably GS, at its N-terminus.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO. 96, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO. 96.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO. 97, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO. 97.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO. 98, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO. 98.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO 99, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO 99.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO. 100, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO. 100.

In one aspect, the present invention provides a recombinant eggAlbumin, the recombinant protein comprises a first ankyrin repeat domain, a second ankyrin repeat domain, a third ankyrin repeat domain, a fourth ankyrin repeat domain, a fifth ankyrin repeat domain and a sixth ankyrin repeat domain, wherein the first ankyrin repeat domain is present in PBS at or below 10 ^- ⁷ Dissociation constant of M (K _D ) Specifically binds human serum albumin, wherein the second ankyrin repeat domain is present in PBS at or below 10 ^-7 Dissociation constant of M (K _D ) Specifically binds human serum albumin, wherein the third ankyrin repeat domain is present in PBS at or below 10 ^-7 Dissociation constant of M (K _D ) Specifically binds to human CD33, wherein the fourth ankyrin repeat domain is present in PBS at or below 10 ^-7 Dissociation constant of M (K _D ) Specifically binds to human CD123, wherein the fifth ankyrin repeat domain is at or below 10 in PBS ^-7 Dissociation constant of M (K _D ) Specifically binds human CD70, wherein the sixth ankyrin repeat domain is at or below 10 in PBS ^-6 Dissociation constant of M (K _D ) Specifically binds human CD3, wherein the first ankyrin repeat domain, the second ankyrin repeat domain, the third ankyrin repeat domain, the fourth ankyrin repeat domain, the fifth ankyrin repeat domain and the sixth ankyrin repeat domain are arranged from N-terminus to C-terminus according to the formula: (first ankyrin repeat domain) - (second ankyrin repeat domain) - (fifth ankyrin repeat domain) - (fourth ankyrin repeat domain) - (third ankyrin repeat domain) - (sixth ankyrin repeat domain). In one embodiment, the first ankyrin repeat domain, the second ankyrin repeat domain, the third ankyrin repeat domain, the fourth ankyrin repeat domain, the fifth ankyrin repeat domain and the sixth ankyrin repeat domain are each at 10 in PBS ^-6 M and 10 ^-12 Dissociation constant between M (K _D ) Specifically bind to its target (as described above). In one embodiment, the binding protein is in the range of 1nM to 400nMEC of (2) ₅₀ Binds human CD3. In one embodiment, the recombinant protein is expressed in an EC of 1nM or less ₅₀ Binding to Molm-13 tumor cells. In one embodiment, the recombinant protein is capable of binding CD3, CD33, CD123 and CD70 simultaneously. In one embodiment, the recombinant protein comprises a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO 96. In another embodiment, the recombinant protein comprises a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO 97. In another embodiment, the recombinant protein comprises a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO. 98. In another embodiment, the recombinant protein comprises a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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% with the amino acid sequence of SEQ ID NO 99 A polypeptide of an amino acid sequence that is at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or 100% identical. In another embodiment, the recombinant protein comprises a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO. 100. In one embodiment, the recombinant protein comprises the amino acid sequence of SEQ ID NO. 96. In one embodiment, the recombinant protein comprises the amino acid sequence of SEQ ID NO. 97. In one embodiment, the recombinant protein comprises the amino acid sequence of SEQ ID NO. 98. In one embodiment, the recombinant protein comprises the amino acid sequence of SEQ ID NO. 99. In one embodiment, the recombinant protein comprises the amino acid sequence of SEQ ID NO. 100. In one embodiment, the recombinant protein further comprises G, S or GS, preferably GS, at its N-terminus.

In one aspect, the invention provides a recombinant protein comprising a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, 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%, or at least about 99% identical to the amino acid sequence of SEQ ID NO. 101, preferably wherein the protein comprises a polypeptide having the amino acid sequence of SEQ ID NO. 101.

In one aspect, the invention provides a recombinant protein comprising a first ankyrin repeat domain, a second ankyrin repeat domain, a third ankyrin repeat domain, a fourth ankyrin repeat domain, a fifth ankyrin repeat domain and a sixth ankyrin repeat domainWherein the first ankyrin repeat domain is at or below 10 in PBS ^- ⁷ Dissociation constant of M (K _D ) Specifically binds human serum albumin, wherein the second ankyrin repeat domain is present in PBS at or below 10 ^-7 Dissociation constant of M (K _D ) Specifically binds human serum albumin, wherein the third ankyrin repeat domain is present in PBS at or below 10 ^-7 Dissociation constant of M (K _D ) Specifically binds to human CD33, wherein the fourth ankyrin repeat domain is present in PBS at or below 10 ^-7 Dissociation constant of M (K _D ) Specifically binds to human CD123, wherein the fifth ankyrin repeat domain is at or below 10 in PBS ^-7 Dissociation constant of M (K _D ) Specifically binds human CD70, wherein the sixth ankyrin repeat domain is at or below 10 in PBS ^-6 Dissociation constant of M (K _D ) Specifically binds human CD3, wherein the first ankyrin repeat domain, the second ankyrin repeat domain, the third ankyrin repeat domain, the fourth ankyrin repeat domain, the fifth ankyrin repeat domain and the sixth ankyrin repeat domain are arranged from N-terminus to C-terminus according to the formula: (first ankyrin repeat domain) - (second ankyrin repeat domain) - (fifth ankyrin repeat domain) - (third ankyrin repeat domain) - (fourth ankyrin repeat domain) - (sixth ankyrin repeat domain). In one embodiment, the first ankyrin repeat domain, the second ankyrin repeat domain, the third ankyrin repeat domain, the fourth ankyrin repeat domain, the fifth ankyrin repeat domain and the sixth ankyrin repeat domain are each at 10 in PBS ^-6 M and 10 ^-12 Dissociation constant between M (K _D ) Specifically bind to its target (as described above). In one embodiment, the binding protein is present in an EC in the range of 1nM to 400nM ₅₀ Binds human CD3. In one embodiment, the recombinant protein is expressed in an EC of 1nM or less ₅₀ Binding to Molm-13 tumor cells. In one embodiment, the recombinant protein is capable of binding CD3, CD33, simultaneously,CD123 and CD70. In one embodiment, the recombinant protein comprises a polypeptide having an amino acid sequence that is at least 80%, such as at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99% or 100% identical to the amino acid sequence of SEQ ID NO 101. In one embodiment, the recombinant protein comprises the amino acid sequence of SEQ ID NO. 101. In one embodiment, the recombinant protein further comprises G, S or GS, preferably GS, at its N-terminus.

The repeat domains of the recombinant binding proteins disclosed herein, preferably the ankyrin repeat domains, preferably comprise an N-terminal and/or C-terminal capping module (hereinafter also referred to as a capping repeat or capping unit). The capping modules are located at the N-terminal and/or C-terminal ends of the ankyrin repeat domains, typically forming a tight tertiary interaction (i.e., tertiary structural interaction) with the ankyrin repeat modules therebetween, thereby providing a cover protecting the hydrophobic core of one side of the ankyrin repeat domain from exposure to solvents. The N-terminal and/or C-terminal capping modules may be derived from capping units or other structural units present in naturally occurring repeat proteins adjacent to the repeat unit. Examples of capping sequences are described in International patent publication Nos. WO 2002/020565 and WO 2012/069655, U.S. patent publication No. US2013/0296221, and Interland et al, J Mol biol.2008, 1 month, 18; 375 (3):837-54. Examples of N-terminal capping modules (i.e., N-terminal capping repeats) are SEQ ID NOS: 16-22, and examples of C-terminal capping modules (i.e., C-terminal capping repeats) are SEQ ID NOS: 23-23.

In one exemplary aspect, the N-terminal capping module comprises the amino acid sequence of any one of SEQ ID NOs 16 to 21, wherein at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 amino acids of any one of SEQ ID NOs 16 to 21 are optionally exchanged for any amino acid.

In one exemplary aspect, the C-terminal capping module comprises the amino acid sequence of any one of SEQ ID NOs 23 to 31, wherein at most 9, at most 8, at most 7, at most 6, at most 5, at most 4, at most 3, at most 2, or at most 1 amino acids of any one of SEQ ID NOs 23 to 31 are optionally exchanged by any amino acid.

Advantageously, in some aspects, certain amino acid residues in the N-terminal end capping module and/or the C-terminal end capping module of the designed ankyrin repeat domains provided herein are altered, resulting in improved pharmacokinetic properties (including extended terminal half-life) of the designed ankyrin repeat domains and recombinant proteins comprising the designed ankyrin repeat domains. The altered amino acid residues are predominantly surface exposed residues. Preferably, the altered amino acid residues are amino acid residues at positions 8 and 15 of the N-terminal capping module, wherein the amino acid at position 8 is Q and the amino acid at position 15 is L, and wherein the position numbers correspond to positions in SEQ ID No. 16; and amino acid residues at positions 14 and 18 of the C-terminal capping module, wherein the amino acid at position 14 is R and the amino acid at position 18 is Q, and wherein the position numbers correspond to positions in SEQ ID No. 16.

For example, an N-terminal capping module having an altered amino acid residue may comprise the following sequence: DLGxxLLQAAxGQLDxVRxLxxxGADVNA (SEQ ID NO: 22), wherein "x" represents any amino acid.

For example, a C-terminal capping module having an altered amino acid residue may comprise the following sequence: xDxxGxTPADxAARxGHQxIAxVLQxAA (SEQ ID NO: 32), wherein "x" represents any amino acid.

Thus, in one aspect, the ankyrin repeat domain of the present invention having binding specificity for CD3 comprises an N-terminal end capping module having the amino acid sequence of SEQ ID No. 22, wherein "x" represents any amino acid. Alternatively or additionally, the ankyrin repeat domain of the invention having binding specificity for CD3 may comprise a C-terminal end capping module having the amino acid sequence of SEQ ID No. 32, wherein "x" represents any amino acid.

Furthermore, the binding domain of the invention may optionally further comprise a "G", "S" or "GS" sequence at its N-terminus. Thus, in some aspects, a binding protein provided herein (i) comprises an amino acid sequence that is at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or 100% identical to any one of SEQ ID NOs 1 to 6, 15, 64 to 70, and (ii) further comprises G, S or GS at its N-terminus. In one exemplary aspect, the binding protein comprises an amino acid sequence having at least 90% identity to any one of SEQ ID NOs 1 to 6, 15, 64 to 70, and further comprises G, S or GS at the N-terminus thereof. In one exemplary aspect, the binding protein comprises an amino acid sequence having at least 95% identity to any one of SEQ ID NOs 1 to 6, 15, 64 to 70, and further comprises G, S or GS at the N-terminus thereof. In one exemplary aspect, the binding protein comprises an amino acid sequence that is identical to any one of SEQ ID NOs 1 to 6, 15, 64 to 70, and further comprises G, S or GS at the N-terminus thereof.

In one aspect, the recombinant binding proteins of the invention further comprise a polypeptide tag. A polypeptide tag is an amino acid sequence attached to a polypeptide/protein, wherein the amino acid sequence can be used to purify, detect or target the polypeptide/protein, or wherein the amino acid sequence improves the physicochemical behavior of the polypeptide/protein, or wherein the amino acid sequence has effector function. The individual polypeptide tags of the binding protein may be linked to other parts of the binding protein directly or via peptide linkers. Polypeptide tags are well known in the art and are fully available to those skilled in the art. Examples of polypeptide tags are small polypeptide sequences, such as His, HA, myc, FLAG or Strep tags; or polypeptides, such as enzymes (e.g., alkaline phosphatase) that allow detection of the polypeptide/protein or polypeptides that can be used for targeting (such as immunoglobulins or fragments thereof) and/or polypeptides that can be used as effector molecules.

In one aspect, the recombinant binding proteins of the invention further comprise a peptide linker. Peptide linkers are amino acid sequences capable of linking, for example, two protein domains, a polypeptide tag and a protein domain, a protein domain and a non-proteinaceous compound or polymer (such as polyethylene glycol), a protein domain and a bioactive molecule, a protein domain and a targeting agent, or two sequence tags. Peptide linkers are known to those skilled in the art. An exemplary list is provided in the specification of patent application WO 2002/020565. In one aspect, the peptide linker for use in the present invention has a length of 1 to 50 amino acids. In another aspect, the peptide linker for use in the present invention has a length of 1 to 30 amino acids. Specific examples of peptide linkers are glycine-serine linkers with variable length and proline-threonine rich linkers. Examples of glycine-serine linkers are amino acid sequence GS and the amino acid sequence of SEQ ID NO. 63, and examples of proline-threonine rich linkers are the amino acid sequence of SEQ ID NO. 37.

In the context of the present invention, a proline-threonine rich linker comprises at least 20% proline residues and at least 20% threonine residues in its amino acid sequence.

In another aspect, the invention relates to a nucleic acid encoding an ankyrin repeat domain of the invention or an amino acid sequence of a recombinant protein. In one aspect, the invention relates to a nucleic acid encoding an amino acid sequence of a recombinant protein of the invention. In one aspect, the invention relates to nucleic acids encoding amino acid sequences selected from SEQ ID NOS.7 to 10 and 58 to 62. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 7. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 8. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 9. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 10. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 58. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 59. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 60. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 61. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 62. Furthermore, the invention relates to a vector comprising any of the nucleic acids of the invention. Nucleic acids are well known to those skilled in the art. In an example, the engineered ankyrin repeat domains or recombinant binding proteins of the invention are produced in e.coli (e.coli) using nucleic acids. Examples of nucleic acids of the invention are provided by SEQ ID NOS 52 to 55 and 90 to 94 encoding the amino acid sequences of SEQ ID NOS 7 to 10 and 58 to 62, respectively.

In another aspect, the invention relates to a nucleic acid encoding an ankyrin repeat domain of the invention or an amino acid sequence of a recombinant protein. In one aspect, the invention relates to a nucleic acid encoding an amino acid sequence of a recombinant protein of the invention. In one aspect, the invention relates to a nucleic acid encoding an amino acid sequence selected from the group consisting of SEQ ID NOS 11 to 14, 78 to 86 and 95 to 101. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 11. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 12. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 13. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 14. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 78. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 79. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 80. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 81. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 82. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 83. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 84. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 85. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 86. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 95. In one embodiment, the nucleic acid encoding the amino acid sequence of SEQ ID NO. 95 is the nucleic acid of SEQ ID NO. 118. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 96. In one embodiment, the nucleic acid encoding the amino acid sequence of SEQ ID NO. 96 is a nucleic acid of SEQ ID NO. 119. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 97. In one embodiment, the nucleic acid encoding the amino acid sequence of SEQ ID NO. 97 is a nucleic acid of SEQ ID NO. 120. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 98. In one embodiment, the nucleic acid encoding the amino acid sequence of SEQ ID NO. 98 is the nucleic acid of SEQ ID NO. 121. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 99. In one embodiment, the nucleic acid encoding the amino acid sequence of SEQ ID NO. 99 is a nucleic acid of SEQ ID NO. 122. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 100. In one embodiment, the nucleic acid encoding the amino acid sequence of SEQ ID NO. 100 is a nucleic acid of SEQ ID NO. 123. In one aspect, the invention relates to a nucleic acid encoding the amino acid sequence of SEQ ID NO. 101. In one embodiment, the nucleic acid encoding the amino acid sequence of SEQ ID NO. 101 is a nucleic acid of SEQ ID NO. 124. Furthermore, the invention relates to a vector comprising any of the nucleic acids of the invention. Nucleic acids are well known to those skilled in the art. In an example, the engineered ankyrin repeat domains or recombinant binding proteins of the invention are produced in e.coli (e.coli) using nucleic acids. Examples of the nucleic acids of the invention are provided by SEQ ID NOS.118 to 124 and these nucleic acids encode the amino acid sequences of SEQ ID NOS.95 to 101, respectively.

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

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

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

The pharmaceutical composition comprises a recombinant binding protein and/or engineered ankyrin repeat domain and/or nucleic acid as described herein, preferably a recombinant binding protein and/or nucleic acid, and a pharmaceutically acceptable carrier, excipient or stabilizer, e.g. as described in Remington's Pharmaceutical Sciences, 16 th edition, osol, a. Edit, 1980.

Suitable carriers, diluents, excipients or stabilizers known to those skilled in the art include, for example, saline, ringer's solution, dextrose solution, hank's solution, fixed oils, ethyl oleate, 5% dextrose saline, substances which enhance isotonicity and chemical stability, buffers and preservatives. Other suitable carriers include any carrier that does not itself induce the production of antibodies harmful to the individual receiving the composition, such as proteins, polysaccharides, polylactic acids, polyglycolic acids, polymeric amino acids, and amino acid copolymers. The pharmaceutical composition may also be a combined preparation comprising an additional active agent, such as an anti-cancer or anti-angiogenic agent, or an additional biologically active compound. The composition to be used for in vivo administration must be sterile or sterile. This is easily accomplished by filtration through sterile filtration membranes.

One aspect of the invention relates to the use of a recombinant protein of the invention comprising a first ankyrin repeat domain having binding specificity for CD3, a second ankyrin repeat domain having binding specificity for TAA1 (preferably CD 33), a third ankyrin repeat domain having binding specificity for TAA2 (preferably CD 123) and/or a fourth ankyrin repeat domain having binding specificity for TAA3 (preferably CD 70) and further comprising an ankyrin repeat domain having binding specificity for human serum albumin, wherein said recombinant protein exhibits an increased terminal half-life, preferably by at least about 5%, preferably at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 60%, about 80%, about 100%, about 250% or about 100%, as compared to a corresponding recombinant protein comprising said first ankyrin repeat domain, said second ankyrin repeat domain and said third ankyrin repeat domain but not comprising said ankyrin repeat domain having binding specificity for serum albumin.

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

In another aspect, the invention provides a method of tumor-targeted activation of T cells in a mammal, including a human, comprising the step of administering to said mammal a recombinant protein of the invention, a nucleic acid of the invention or a pharmaceutical composition of the invention.

In another aspect, the invention provides a method of treating a medical condition, the method comprising the step of administering to a patient in need thereof a therapeutically effective amount of a recombinant protein of the invention, a nucleic acid of the invention, or a pharmaceutical composition of the invention.

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

In one aspect, the invention relates to the use of a pharmaceutical composition, recombinant protein or nucleic acid according to the invention for the treatment of a disease. For this purpose, the pharmaceutical composition, nucleic acid or recombinant binding protein according to the invention is administered to a patient in need thereof in a therapeutically effective amount. Administration may include topical administration, oral administration, and parenteral administration. A typical route of administration is parenteral. In parenteral administration, the pharmaceutical compositions of the invention will be formulated in unit-dose injectable forms, such as solutions, suspensions or emulsions, in association with the pharmaceutically acceptable excipients described above. The dosage and mode of administration will depend on the individual to be treated and the particular disease.

In addition, any of the above-mentioned pharmaceutical compositions, nucleic acids or recombinant proteins are contemplated for use in treating a disorder.

In one aspect, the recombinant protein or other such pharmaceutical composition described herein is administered intravenously. For parenteral administration, the recombinant protein or the pharmaceutical composition may be injected in a therapeutically effective amount by bolus injection, either rapidly or by slow infusion.

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

In one aspect, the invention also provides the use of such recombinant proteins for treating a medical condition in a subject in need thereof.

As used herein, the medical condition or disease is a cancer, preferably a liquid tumor, more preferably leukemia, even more preferably Acute Myelogenous Leukemia (AML).

The recombinant proteins of the invention, the nucleic acids of the invention or the pharmaceutical compositions of the invention may also be used in combination with one or more other therapies known in the art. The term "used in conjunction with … …" as used herein shall refer to co-administration under a given regimen. This includes simultaneous administration of different compounds as well as staggered administration of different compounds (e.g., compound a is administered once followed by compound B administered multiple times, and vice versa, or both compounds are administered simultaneously and one of the compounds is also administered in a subsequent stage).

In one aspect, the invention relates to a kit comprising a recombinant protein of the invention. In one aspect, the invention relates to a kit comprising a nucleic acid encoding a recombinant protein of the invention. In one aspect, the invention relates to a kit comprising the pharmaceutical composition of the invention. In one aspect, the invention relates to a kit comprising a recombinant protein of the invention, and/or a nucleic acid of the invention, and/or a pharmaceutical composition of the invention. In one aspect, the invention relates to a kit comprising a recombinant protein of the invention comprising any one of SEQ ID NOs 7 to 10 and 58 to 62, and/or a nucleic acid encoding said recombinant protein comprising any one of SEQ ID NOs 52 to 55 and 90 to 94, and/or a pharmaceutical composition comprising a recombinant protein comprising any one of SEQ ID NOs 7 to 10 and 58 to 62. In one aspect, the invention relates to a kit comprising a recombinant protein comprising any of the amino acid sequences of SEQ ID NOs 7 to 10 and 58 to 62, and/or a nucleic acid encoding said recombinant protein, and/or a pharmaceutical composition comprising said recombinant protein.

In one aspect, the invention relates to a kit comprising a recombinant protein of the invention. In one aspect, the invention relates to a kit comprising a nucleic acid encoding a recombinant protein of the invention. In one aspect, the invention relates to a kit comprising the pharmaceutical composition of the invention. In one aspect, the invention relates to a kit comprising a recombinant protein of the invention, and/or a nucleic acid of the invention, and/or a pharmaceutical composition of the invention. In one aspect, the invention relates to a kit comprising a recombinant protein of the invention comprising any one of SEQ ID NOs 11 to 14, 78 to 82 and 95 to 101, and/or a nucleic acid encoding said recombinant protein comprising any one of SEQ ID NOs 118 to 124, and/or a pharmaceutical composition comprising a recombinant protein comprising any one of SEQ ID NOs 11 to 14, 78 to 82 and 95 to 101. In one aspect, the invention relates to a kit comprising a recombinant protein comprising any of the amino acid sequences of SEQ ID NOs 11 to 14, 78 to 82 and 95 to 101, and/or a nucleic acid encoding said recombinant protein, and/or a pharmaceutical composition comprising said recombinant protein.

In one aspect, the invention relates to a method for producing a recombinant protein of the invention. In one aspect, the invention provides a method for producing a recombinant binding protein (e.g., a recombinant protein comprising the amino acid sequences of SEQ ID NOS: 7 to 10 and 58 to 62), the method comprising the steps of: (i) Expressing the recombinant binding protein in a suitable host cell (e.g., bacteria), and (ii) purifying the recombinant binding protein (e.g., using chromatography). The method may comprise additional steps. Such a method of producing the recombinant binding proteins of the invention is described in example 1.

In one aspect, the invention relates to a method for producing a recombinant protein of the invention. In one aspect, the invention relates to a method for producing a recombinant binding protein (e.g., a recombinant protein comprising the amino acid sequences of SEQ ID NOs 11 to 14, 78 to 82 and 95 to 101), comprising the steps of: (i) Expressing the recombinant binding protein in a suitable host cell (e.g., bacteria), and (ii) purifying the recombinant binding protein (e.g., using chromatography). The method may comprise additional steps. Such a method of producing the recombinant binding proteins of the invention is described in example 1.

All amino acid sequences described herein may be substituted with one or more amino acids. In some aspects, up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, 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 substitutions are made in any of the amino acid sequences described herein.

In some aspects, amino acid substitutions are all made at framework positions. In some aspects, amino acid substitutions are all made at non-random positions. The localization of random positions in the designed ankyrin repeat domain is disclosed, for example, in Binz et al, nature Biotech.22 (5): 575-582 (2004).

In some aspects, amino acid substitutions alter the KD value of the binding agent by no more than about 1000-fold, no more than about 100-fold, or no more than about 10-fold as compared to the KD value of the unsubstituted binding agent. For example, in some aspects, K is a binding agent to any one of the sequences comprising SEQ ID NOS: 7 to 10 and 58 to 62 _D Amino acid substitutions result in K compared to the values _D The value changes by no more than about 1000-fold, no more than about 300-fold, no more than about 100-fold, no more than about 50-fold, no more than about 25-fold, no more than about 10-fold, or no more than about 5-fold.

In certain aspects, the amino acid substitutions in the binding moiety are conservative substitutions according to table 1 below.

TABLE 1 amino acid substitutions

Initial residues	Conservative substitutions	Exemplary substitution
			Ala(A)	Val	Val；Leu；Ile
Arg(R)	Lys	Lys；Gln；Asn
			Asn(N)	Gln	Gln；His；Asp、Lys；Arg
Asp(D)	Glu	Glu；Asn
			Cys(C)	Ser	Ser；Ala
Gln(Q)	Asn	Asn；Glu
			Glu(E)	Asp	Asp；Gln
Gly(G)	Ala	Ala
			His(H)	Arg	Asn；Gln；Lys；Arg
Ile(I)	Leu	Leu; val; met; ala; phe; norleucine (N-leucine)
			Leu(L)	Ile	Norleucine; ile; val; met; ala; phe (Phe)
Lys(K)	Arg	Arg；Gln；Asn
			Met(M)	Leu	Leu；Phe；Ile
Phe(F)	Tyr	Leu；Val；Ile；Ala；Tyr

When the binding agent is an ankyrin repeat domain, in some aspects, the substitution may be outside of the structural core residues of the ankyrin repeat domain (e.g., in the β loop joining the α -helix). In other aspects, substitutions may be made within the structural core residues of the ankyrin repeat domain. For example, the ankyrin domain may comprise a consensus sequence: xdxgxtpllhaxxxgxxxlxvxvllxxgadvna, wherein "x" represents any amino acid (preferably not cysteine, glycine or proline); or xdxxgxtpllhaxxghleiv llkzgadvna, wherein "x" represents any amino acid (preferably not cysteine, glycine or proline) and "z" is selected from asparagine, histidine or tyrosine. In one aspect, a residue designated "x" is substituted. In another aspect, substitutions are made outside of the residue designated "x".

The invention is not limited to the specific aspects described in the examples. The present specification relates to a number of amino acid sequences, nucleic acid sequences, and SEQ ID NOs disclosed in the accompanying sequence Listing, which is incorporated herein by reference in its entirety.

Definition of the definition

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

Unless otherwise indicated, the terms "comprising," "having," "including," and "containing" are to be construed as open-ended terms. Aspects of the invention that "consist of" or "consist essentially of the feature are also contemplated if they are described as" comprising "the feature. Any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure. Except in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as being modified in all instances by the term "about" as will be understood by those skilled in the relevant art. The term "about" as used herein is equivalent to ± 10% of a given value unless otherwise indicated.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value and each endpoint falling within the range, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein.

In the context of the present invention, the term "protein" refers to a molecule comprising a polypeptide, wherein at least a portion of the polypeptide has or is capable of achieving a defined three-dimensional arrangement by forming secondary, tertiary and/or quaternary structures within a single polypeptide chain and/or between multiple polypeptide chains. If a protein comprises two or more polypeptide chains, a single polypeptide chain may be non-covalently linked or covalently linked, for example, by disulfide bonds between the two polypeptides. Protein moieties that have or are able to acquire a defined three-dimensional arrangement by forming secondary and/or tertiary structures alone are referred to as "protein domains". Such protein domains are well known to those skilled in the art.

The term "recombinant" as used in the context of recombinant proteins, recombinant polypeptides, and the like means that the protein or polypeptide is produced using recombinant DNA techniques 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 DNA capable of in vitro expression. If, for example, such recombinant bacterial expression plasmids are inserted into an appropriate bacterium, for example, E.coli (Escherichia coli), these bacteria can produce the polypeptide encoded by the recombinant DNA. The corresponding polypeptide or protein produced is referred to as a recombinant polypeptide or recombinant protein.

In the context of the present invention, the term "binding protein" refers to a protein comprising a binding domain. The binding protein may also comprise two, three, four, five or more binding domains. Preferably, the binding protein is a recombinant binding protein. The binding proteins of the invention comprise an ankyrin repeat domain with binding specificity for CD3, an ankyrin repeat domain with binding specificity for CD33, and an ankyrin repeat domain with binding specificity for CD 123.

In addition, any such binding protein may comprise additional polypeptides (e.g., polypeptide tags, peptide linkers, fusion to other protein domains with binding specificity, cytokines, hormones, or antagonists), or chemical modifications well known to those skilled in the art (such as coupling to polyethylene glycol, toxins (e.g., DM1 from Immunogen), small molecules, antibiotics, etc.). Binding proteins of the invention may comprise a targeting agent molecule.

The term "binding domain" means a protein domain that exhibits binding specificity for a target. Preferably, the binding domain is a recombinant binding domain.

The term "target" refers to a single molecule, such as a nucleic acid molecule, polypeptide or protein, carbohydrate or any other naturally occurring molecule, including any portion of such a single molecule, or a complex of two or more such molecules, or an entire cell or tissue sample, or any unnatural compound. Preferably, the target is a naturally occurring or non-natural polypeptide or protein, or a polypeptide or protein that contains a chemical modification (e.g., natural or non-natural phosphorylation, acetylation, or methylation). In the context of the present invention, T cells are targets for CD3 specific binding proteins and targeting proteins for the targeting agent, and cells and tissues are targets for the targeting agent.

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

Patent applications WO2002/020565 and Forrer et al, 2003 (Forrer, p., stumpp, m.t., binz, h.k., pluckthun, a.,2003.FEBS Letters 539,2-6) contain general descriptions of repeat protein features and repeat domain features, techniques and applications. The term "repeat protein" refers to a protein comprising one or more repeat domains. Preferably, the repeat protein comprises one, two, three, four, five or six repeat domains. In addition, the repeat protein may comprise additional non-repeat protein domains, polypeptide tags, and/or peptide linkers. The repeat domain may be a binding domain.

The term "repeat domain" refers to a protein domain comprising two or more consecutive repeat modules as structural units, wherein the repeat modules have structural and sequence homology. Preferably, the repeat domain further comprises an N-terminal and/or C-terminal capping module. For clarity, the end-capping module may be a repeating module. Such repeat domains, repeat modules and end-capping modules, sequence motifs and structural and sequence homologies are well known to those skilled in the art from the following examples: ankyrin repeat domain (WO 2002/020565), leucine rich repeat domain (WO 2002/020565), thirty-four peptide repeat domain (Main, e.r., xiong, Y., cocco, M.J., D' Andrea, L., regan, L., structure 11 (5), 497-508, 2003) and armadine repeat domain (WO 2009/040338). It is also well known to those skilled in the art that such repeat domains differ from proteins comprising repeat amino acid sequences, wherein each of the repeat amino acid sequences is capable of forming a single domain (e.g., the FN3 domain of fibronectin).

The term "ankyrin repeat domain" refers to a repeat domain comprising two or more consecutive ankyrin modules as building blocks. The ankyrin repeat domains may be assembled modularly into larger ankyrin repeat proteins, optionally with half-life extending domains, using standard recombinant DNA repeat techniques (see e.g. Forrer, p. Et al, FEBS letters, volume 539, pages 2-6, 2003, WO2002/020565, WO2016/156596, WO 2018/054971).

As used in the context of engineered repeat proteins, engineered repeat domains, and the like, the term "engineered" refers to the property of such repeat proteins and repeat domains, respectively, that are artificial and do not exist in nature. The binding proteins of the invention are designed repeat proteins and they comprise at least one designed ankyrin repeat domain. Preferably, the designed repeat domain is a designed ankyrin repeat domain.

The term "target interaction residue" refers to an amino acid residue of a repeat module that facilitates direct interaction with a target.

The term "framework residue" refers to an amino acid residue of a repeat module that contributes to the folding topology, i.e., to the folding of the repeat module or to interactions with neighboring modules. Such contributions may be interactions with other residues in the repeat module, or effects on the conformation of the polypeptide backbone as present in the alpha-helix or beta-sheet, or amino acid extensions involved in the formation of 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 and relevant structural information well known to practitioners in the field of structural biology and/or bioinformatics.

The term "repeat module" refers to the repeat amino acid sequence and structural units of a designed repeat domain that originally originated from the repeat unit of a naturally occurring repeat protein. Each repeat module contained in a repeat domain is derived from one or more repeat units of a naturally occurring family or subfamily of repeat proteins (e.g., the ankyrin family of repeat proteins). Furthermore, each repeat module comprised in a repeat domain may comprise a "repeat sequence motif" derived from a homologous repeat module obtained from a repeat domain selected on the target, e.g. as described in example 1, and having the same target specificity.

Thus, the term "ankyrin repeat module" refers to a repeat module that is originally derived from a repeat unit of a naturally occurring ankyrin repeat protein. Ankyrin repeat proteins are well known to those skilled in the art. Designed ankyrin repeat proteins have been previously described; see, for example, international patent publication nos. WO2002/020565, WO2010/060748, WO2011/135067, WO2012/069654, WO2012/069655, WO2014/001442, WO2014/191574, WO2014/083208, WO2016/156596, and WO2018/054971, all of which are incorporated by reference in their entirety. Typically, an ankyrin repeat module comprises about 31 to 33 amino acid residues that form two alpha helices separated by a loop.

The repeat module may comprise positions having amino acid residues that have not been randomized in the library for selection of the target-specific repeat domain ("non-randomized positions") and positions having amino acid residues that have been randomized in the library for selection of the target-specific repeat domain ("randomized positions"). The non-randomized positions comprise framework residues. The randomized positions comprise target interaction residues. By "randomized" is meant that two or more amino acids are allowed at the amino acid positions of the repeat module, e.g., wherein any of the typically twenty naturally occurring amino acids are allowed, or wherein a majority of the twenty naturally occurring amino acids are allowed, such as amino acids other than cysteine, or amino acids other than glycine, cysteine, and proline.

The term "repeat motif" refers to an amino acid sequence derived from one or more repeat modules. Preferably, the repeat module is from a repeat domain having binding specificity for the same target. Such repeat sequence motifs comprise framework residue positions and target interaction residue positions. The framework residue positions correspond to framework residue positions of the repeat module. Likewise, the target interaction residue position corresponds to the position of the target interaction residue of the repeat module. The repeat motif comprises non-randomized positions and randomized positions.

The term "repeat unit" refers to an amino acid sequence comprising the sequence motif of one or more naturally occurring proteins, wherein the "repeat unit" is present in multiple copies and exhibits a defined folding topology that is common to all of the motifs that determine protein folding. Examples of such repeating units include leucine-rich repeating units, ankyrin repeating units, armadillo repeating units, thirty-tetrapeptide repeating units, HEAT repeating units, and leucine-rich variant repeating units.

The terms "bind specifically to a target," "specifically bind to a target," "bind with high specificity to a target," "specifically to a target," "target-specific," or "specifically bind" and the like mean that a binding protein or binding domain binds to a target in PBS with a lower dissociation constant (i.e., it binds with higher affinity) than it binds to an unrelated protein such as e.coli Maltose Binding Protein (MBP). Preferably, the dissociation constant ("K") of the target in PBS _D ") is at least 1/102, more preferably at least 1/103, more preferably at least 1/104 or more preferably at least 1/105 of the corresponding dissociation constant of MBP. Methods for determining 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. Measurement of specific protein-protein interactions K if the measurements are performed under different conditions (e.g., salt concentration, pH) _D The values may be varied. Thus, K is preferably performed using a standardized protein solution and a standardized buffer (such as PBS) _D Measurement of the values. Dissociation constants (K) of the recombinant binding proteins of the invention with binding specificity for CD3, CD33 and CD123 by Surface Plasmon Resonance (SPR) analysis _D ) Is described in example 4. A variety of assay formats may be used to select or characterize binding moieties that specifically bind to a drug molecule of interest. For example, solid phase ELISA immunoassays, immunoprecipitation, BIAcore ^TM (GE Healthcare, piscataway, NJ), fluorescence Activated Cell Sorting (FACS), octet ^TM (ForteBio Inc., menlo Park, calif.) and Western blot analysis are a number of assays that can be used to identify binding moieties that specifically bind to target drug molecules. Typically, the specific or selective binding will be at least twice the background signal or noise, and more typically more than 10 times the background signal. Even particularly when the equilibrium dissociation constant (K _D ) Value of<1 mu m, such as<500nΜ、<100nM、<10nM、<1nM、<100pM or<At 10pM, the binding agent is considered to "specifically bind" to the target.

The term "binding agent" refers to any molecule capable of specifically binding to a target molecule. Binding agents include, for example, antibodies, antibody fragments, aptamers, peptides (e.g., williams et al, J Biol Chem 266:5182-5190 (1991)), antibody mimetics, repeat proteins (e.g., engineered ankyrin repeat proteins), receptor proteins, and any other naturally occurring interaction partner of a target molecule, and may include natural proteins and proteins modified or genetically engineered (e.g., to contain non-natural residues and/or to lack natural residues).

The term "PBS" refers to a phosphate buffered saline solution containing 137mM NaCl, 10mM phosphate, and 2.7mM KCl and having a pH of 7.4.

The term "tumor-associated antigen" or TAA as used herein refers to an antigen found on tumor cells that is not qualitatively different in structure from the antigen found on normal cells. However, the number of tumor-associated antigens found on the surface of tumor cells is greater than that found on the surface of most healthy cells. The tumor-associated antigen may be tumor-type specific, but may also be expressed in several tumor types. For example, TAA MUC1 is associated with colon, breast, ovarian, lung and pancreatic cancer.

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

Preferably, clearance and/or exposure and/or terminal half-life is assessed in a mammal, more preferably a mouse and/or a cynomolgus monkey, more preferably a cynomolgus monkey. Preferably, when measuring clearance, and/or exposure and/or terminal half-life in mice, evaluation is performed taking into account data up to 48 hours after injection. More preferably, in evaluating the terminal half-life in mice, calculations are performed with data from 24 hours to 48 hours. Preferably, when measuring clearance and/or exposure and/or terminal half-life in cynomolgus monkeys, evaluation is performed taking into account data up to day 7 after injection. More preferably, the calculation is performed with data from day 1 to day 5 when evaluating the terminal half-life in cynomolgus monkeys. Those skilled in the art are also able to recognize effects such as target-mediated clearance and consider them in calculating the terminal half-life. The term "terminal half-life" of a drug, such as a recombinant binding protein of the invention, refers to the time required for the plasma concentration of the drug to reach half of that drug concentration administered to a mammal after reaching pseudo-equilibrium (e.g., calculated in mice with data from 24 hours to 48 hours, or in cynomolgus monkeys with data from day 1 to day 5). The terminal half-life is not defined as the time required for half of the dose of drug administered to a mammal to be eliminated. The term "terminal half-life" is well known to those skilled in the art. Preferably, the pharmacokinetic comparison is performed at any dose, more preferably at an equivalent dose (i.e. the same mg/kg dose) or at an equimolar dose (i.e. the same mol/kg dose), more preferably at an equimolar dose (i.e. the same mol/kg dose). It will be appreciated by those skilled in the art that the experimental dose of equivalent and/or equimolar administration in an animal will vary by 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 for pharmacokinetic measurements is selected from about 0.001mg/kg to about 1000mg/kg, more preferably about 0.01mg/kg to about 100mg/kg, more preferably about 0.1mg/kg to about 50mg/kg, more preferably about 0.5mg/kg to about 10mg/kg.

The term "CD3" or "cluster of differentiation 3" refers to a multimeric protein complex consisting of four different polypeptide chains epsilon, gamma and zeta (ζ) assembled into three pairs epsilon gamma, epsilon delta and zeta. The CD3 complex acts as a T cell co-receptor non-covalently associated with the T cell receptor. It may refer to any form of CD3, as well as variants, isoforms and species homologs thereof that retain at least a portion of the CD3 activity. Thus, a binding protein as defined and disclosed herein may also bind CD3 from a species other than human. In other cases, the binding protein may be entirely specific for human CD3 and may not exhibit species or other types of cross-reactivity. Unless otherwise indicated, such as by specific reference to human CD3, CD3 includes all mammalian species of native sequence CD3, e.g., human, canine, feline, equine, and bovine. The amino acid sequences of the human CD3 gamma, delta and zeta chains are shown in NCBI (www.ncbi.nlm.nih.gov /) Ref.seq. NP-000064.1, NP-000723.1 and NP-932170.1, respectively.

As used herein, the term "CD3 expressing cell" refers to any cell that expresses CD3 (cluster of differentiation 3) on the cell surface, including but not limited to T cells such as cytotoxic T cells (cd8+ T cells) and T helper cells (cd4+ T cells).

The term "CD33" refers to the bone marrow cell surface antigen CD33, which is a sialic acid-binding immunoglobulin-like lectin (Siglec) that plays a role in mediating cell-cell interactions and maintaining immune cells in a dormant state. The amino acid sequence of human CD33 (hCD 33) is shown in UniProt (www.uniprot.org) reference number P20138.

The term "CD70" refers to the CD70 antigen, which is a cytokine that functions as a ligand for CD 27. The CD70-CD27 pathway plays an important role in the generation and maintenance of T cell immunity, particularly during antiviral responses. The amino acid sequence of human CD70 (hCD 70) is shown in UniProt (www.uniprot.org) reference number P32970.

The term "CD123" refers to interleukin-3 receptor subunit α. This is the receptor for interleukin-3. The amino acid sequence of human CD123 (hCD 123) is shown in UniProt (www.uniprot.org) reference number P26951.

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

Furthermore, the term "peptide" also encompasses peptides modified by, for example, glycosylation, as well as proteins comprising two or more polypeptide chains each 4 to 600 amino acids long, crosslinked by, for example, disulfide bonds, such as, for example, insulin and immunoglobulins. The term "chemical or biochemical agent" is intended to include any naturally occurring or synthetic compound that can be administered to a recipient. In a preferred aspect, the targeting agent is a target specific ankyrin repeat domain.

The term "medical condition" (or disorder or disease) includes autoimmune disorders, inflammatory disorders, retinopathies (particularly proliferative retinopathies), neurodegenerative disorders, infections, metabolic diseases, and oncological diseases. Any of the recombinant binding proteins described herein can be used in the manufacture of a medicament for the treatment of such disorders, in particular disorders such as neoplastic diseases. A "medical condition" may be a condition characterized by inappropriate cell proliferation. The medical condition may be a hyperproliferative condition. The present invention relates in particular to a method of treating a medical condition comprising the step of administering to a patient in need of such treatment a therapeutically effective amount of a recombinant binding protein of the invention or said pharmaceutical composition. In a preferred aspect, the medical condition is a neoplastic disease. As used herein, the term "neoplastic disease" refers to an abnormal state or condition of a cell or tissue characterized by cell growth or rapid proliferation of a tumor. In one aspect, the medical condition is a malignant tumor disease. In one aspect, the medical condition is cancer, preferably leukemia, more preferably acute myelogenous leukemia. The term "therapeutically effective amount" refers to an amount sufficient to produce the desired effect on the patient.

The term "antibody" means not only an intact antibody molecule but also any fragment or variant of an antibody molecule that retains the ability to bind an immunogen. Such fragments and variants are also well known in the art and are often used in vitro and in vivo. Thus, the term "antibody" encompasses intact immunoglobulin molecules, antibody fragments such as, for example, fab ', F (ab') 2, and single chain V region fragments (scFv), bispecific antibodies, chimeric antibodies, antibody fusion polypeptides, and non-conventional antibodies.

The terms "cancer" and "cancerous" are used herein to refer to or describe the physiological condition in mammals that is typically characterized by unregulated cell growth. Cancers encompass solid and liquid tumors, and primary tumors and metastases. A "tumor" comprises one or more cancer cells. Solid tumors also typically comprise tumor stroma. Examples of cancers include, but are not limited to, primary and metastatic cancers, lymphomas, blastomas, sarcomas, and leukemias, as well as any other epithelial and lymphoid malignancies. More specific examples of such cancers include brain cancer, bladder cancer, breast cancer, ovarian cancer, clear cell kidney cancer, head/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 Myelogenous Leukemia (AML), chronic Lymphocytic Leukemia (CLL), chronic Myelogenous Leukemia (CML), diffuse large B-cell lymphoma (DLBCL), follicular lymphoma, hodgkin Lymphoma (HL), mantle Cell Lymphoma (MCL), multiple Myeloma (MM), myelodysplastic syndrome (MDS), non-hodgkin lymphoma (NHL), head and neck Squamous Cell Carcinoma (SCCHN), chronic Myelogenous Leukemia (CML), small Lymphocytic Lymphoma (SLL), malignant mesothelioma, colorectal cancer, or gastric cancer.

Examples

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.

Material

Chemicals were purchased from Sigma-Aldrich (USA). The oligonucleotides were from Microsynth (Switzerland). Unless otherwise indicated, DNA polymerase, restriction enzyme and buffer were from New England Biolabs (USA) or Fermentas/Thermo Fisher Scientific (USA). Inducible E.coli expression strains are used for cloning and protein production, for example E.coli XL1-blue (Stratagene, USA) or BL21 (Novagen, USA). Proteins with an N-terminal His tag (such as SEQ ID NO: 33) are often produced to facilitate purification, if appropriate. Two reference T cell conjugates were generated and used as controls in the various experiments described in the examples. These reference T cell conjugates are similar to AMG330 and Fu Tuozhu mab, respectively, and are referred to throughout the examples, descriptions of the figures, and figures as AMG330 or AMG330 analogs and as pertuzumab or pertuzumab analogs.

Molecular biology

Unless otherwise indicated, methods were performed according to known protocols (see, e.g., sambrook j., fritsch e.f. and manitis t. Molecular Cloning: A Laboratory Manual, cold Spring Harbor Laboratory 1989, new York).

Engineered ankyrin repeat libraries

Methods for generating designed ankyrin repeat protein libraries have been described in the following documents: for example, U.S. patent No. 7,417,130; binz et al, J.mol.biol.332,489-503,2003; binz et al 2004, supra. By such methods, a library of engineered ankyrin repeat proteins with randomized ankyrin repeat modules and/or randomized end capping modules can be constructed. For example, such libraries can thus be assembled based on immobilized N-terminal capping modules (e.g., N-terminal capping modules of SEQ ID NO:16, 17, 18, 19, 20, or 21) or randomized N-terminal capping modules according to SEQ ID NO:22, and immobilized C-terminal capping modules (e.g., C-terminal capping modules of SEQ ID NO:23, 24, 25, 26, 27, 28, 29, 30, or 31) or randomized C-terminal capping modules according to SEQ ID NO: 32. Preferably, such libraries are assembled without any of amino acids C, G, M, N (preceding the G residue) and P at randomized positions of the repeat or end-capping module.

Furthermore, such randomized modules in such libraries may comprise additional polypeptide loop insertions with randomized amino acid positions. Examples of such polypeptide loop insertions are Complementarity Determining Region (CDR) loop libraries of antibodies or de novo generated peptide libraries. For example, such loop insertions can be designed using the structure of the N-terminal ankyrin repeat domain of human ribonuclease L (Tanaka, N., nakanishi, M, kusakabe, Y, goto, Y., kitade, Y, nakamura, K.T., EMBO J.23 (30), 3929-3938, 2004) as a guide. Similar to such ankyrin repeat domains in which ten amino acids are inserted in β -turns that exist near the boundary of two ankyrin repeats, the ankyrin repeat library may comprise randomized loops (with fixed and randomized positions) of variable length (e.g., 1 to 20 amino acids) inserted in one or more β -turns of the ankyrin repeat domain.

Any such N-terminal capping module of the ankyrin repeat library preferably has a RILLAA, RILLKA or RELLKA motif (e.g., present at positions 21 to 26 in SEQ ID NO: 1), and any such C-terminal capping module of the ankyrin repeat library preferably has a KLN, KLA or KAA motif (e.g., present at the last three amino acids in SEQ ID NO: 1). Examples of N-terminal capping modules comprising a RILLAA, RILLKA or RELLKA motif are provided in SEQ ID NO 16, 17, 18, 19, 20 or 21, and examples of C-terminal capping modules comprising a KLN, KLA or KAA motif are provided in SEQ ID NO 23, 24, 25, 26, 27, 28, 29, 30 or 31.

The design of such ankyrin repeat libraries can be guided by the known structure of ankyrin repeat domains that interact with the target. Examples of such structures identified by their Protein Database (PDB) unique accession number or identification code (PDB-ID) are 1WDY, 3V31, 3V30, 3V2X, 3V2O, 3UXG, 3TWQ-3TWX, 1N11, 1S70 and 2ZGD.

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

The nomenclature used to define the repeat units and the positions within the modules is based on Binz et al, 2004 (supra), modified in that the boundaries of the ankyrin repeat modules and ankyrin repeat units are shifted by one amino acid position. For example, position 1 of the ankyrin repeat module of Binz et al 2004 (supra) corresponds to position 2 of the ankyrin repeat module of the disclosure, and thus position 33 of the ankyrin repeat module of Binz et al 2004 (supra) corresponds to position 1 of the following ankyrin repeat module of the disclosure.

All DNA sequences were confirmed by sequencing and the calculated molecular weight of the selected proteins was confirmed by mass spectrometry.

Example 1: selection of ankyrin repeat structures comprising binding specificities for CD3, CD33, CD70 or CD123 Domain binding proteins.

A. Selecting a binding protein comprising an ankyrin repeat domain with binding specificity for CD3

Ribosome display (Hanes, J. And Pluckthun, A., PNAS 94,4937-42,1997) was used, from analogy to that described in Binz et al 2004 (supra)A number of ankyrin repeat proteins were selected from the library that have binding specificity for human scCD 3. Assessment of binding of selected clones to recombinant human CD3 targets by crude extract Homogeneous Time Resolved Fluorescence (HTRF) suggests successful selection of hundreds of human scCD3 specific binding proteins. For example, the ankyrin repeat domain of SEQ ID NO. 2 constitutes the amino acid sequence of a selected binding protein comprising an ankyrin repeat domain having binding specificity for scCD 3.

Human recombinant CD3 target preparation

The target form selected is based on a single-chain form consisting of human CD3 epsilon and CD3 gamma heterodimers linked by a 26 amino acid linker (scCD 3 epsilon gamma) and a C-terminal Avi tag for site-directed biotinylation. The target protein contains only the CD3 extracellular domain, lacks the C-terminal cysteine "knob" and the entire transmembrane and cytoplasmic regions.

The extracellular domain of human scCD3 εγ (SEQ ID NO:38_scCD3 εγ_Avi-Bio) was expressed in E.coli in a single-stranded form similar to that described previously (Kjer-Nielsen et al, PNAS,2004,101 (20): 7675-7680), then refolded from inclusion bodies and purified by preparative Size Exclusion Chromatography (SEC). The material was concentrated to 3.4mg/ml in 10mM Tris-HCl, 50mM NaCl, pH 8.0 and biotinylated in vitro using recombinant BirA. To isolate the functional target material, the material was repurified using OKT3 loaded columns (GE HiTrap NHS activated HP columns). The final material was monomeric in size exclusion and stored in 10mM Tris, 100mM NaCl, pH 8.0, 10% glycerol at a final concentration of 0.39 mg/ml.

A review of the preparation of recombinant human scCD3 targets is described in Dunstone et al, acta Crystallographica 2004.

Selection of CD3 specific ankyrin repeat proteins by ribosome display

The selection of CD 3-specific ankyrin repeat proteins was performed by ribosome display technology (Hanes and Pluckthun, supra) using part of the extracellular domain of CD3 (SEQ ID NO: 38) as target protein, the ankyrin repeat protein library described above and established protocols (see, e.g., zahnd, C., amstutz, P. And Pluckthun, A., nat. Methods 4,69-79,2007). After each round of selection, the number of Reverse Transcription (RT) -PCR cycles was continuously reduced from 45 to 28, thereby adjusting the yield due to enrichment of the conjugate. The first four rounds of selection employed standard ribosome display selection, using reduced target concentrations (400 nM, 133nM, 45nM and 15nM, respectively) and increased wash stringency to increase selection pressure from round 1 to round 4 (Binz et al, 2004, supra). In rounds 2-4, mRNA was recovered by competitive elution using excess CD3 binding antibody OKT3 (in each round the competitor excess was constantly added from 35-fold to 300-fold).

Selected clones activate T cells in a bivalent form

Individual ankyrin repeat clones that bound to the CD3 target were selected by ribosome display, cloned into a derivative of the pQE30 (Qiagen) expression vector, transformed into e.coli XL1-Blue (Stratagene), plated on LB-agar (containing 1% glucose and 50 μg/ml ampicillin) and then incubated overnight at 37 ℃. The expression vector (Jun leucine zipper construct with His-tag and Myc-tag and CD3 specific ankyrin repeat domain) was used to screen in bivalent form (with respect to CD3 specific binding domain) which allowed testing of function by cross-linking of T cells. Individual colonies were picked into 96-well plates (each clone in a single well) containing 160 μl of growth medium (TB containing 1% glucose and 50 μg/ml ampicillin) and incubated overnight at 37 ℃ with shaking at 800 rpm. 150 μl of fresh TB medium containing 50 μg/ml ampicillin was inoculated with 8.5 μl of overnight culture in a fresh 96 well plate. After 120 min incubation at 37℃and 850rpm, expression was induced with IPTG (0.5 mM final concentration) and continued for 4 hours. Cells were harvested and the pellet was frozen overnight at-20℃and then resuspended in 8.5. Mu. l B-PERII (Thermo Scientific) and incubated for one hour with shaking (600 rpm) at room temperature. Then 160 μl PBS was added and cell debris was removed by centrifugation (3220 g for 15 min) and stored at-20℃for future use.

In the first step, T cell activation screening was performed using BK112 cd8+ monoclonal T cells. Extracts of each lysed clone were applied to anti-penta-histidine antibody (Qiagen) -coated 96-well plates in 1:20 dilutions (final concentration) in PBSB (PBS pH7.4 supplemented with 12% (w/v) FBS) and incubated overnight at 4 ℃. Plates were washed five times with PBS and then 100. Mu.l of 100,000BK 112T cells were added to each well and incubated in T cell assay medium RPMI-1640+10% FBS+1% L-glutamine+ 1%Pen Strep+200IU IL2. Golgi Stop was added at 0.1. Mu.g/100. Mu.L and the plate was centrifuged at 20g for 3 minutes at room temperature and then at 37℃in CO ₂ Incubate in incubator for 4-5 hours. The cells were centrifuged at 350g for 5 min at 4℃and decanted. Cells were stained for surface CD8 expression, then the cells were stored using BD Cytofix and incubated overnight at 4 ℃. Cells were washed with 1xPBS+2% FBS, and intracellular IFN gamma was stained by adding 50. Mu.l of IFN gamma-APC antibody to Cell perm (BD) and incubating at 4℃for 30 minutes. Cells were again washed in PBS and analyzed using a cell counter FACS Canto II from BD.

Selected clones showed binding to CD3 (shown by HTRF and OKT3 competition) and bispecific forms of work Can be used for

The identified functionally designed ankyrin repeat domain hits (hits) were subcloned into pQE30 (Qiagen) expression vector derivatives containing an N-terminal His tag, a Tumor Associated Antigen (TAA) specific ankyrin repeat domain and a CD3 specific ankyrin repeat domain in order to generate T Cell Engager (TCE) constructs. The construct was expressed in E.coli cells and purified using its His tag according to standard protocols. 500ml of the culture (TB, 50. Mu.g/ml ampicillin, 37 ℃) was inoculated with 25ml of the fixed overnight culture (TB, 1% glucose, 50. Mu.g/ml ampicillin; 37 ℃). Cultures were induced with 0.5mM IPTG at an absorbance of 1.0 to 1.5 at 600nm and incubated at 37℃for 4-5 hours with shaking. The culture was centrifuged and the resulting pellet was resuspended in 25ml TBS ₅₀₀ (50 mM Tris-HCl,500mM NaCl,pH8) and cleaved (sonicated). After lysis, the samples were mixed with 50KU DNase/ml, incubated for 15 minutes, then heat treated at 62.5℃for 30 minutes, centrifuged and the supernatant collected and filtered. Triton X100 (1% (v/v) final concentration) and imidazole (20 mM final concentration) were added to the homogenate. The protein was purified on a nickel-nitrilotriacetic acid (Ni-NTA) column according to standard protocols and resins known to those skilled in the art, followed by Size exclusion chromatography was performed on the system.

In a first step, binding to recombinant proteins is tested using HTRF assay. Titration of ankyrin repeat protein (5 nM-640 nM) in PBS-TC (PBS supplemented with 0.1% (w/v) Casein and 0.1% Tween20, pH 7.4) was performed against 48nM (final concentration) of human biotinylated scCD3 εγ, 1:100 (final concentration) of anti-6 His-D2 HTRF antibody-FRET acceptor conjugate (Cisbio) and 1:100 (final concentration) of anti-strep-Tb antibody FRET donor conjugate (Cisbio) in wells of 384-well plates and incubated at room temperature for 120 min. HTRF was read out on Tecan M1000 using 340nm excitation wavelength and 665±10nm emission filter. Several candidates showed dose-dependent binding and were used for further evaluation. For all these constructs, the binding signal was at background levels when competing with a 20-fold excess of CD3 binding antibody (OKT 3 variant containing human Fc region, final concentration 2.4 mM), which binds to the conformational epitope of CD3 epsilon (Kjer-Nielsen et al, PNAS,2004,101 (20): 7675-7680). Dose-dependent in vitro T cell activation was confirmed using BK 112T cell activation assay (BK 112 CD8 monoclonal T cells pre-activated with CD3/CD28 Dynabeads) in the presence of TAA1 expressing tumor cells. Intracellular IFNy levels on cd8+ or cd4+ T cells were measured after 5 hours incubation of BK112 and SKOV3 cells (E: t=1:10) in the presence of 1pM-100,000pM bispecific TCE construct. For CD4+ and CD8+ cells, the most potent constructs showed EC50 values of 0.5nM and 0.4nM, respectively.

Affinity maturation and rational design of selected CD 3-specific ankyrin repeat proteins

Several rounds of affinity maturation combined with rational design were applied to parental low affinity binding CD3 specific ankyrin repeat proteins (designated precursor a) and four higher affinity CD3 specific ankyrin repeat proteins (designated precursors B, C and D) were obtained. These precursor molecules are then finally engineered into CD 3-specific ankyrin repeat proteins, e.gProtein #2.

Diversity was introduced by using error-prone PCR and DNA I shuffling as described in Zahnd et al, nat Methods,2007,4:269-279, affinity maturation was performed on one of the parent CD 3-specific ankyrin repeat proteins (precursor A) that was selected taking into account its sufficient binding capacity for the CD3 target and its ability to activate T cells in vitro effectively. Briefly: three rounds of ribosome display were performed using different concentrations of dNTP analogues (mutagenesis kit from Jena Biosciences, using 5 μm-10 μm 8-oxo-dGTP and dtpp) to introduce about 1-2 mutations per CD 3-specific ankyrin repeat protein/round (wash step increased from round 1 (3 x 15 min) to 3 x 30 min (round 2) to 3 x 45 min (round 3)) with target concentration kept constant at 5nM with increasing selection pressure. In the second step, DNA shuffling and backcrossing of DNA libraries using parental clones was performed using DNAse I incubation time of 90 seconds and DNA polymerase HotStarTaq DNA polymerase (Qiagen) in one or two rounds of ribosome display, as previously described (Cadwell and Joyce, PCR Methods Appl,1992,2:28-33;Stemmer,Nature,1994 370:389-391; zaccolo et al, J Mol Biol 1996, 255:589-603). The affinity matured CD 3-specific ankyrin repeat protein library was subcloned into a derivative of the pQE30 (Qiagen) expression vector, which ultimately contained an N-terminal His-Flag tag, HSA binding ankyrin repeat domain for half-life extension, TAA1 binding ankyrin repeat domain and CD 3-specific ankyrin repeat domain, and HTRF binding to recombinant sccd3εγ was expressed and screened as previously described. The leader clone (precursor B, generated after 5 rounds of affinity maturation, including 3 rounds of error-prone PCR and two rounds of DNA shuffling and backcrossing using 10 μm dNTP analogues in all steps) was selected.

In the same process, several potentially beneficial mutations for increased T cell activation were identified by% ifnγ+ T cells in the BK112 assay, including the N cap mutation at positions 5 and 20, the repeat mutation inside position 1 of positions 2 and 4, the repeat mutation inside position 2, 5 and 20, and the C cap mutation at positions 1 and 18. The combination of these mutations (while keeping the N-cap and C-cap framework mutations at a minimum to maintain thermostability) resulted in a set of designed variants that were retested for improved T cell activation in the same format. Thus, a new further mature variant (precursor C) is generated, which has a higher T cell activation potency than the parent and the originally mature variant.

To further increase CD3 affinity and T cell activation efficacy, precursor C clones were subjected to a second affinity maturation similar to that described above. Briefly: four-wheeled ribosome display was performed. In the first three rounds, mutations were introduced by error-prone PCR with 7.5. Mu.M dNTP analogues. In the first and third rounds, dissociation rate selection was performed using the further mature cloned as such or non-biotinylated CD3 target protein to achieve competition. The 1nM target was used in rounds 1 and 2, while the 5nM target was used in the less stringent rounds 3 and 4. CD3 variants, comprising an HSA binding domain and a TAA1 binding ankyrin repeat domain, were screened in a trispecific format using a dissociation rate HTRF assay with 250-fold further mature clones as competitors. A total of 3x96 clones with the highest remaining HTRF signal after competition were sequenced. The beneficial mutations identified for improved binding (including N cap position 16, first internal repeat positions 1, 12, 18, 19, 26, 30 and 33, second internal repeat positions 2, 3, 7, 20, 21, 26 and 32, and C cap positions 9, 11 and 18) or for reduced domain interactions (including N cap positions 11, 18, 19, 26, and C cap positions 3, 19 and 22) were first tested as separate or paired mutations on purified protein variants for BK112T cell activation. The most beneficial variants are then recombined in a second step and the variants with the highest T cell activation are selected, which results in the identification of precursor D.

In the final step, variants were generated based on CD3 specific ankyrin repeat protein precursor A, B, C and D, respectivelyProtein #6 in order to improve serum half-life and biophysical properties. Thus, an N-cap mutation was introduced at positions 23 and/or 26, while some of the framework mutations were removed (positions 19, 18).

As described below, the affinity-matured ankyrin repeat domain with binding specificity for human CD3 was cloned into a pQE (QIAgen, germany) -based expression vector, providing an N-terminal His tag (SEQ ID NO: 33) to facilitate simple protein purification. For example, an expression vector encoding the following ankyrin repeat proteins is constructed:

protein #1 (SEQ ID NO:1 having His tag fused to its N-terminus (SEQ ID NO: 33))

Protein #2 (SEQ ID NO:2 having His tag fused to its N-terminus (SEQ ID NO: 33))

Protein #3 (SEQ ID NO:3 having His tag fused to its N-terminus (SEQ ID NO: 33))

Protein #4 (SEQ ID NO:4 having His tag fused to its N-terminus (SEQ ID NO: 33))

High level and soluble expression of CD3 specific ankyrin repeat proteins

For further analysis, selected clones exhibiting specific CD3 binding were expressed in E.coli cells as monovalent or in combination with TAA and/or HSA binding ankyrin repeat domains and purified using their His tag, followed by purification in accordance with standard protocols and resins known to those skilled in the art Size exclusion chromatography analysis was performed on the system. For monovalent and multivalent constructs, the proteins were monomeric and soluble when concentrated to 10mg/ml in TBS pH 8.0 (50mM Tris,500mM NaCl) or PBS pH 7.4, respectively. A representative example of such SDS-PAGE analysis is shown in fig. 19.

B. Selecting a binding protein comprising an ankyrin repeat domain with binding specificity for CD33

Ribosome display (Hanes, J. And Pluckthun, A., PNAS 94,4937-42,1997) was used, from analogy to that described in Binz et al 2004 (supra)A number of ankyrin repeat proteins were selected from the library that have binding specificity for human CD33 (hCD 33). Assessment of binding of selected clones to recombinant human CD33 targets (full length and splice variants of ECD of CD 33) by crude extract Homogeneous Time Resolved Fluorescence (HTRF) suggests successful selection of hundreds of hCD 33-specific binding proteins. For example, the number of the cells to be processed,the ankyrin repeat domains of SEQ ID NOS 15 and 67-70 constitute the amino acid sequence of a selected binding protein comprising an ankyrin repeat domain having binding specificity for hCD 33.

Selection of CD 33-specific ankyrin repeat proteins by ribosome display

Selection of hCD 33-specific ankyrin repeat proteins was performed by ribosome display technology (Hanes and Pluckthun, supra) using the biotinylated extracellular domain of human CD33 (SEQ ID NO: 87) as target protein, ankyrin repeat protein library as described above and established protocols (see, e.g., zahnd, C., amstutz, P. And Pluckthun, A., nat. Methods 4,69-79,2007). The CD33 target (evatric) contains a C-terminal Fc tag and Avi tag and is biotinylated using the enzyme BirA-GST. Two different forms of CD33 have been used to select: full length ECD of CD33 by using residues 18-259 to cover both the variable and constant domains of CD33 and CD33 splice variants by using residues 120-259 to cover only the constant domains of CD 33. With a total of four rounds of standard ribosome selection, the selection pressure from round 1 to round 4 was increased using decreasing target concentration and increasing wash stringency (Binz et al, 2004, supra). The deselection strategy was applied in each round by using streptavidin and Neutravidin (Neutravidin) beads bound to biotinylated non-CD 33 Fc domain. After each round of selection, the number of Reverse Transcription (RT) -PCR cycles was continuously reduced from 45 to 28, thereby adjusting the yield due to enrichment of the conjugate.

To enrich for high affinity CD 33-specific ankyrin repeat proteins, the output from the fourth round of standard ribosome display selection (above) was subjected to rounds of dissociation rate selection with increased selection stringency (Zahnd, 2007, supra). A final standard selection round is performed after the dissociation rate selection round to amplify and recover the dissociation rate selected binding protein. In these last two rounds of selection, the number of RT-PCR cycles was 30 and 35, respectively.

As described above, a total of three different selection methods have been performed, which differ as follows: in the first approach, ECD was selected only for full length CD33 protein. In the second method, at each ofTargets for alternate full length CD33 and splice variants in the wheel. In the third method, the conditions of the first method were used by adding HIM-3-4CD33 binding antibody (BD Pharmingen ^TM ) To apply a competitive elution step. From each method, conjugates directed against full-length CD33 and/or splice variants thereof were generated.

As shown by the crude extract HTRF, selected clones specifically bind to human and cynomolgus monkey CD33

Each selected ankyrin repeat protein that specifically binds hCD33 in solution was identified by a Homogeneous Time Resolved Fluorescence (HTRF) assay using a crude extract of escherichia coli cells expressing the ankyrin repeat protein using standard protocols. The ankyrin repeat selected by ribosome display was cloned into a derivative of pQE30 (Qiagen) expression vector (pMPAG 06), this derivative was transformed into E.coli XL1-Blue (Stratagene), which was plated on LB-agar (containing 1% glucose and 50. Mu.g/ml ampicillin) and then incubated overnight at 37 ℃. Individual colonies were picked into 96-well plates (each clone in a single well) containing 160 μl of growth medium (TB containing 1% glucose and 50 μg/ml ampicillin) and incubated overnight at 37 ℃ with shaking at 800 rpm. Mu.l of fresh TB medium containing 50. Mu.g/ml ampicillin was inoculated with 50. Mu.l of overnight culture in fresh 96-well plates. After 120 min incubation at 37℃and 700rpm, expression was induced with IPTG (0.5 mM final concentration) for 4 hours. Cells were harvested and the pellet was frozen overnight at-20℃and then resuspended in 50. Mu. l B-PERII (Thermo Scientific) and incubated for 15 minutes with shaking (900 rpm) at room temperature. Then, 950. Mu.l of PBS was added and cell debris was removed by centrifugation (3220 g for 15 min).

The extract of each lysed clone was used as PBSTB (supplemented with 0.1% tweenAnd 0.2% (w/v) BSA in PBS, pH 7.4) with 12nM (final concentration) biotinylated hCD33, 1:200 (final concentration) anti-strep-D2 HTRF antibody-FRET receptor conjugate (Cisbio) and 1:200 (final concentration) anti-6HThe is-Tb antibody FRET donor conjugate (Cisbio) was applied together in the wells of 384-well plates and incubated for 90 minutes at room temperature. HTRF was read out on Tecan M1000 using 340nm excitation wavelength and 665±10nm emission filter. Hundreds of clones were screened by HTRF of such crude cell extracts, and ankyrin repeat domains specific for hCD33 were found. The amino acid sequence of a selected ankyrin repeat domain that specifically binds hCD33 is provided in SEQ ID No. 15.

Protein #15 (SEQ ID NO:15)；

Similarly, each selected ankyrin repeat protein in solution that specifically binds hCD33 was identified by a Homogeneous Time Resolved Fluorescence (HTRF) assay using a standard protocol using a crude extract of e.coli cells expressing ankyrin repeat proteins. The ankyrin repeat clones selected by ribosome display were cloned into a derivative of pQE30 (Qiagen) expression vector (pMPDV 045), which contains the engineered ankyrin repeat protein with C-terminal binding to CD3 and subsequent Flag tag, which was transformed into E.coli XL1-Blue (Stratagene), which was plated on LB-agar (containing 1% glucose and 50. Mu.g/ml ampicillin) and then incubated overnight at 37 ℃. Individual colonies were picked into 96-well plates (each clone in a single well) containing 160 μl of growth medium (TB containing 1% glucose and 50 μg/ml ampicillin) and incubated overnight at 37 ℃ with shaking at 800 rpm. 150 μl of fresh TB medium containing 50 μg/ml ampicillin was inoculated with 8.5 μl of overnight culture in a fresh 96 well plate. After 120 min incubation at 37℃and 700rpm, expression was induced with IPTG (0.5 mM final concentration) for 4 hours. Cells were harvested and the pellet was frozen overnight at-20℃and then resuspended in 8. Mu. l B-PERII (Thermo Scientific) and incubated for 1 hour with shaking (900 rpm) at room temperature. Then 160. Mu.l PBS was added and cell debris was removed by centrifugation (3220 g for 15 min).

The extract of each lysed clone was used as PBSTB (supplemented with 0.1% tweenAnd 0.2% (w/v) BSA in PBS, pH 7.4) 1:1000 dilutions (final concentration) were applied to wells of 384 well plates with 6nM (final concentration) biotinylated hCD33 (full length or splice variant of ECD CD 33), 1:400 (final concentration) anti-strep-Tb HTRF antibody-FRET donor conjugate (Cisbio) and 1:400 (final concentration) anti-6 His-D2 antibody FRET acceptor conjugate (Cisbio) and incubated at room temperature for 60 min. HTRF was read out on Tecan M1000 using 340nm excitation wavelength and 665±10nm emission filter. Hundreds of clones were screened by HTRF of such crude cell extracts, and ankyrin repeat domains specific for hCD33 were found. Amino acid sequence of selected ankyrin repeat domain which specifically binds hCD33 is for +.>Protein #37 is provided in SEQ ID NO. 68 and is for +.>Protein #38 is provided in SEQ ID NO. 69.

Engineering of additional ankyrin repeat proteins with binding specificity for hCD33

SEQ ID NOS 67 to 70 are engineered based on the sequences of SEQ ID NOS 68 and 69, respectively.

For the followingProtein #37 and->Protein #38, the sequence was modified to reduce the amount of aromatic residues and alter the surface charge. In both N-terminal capping modules, the RILLAA motif is replaced by RILLKA and the aspartic acid (position 18) is replaced by leucine. In both C-terminal capping modules, glutamic acid (position 18) is replaced with glutamine. For the following Protein #12, the additional phenylalanine (position 14) in the N-terminal end-capping module was replaced with valine. For the followingProtein #37, in which the additional tryptophan (position 7) was replaced with valine and the EDIA motif in the second internal repeat (positions 18-21) was replaced with LEIV. After 48h incubation with PanT cells and MOLM-13 cells at a ratio of 5:1, the engineered variants altered T cell killing (evaluated in combination with DARPin molecules binding other TAA and CD 3) by no more than a factor of 2 compared to the measured parent form in the standard LDH killing assay.

Expression of CD 33-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 and purified using His-tag according to standard protocols. 0.11ml of the fixed overnight culture (TB, 1% glucose, 50mg/l ampicillin; 37 ℃) was used to inoculate 0.99ml of the culture in 96-well plates (TB, 50mg/l ampicillin, 37 ℃). After incubation for 2 hours at 37 ℃ (700 rpm), the cultures were induced with 0.5mM IPTG and incubated for 6h with shaking (900 rpm) at 37 ℃. Cells were harvested and the pellet was frozen overnight at-20℃and then resuspended in 50. Mu. l B-PERII (Thermo Scientific) supplemented with DNase I (200 units/ml) and Lysozyme (0.4 mg/ml) and incubated for one hour at room temperature with shaking (900 rpm). Then, 60. Mu.l of low-salt sodium phosphate buffer was added, and cell debris was removed by centrifugation (3220 g for 15 min). A total of 8 separate expressions were pooled before removal of cell debris by centrifugation (3,200 g, 60min at 4 ℃). Supernatants were filtered using MultiScreen filter plates (Millipore), then purified using 96-well Thermo HisPur cobalt centrifuge plates, and protein solutions were re-buffered in PBS using 96-well Thermo Zeba centrifuge desalter plates. Proteins purified on an Agilent 1200HPLC system using a standard Sephadex 150/5 column were soluble and monomeric in PBS.

Producing affinity matured CD33 specific binding proteins: derived from Protein #36 (SEQ ID NO: 67 Of the components of the system) is provided/> Protein #72 (SEQ ID NO: 111) and/> protein #73 (SEQ ID NO: 112)

In further development of the CD33 specific binding proteins initially identified, affinity maturation was used to generate new variants with very high affinity for the target protein and/or very low dissociation rates from the target protein. Thus, one of the initially identified CD 33-specific binding proteins is selectedProtein #36 ("parent" binding protein) serves as a suitable starting point for affinity maturation. The affinity maturation procedure involves saturation mutagenesis of each random position of the ankyrin repeat domain that serves as a starting point. Sequences generated by the affinity maturation procedure were screened for lower dissociation rates by competing HTRF. Briefly: a single amino acid point mutant variant was generated by performing a standard QuikChange PCR on the parental plasmid (pMCHE 1190) using primers with a single NNK degenerate codon to introduce all 20 amino acids at potential binding positions. Crude Extracts (CE) of protein variants (eventually containing an N-terminal His tag followed by a CD33 specific binding domain) were produced from standard e.coli expression cultures. Incubating the diluted CE with biotinylated target, followed by adding excess unlabeled parent CD33 binding protein (++ >Protein # 36) and measure HTRF signal over time. Beneficial mutations identified based on higher HTRF signals compared to parental clones were passed through the eggWhite matter engineering is combined in binding proteins.

Engineered CD33 specific hits were subcloned into derivatives of pQE30 (Qiagen) expression vectors to express the protein in bivalent (2D) and monovalent (1D) form, respectively, one vector eventually contained an N-terminal His tag followed by the CD33 specific binding domain of SEQ ID NO:111 or 112 and the C-terminal CD3 binding domain of SEQ ID NO:3, and one vector eventually contained an N-terminal His tag followed by the CD33 specific binding domain of SEQ ID NO:111 or 112. All constructs were expressed in E.coli cells and purified using their His tag according to standard protocols.

C. Selecting a binding protein comprising an ankyrin repeat domain with binding specificity for CD123

Ribosome display (Hanes, J. And Pluckthun, A., PNAS 94,4937-42,1997) was used, from analogy to that described in Binz et al 2004 (supra)A number of ankyrin repeat proteins were selected from the library that have binding specificity for human CD123 (hCD 123) and cynomolgus monkey CD123 (CD 123). The binding of selected clones to recombinant human and cynomolgus monkey CD123 targets was assessed by crude extract Homogeneous Time Resolved Fluorescence (HTRF), indicating successful selection of hundreds of hCD123 specific binding proteins. For example, the ankyrin repeat domains of SEQ ID NOS 6 and 65 to 66 constitute the amino acid sequence of a selected binding protein comprising ankyrin repeat domains having binding specificity for hCD123 and cCD 123.

Selection of CD123 specific ankyrin repeat proteins by ribosome display

The selection of hCD123 specific ankyrin repeat proteins (SEQ ID NO: 6) was performed by ribosome display (Hanes and plackthun, supra) using the biotinylated extracellular domain of human CD123 (uniprot ID 26951) and cynomolgus CD123 (uniprot ID G8F3K 3) as target proteins, an ankyrin repeat library as described above and established protocols (see e.g. Zahnd, c., amstuz, p. And plackthun, a., nat. Methods 4,69-79,2007). All target molecules were produced in Fc format. With a total of four rounds of standard ribosome selection, the selection pressure from round 1 to round 4 was increased using decreasing target concentration and increasing wash stringency (Binz et al, 2004, supra). Specifically, cynomolgus monkey targets were used in round 1, round 2 and round 4, and human CD123 was used in round 3. The deselection strategy was applied starting from the second round with an excess of non-CD 123-Fc protein. After each round of selection, the number of Reverse Transcription (RT) -PCR cycles was continuously reduced from 45 to 31, thereby adjusting the yield due to enrichment of the conjugate.

The selection of hCD123 specific ankyrin repeat proteins (SEQ ID NOs: 65 to 66) was performed by ribosome display technology (Hanes and plinckthun, supra) using the biotinylated extracellular domain of human CD123 (SEQ ID NO: 88) as target protein, ankyrin repeat protein library as described above and established protocols (see e.g. Zahnd, c., amstuz, p. And plinckthun, a., nat. Methods 4,69-79,2007). The CD123 target (evatric) contains a C-terminal Fc tag and Avi tag and is biotinylated using the enzyme BirA-GST. With a total of four rounds of standard ribosome selection, the selection pressure from round 1 to round 4 was increased using decreasing target concentration and increasing wash stringency (Binz et al, 2004, supra). The deselection strategy was applied in each round by using streptavidin and neutravidin beads. After each round of selection, the number of Reverse Transcription (RT) -PCR cycles was continuously reduced from 45 to 28, thereby adjusting the yield due to enrichment of the conjugate.

To enrich for high affinity CD 123-specific ankyrin repeat proteins, the output from the fourth round of standard ribosome display selection (above) was subjected to rounds of dissociation rate selection with increased selection stringency (Zahnd, 2007, supra). A final standard selection round is performed after the dissociation rate selection round to amplify and recover the dissociation rate selected binding protein. In rounds 5 and 6, the number of RT-PCR cycles was 30 and 35, respectively.

In addition, two additional selection methods were performed following standard methods (as described above) to expand potential epitope space by applying a competitive elution step using a CD 123-binding T cell conjugate (epitope of patent US9822181B2-7G 3) or by co-incubating hCD 123-specific ankyrin repeat protein (with a different epitope than antibody 7G 3) with CD123 target prior to selection. From the methods of each application, conjugates directed to CD123 were generated.

Clones selected as indicated by crude extract HTRF specifically bind human CD123

Each selected ankyrin repeat protein that specifically binds hCD123 in solution was identified by a Homogeneous Time Resolved Fluorescence (HTRF) assay using a crude extract of escherichia coli cells expressing the ankyrin repeat protein using standard protocols. The ankyrin repeat clones selected by ribosome display were cloned into a derivative of pQE30 (Qiagen) expression vector (pMPDV 045), which contains the engineered ankyrin repeat protein with C-terminal binding to CD3 and subsequent Flag tag, which was transformed into E.coli XL1-Blue (Stratagene), which was plated on LB-agar (containing 1% glucose and 50. Mu.g/ml ampicillin) and then incubated overnight at 37 ℃. Individual colonies were picked into 96-well plates (each clone in a single well) containing 160 μl of growth medium (TB containing 1% glucose and 50 μg/ml ampicillin) and incubated overnight at 37 ℃ with shaking at 800 rpm. 150 μl of fresh TB medium containing 50 μg/ml ampicillin was inoculated with 8.5 μl of overnight culture in a fresh 96 well plate. After 120 min incubation at 37℃and 700rpm, expression was induced with IPTG (0.5 mM final concentration) for 4 hours. Cells were harvested and the pellet was frozen overnight at-20℃and then resuspended in 8. Mu. l B-PERII (Thermo Scientific) and incubated for 1 hour with shaking (900 rpm) at room temperature. Then 160. Mu.l PBS was added and cell debris was removed by centrifugation (3220 g for 15 min).

The extract of each lysed clone was used as PBSTB (supplemented with 0.1% tweenAnd 0.2% (w/v) BSA in PBS, pH 7.4) with 6nM (final concentration) biotinylated human or cynomolgus monkey CD123, 1:300 (final concentration) anti-6 His-D2HTRF antibody-FRET receptor conjugate (Cisbio) and 1:300 (final concentration) anti-strep-Tb antibodyFRET donor conjugates (Cisbio) were applied together in the wells of 384-well plates and incubated for 120 min at room temperature. HTRF was read out on Tecan M1000 using 340nm excitation wavelength and 665±10nm emission filter. Hundreds of clones were screened by HTRF of such crude cell extracts, and ankyrin repeat domains specific for human and cynomolgus CD123 were found. Amino acid sequences of selected ankyrin repeat domains that specifically bind human and cynomolgus monkey CD123 are provided in SEQ ID NOs 6, 102 and 103.

Protein #6 (SEQ ID NO: 6);

protein #63 (SEQ ID NO: 102)

Protein #64 (SEQ ID NO: 103)

Alternatively, the extract of each lysed clone was used as PBSTB (supplemented with 0.1% tweenAnd 0.2% (w/v) BSA in PBS, pH 7.4) in 1:1000 dilution (final concentration) was applied to the wells of 384 well plates together with 4nM (final concentration) biotinylated hCD123, 1:400 (final concentration) anti-strep-Tb HTRF antibody-FRET donor conjugate (Cisbio) and 1:400 (final concentration) anti-6 His-D2 antibody FRET acceptor conjugate (Cisbio) and incubated at room temperature for 60 minutes. HTRF was read out on Tecan M1000 using 340nm excitation wavelength and 665±10nm emission filter. Hundreds of clones were screened by HTRF of such crude cell extracts, and ankyrin repeat domains specific for hCD123 were found. The amino acid sequence of a selected ankyrin repeat domain that specifically binds hCD123 is provided in SEQ ID No. 66.

Protein #35 (SEQ ID NO: 66).

Engineering of additional ankyrin repeat proteins with binding specificity for hCD123

SEQ ID NO. 65 is further engineered based on the sequence of SEQ ID NO. 66.

ModificationProtein #35 (SEQ ID NO: 66) to alter surface charge. In the N-terminal capping module, aspartic acid (position 18) is replaced with leucine, and in the C-terminal capping module, glutamic acid (position 18) is replaced with glutamine. After 48h incubation with PanT cells and MOLM-13 cells at a ratio of 5:1, the engineered variants did reduce T cell killing (evaluated in combination with DARPin molecules of bound CD 3) compared to the parent measured in the standard LDH killing assay.

Expression of CD123 specific ankyrin repeat proteins

For further analysis, selected clones exhibiting specific human CD123 binding in crude cell extract HTRF as described above were expressed in e.coli cells and purified using His-tag according to standard protocols. 0.11ml of the fixed overnight culture (TB, 1% glucose, 50mg/l ampicillin; 37 ℃) was used to inoculate 0.99ml of the culture in 96-well plates (TB, 50mg/l ampicillin, 37 ℃). After incubation for 2 hours at 37 ℃ (700 rpm), the cultures were induced with 0.5mM IPTG and incubated for 6h with shaking (900 rpm) at 37 ℃. Cells were harvested and the pellet was frozen overnight at-20℃and then resuspended in 50. Mu.l of B-PERII (Thermo Scientific) supplemented with DNase I (200 units/ml) and Lysozyme (0.4 mg/ml) and incubated for one hour at room temperature with shaking (900 rpm). Then, 60. Mu.l of low-salt sodium phosphate buffer was added, and cell debris was removed by centrifugation (3220 g for 15 min). A total of 8 separate expressions were pooled before removal of cell debris by centrifugation (3,200 g, 60min at 4 ℃). Supernatants were filtered using MultiScreen filter plates (Millipore), then purified using 96-well Thermo HisPur cobalt centrifuge plates, and protein solutions were re-buffered in PBS using 96-well Thermo Zeba centrifuge desalter plates. Proteins purified on an Agilent 1200HPLC system using a standard Sephadex 150/5 column were soluble and monomeric in PBS.

Producing affinity matured CD123 specific binding proteins: derived from Protein #34 Protein #66/> Protein #67

In further development of the CD123 specific binding proteins initially identified, affinity maturation was used to produce variants with very high affinity for the target protein and/or very low dissociation rates from the target protein. Thus, one of the initially identified binding proteins is selectedProtein #34 (SEQ ID NO:65, "parent" binding protein) serves as a suitable starting point for affinity maturation. The affinity maturation procedure involves saturation mutagenesis of each random position of the ankyrin repeat domain that serves as a starting point. Sequences generated by the affinity maturation procedure were screened for lower dissociation rates by competing HTRF. Briefly: a single amino acid point mutant variant was generated by performing a standard QuikChange PCR on a parental plasmid using primers with single NNK degenerate codons to introduce all 20 amino acids at potential binding sites. Crude Extracts (CE) of protein variants (eventually containing an N-terminal His tag followed by CD123 specific binding domain of SEQ ID NO:105 or 106) were produced from standard E.coli expression cultures. The diluted CE was incubated with biotinylated target, then excess unlabeled parent CD123 specific binding domain of SEQ ID NO:65 was added and HTRF signal was measured over time. Based on and parent of The beneficial mutations identified by the higher HTRF signals of proteins compared to proteins are combined in the binding protein by protein engineering.

In a first step, the engineered CD123 specific hits are subcloned into a derivative of the pQE30 (Qiagen) expression vector, which ultimately contains an N-terminal His tag, followed by the CD123 specific binding domain of SEQ ID NO:105 or 106 and the C-terminal CD3 specific binding domain of SEQ ID NO: 3. The construct was expressed in E.coli cells and purified using its His tag according to standard protocols.

D. Selecting a binding protein comprising an ankyrin repeat domain with binding specificity for CD70

Ribosome display (Hanes, J. And Pluckthun, A., PNAS 94,4937-42,1997) was used, from analogy to that described in Binz et al 2004 (supra)A number of ankyrin repeat proteins were selected from the library that have binding specificity for human CD70 (hCD 70). The binding of selected clones to recombinant human CD70 targets was assessed by crude extract Homogeneous Time Resolved Fluorescence (HTRF), indicating successful selection of hundreds of hCD 70-specific binding proteins. For example, the ankyrin repeat domain of SEQ ID NO. 64 constitutes the amino acid sequence of a selected binding protein comprising an ankyrin repeat domain having binding specificity for hCD 70.

Selection of CD 70-specific ankyrin repeat proteins by ribosome display

Selection of hCD 70-specific ankyrin repeat proteins was performed by ribosome display technology (Hanes and Pluckthun, supra) using the biotinylated extracellular domain of human CD70 (SEQ ID NO: 89) as target protein, ankyrin repeat protein library as described above and established protocols (see e.g., zahnd, C., amstutz, P. And Pluckthun, A., nat. Methods 4,69-79,2007). CD70 targets (ACROBiosystems) contain a C-terminal Fc tag and are chemobiotinylated using a 5-fold excess of biotin. With a total of four rounds of standard ribosome selection, the selection pressure from round 1 to round 4 was increased using decreasing target concentration and increasing wash stringency (Binz et al, 2004, supra). The deselection strategy was applied in each round by using streptavidin and neutravidin beads. After each round of selection, the number of Reverse Transcription (RT) -PCR cycles was continuously reduced from 45 to 28, thereby adjusting the yield due to enrichment of the conjugate.

To enrich for high affinity CD 70-specific ankyrin repeat proteins, the output from the fourth round of standard ribosome display selection (above) was subjected to rounds of dissociation rate selection with increased selection stringency (Zahnd, 2007, supra). A final standard selection round is performed after the dissociation rate selection round to amplify and recover the dissociation rate selected binding protein. In rounds 5 and 6, the number of RT-PCR cycles was 30 and 35, respectively.

Clones selected as indicated by crude extract HTRF specifically bind human CD70

Each selected ankyrin repeat protein that specifically binds hCD70 in solution was identified by a Homogeneous Time Resolved Fluorescence (HTRF) assay using a crude extract of escherichia coli cells expressing the ankyrin repeat protein using standard protocols. The ankyrin repeat selected by ribosome display was cloned into a derivative of pQE30 (Qiagen) expression vector (pMPDV 25), this derivative was transformed into E.coli XL1-Blue (Stratagene), which was plated on LB-agar (containing 1% glucose and 50. Mu.g/ml ampicillin) and then incubated overnight at 37 ℃. Individual colonies were picked into 96-well plates (each clone in a single well) containing 160 μl of growth medium (TB containing 1% glucose and 50 μg/ml ampicillin) and incubated overnight at 37 ℃ with shaking at 800 rpm. 150 μl of fresh TB medium containing 50 μg/ml ampicillin was inoculated with 8.5 μl of overnight culture in a fresh 96 well plate. After 120 min incubation at 37℃and 700rpm, expression was induced with IPTG (0.5 mM final concentration) for 4 hours. Cells were harvested and the pellet was frozen overnight at-20℃and then resuspended in 8. Mu. l B-PERII (Thermo Scientific) and incubated for 1 hour with shaking (900 rpm) at room temperature. Then 160 μl PBS was added and cell debris was removed by centrifugation (3220 g for 15 min).

Each of the lysed clones was clonedExtracts as PBSTB (supplemented with 0.1% TweenAnd 0.2% (w/v) BSA in PBS, pH 7.4) in 1:2000 dilution (final concentration) was applied to the wells of 384 well plates together with 2nM (final concentration) biotinylated hCD70, 1:400 (final concentration) anti-strep-Tb HTRF antibody-FRET donor conjugate (Cisbio) and 1:400 (final concentration) anti-6 His-D2 antibody FRET acceptor conjugate (Cisbio) and incubated at room temperature for 60 minutes. HTRF was read out on Tecan M1000 using 340nm excitation wavelength and 665±10nm emission filter. Hundreds of clones were screened by HTRF of such crude cell extracts, and ankyrin repeat domains specific for hCD70 were found. Amino acid sequences of selected ankyrin repeat domains that specifically bind hCD70 are provided in SEQ ID NOs 107 and 108:

protein #68 (SEQ ID NO: 107);

protein #69 (SEQ ID NO: 108);

engineering of additional ankyrin repeat proteins with binding specificity for hCD70

SEQ ID NO. 64 is a sequence of the selected designed ankyrin repeat domain based on the previous step (i.eProtein #69 (SEQ ID NO: 108)).

This selected sequence is modified to alter the surface charge. In the N-terminal capping module, the rilla motif is replaced with RILLKA and the aspartic acid (position 18) is replaced with leucine. In addition, for the C-terminal capping module, the modification is to replace serine (position 16) with glycine and glutamic acid (position 18) with glutamine.

CD70 specificityExpression of ankyrin repeat proteins

For further analysis, selected clones exhibiting specific human CD70 binding in crude cell extract HTRF as described above were expressed in e.coli cells and purified using His-tag according to standard protocols. 0.11ml of the fixed overnight culture (TB, 1% glucose, 50mg/l ampicillin; 37 ℃) was used to inoculate 0.99ml of the culture in 96-well plates (TB, 50mg/l ampicillin, 37 ℃). After incubation for 2 hours at 37 ℃ (700 rpm), the cultures were induced with 0.5mM IPTG and incubated for 6h with shaking (900 rpm) at 37 ℃. Cells were harvested and the pellet was frozen overnight at-20℃and then resuspended in 50. Mu.l of B-PERII (Thermo Scientific) supplemented with DNase I (200 units/ml) and Lysozyme (0.4 mg/ml) and incubated for one hour at room temperature with shaking (900 rpm). Then, 60. Mu.l of low-salt sodium phosphate buffer was added, and cell debris was removed by centrifugation (3220 g for 15 min). A total of 8 separate expressions were pooled before removal of cell debris by centrifugation (3,200 g, 60min at 4 ℃). Supernatants were filtered using MultiScreen filter plates (Millipore), then purified using 96-well Thermo HisPur cobalt centrifuge plates, and protein solutions were re-buffered in PBS using 96-well Thermo Zeba centrifuge desalter plates. Proteins purified on an Agilent 1200 HPLC system using a standard Sephadex 150/5 column were soluble and monomeric in PBS.

Generation of affinity matured CD 70-specific ankyrin repeat proteins: derived from parent proteins, respectively Protein #69 (SEQ ID NO: 108) and/> protein #68 (SEQ ID NO: 107)/> Proteins Mass #70 (SEQ ID NO: 109) and/> protein #71 (SEQ ID NO: 110).

In further development of the CD 70-specific binding proteins initially identified, affinity maturation was used to generate binding domains with very high affinity for the target protein and/or very low dissociation rates from the target protein. Thus, two initially identified binding proteins ("parent" binding proteins) were selectedProtein #68 and->Protein # 69) served as a suitable starting point for affinity maturation. The affinity maturation procedure involves saturation mutagenesis of each random position of the ankyrin repeat domain that serves as a starting point. Sequences generated by the affinity maturation procedure were screened for lower dissociation rates by competing HTRF. Briefly: crude extracts of ankyrin repeat proteins containing an N-terminal His tag were incubated with biotinylated targets, then excess unlabeled parent CD70 specific binding protein was added and HTRF signal was measured over time. The beneficial mutations identified based on higher HTRF signals compared to the parental clones are combined in the binding protein by protein engineering.

In a first step, the engineered CD70 specific binding domain is subcloned into a derivative of pQE30 (Qiagen) expression vector pMPTLO847, which ultimately contains an N-terminal His tag, followed by a CD70 specific binding domain (SEQ ID NO:109 or 110) and a C-terminal CD3 specific binding domain (SEQ ID NO: 3). The construct was expressed in E.coli cells and purified using its His tag according to standard protocols. The proteins were tested for dose-dependent in vitro T cell activation and killing assays using primary T cells and Molm-13-N1 tumor cells (E: T ratio 5:1) isolated from healthy donor PBMC. Assay the co-cultures were incubated for 48 hours and analyzed by flow cytometry and LDH release.Protein #70 (SEQ ID NO: 109) and +.>Protein #71 (SEQ ID NO: 110) was selected based on: the EC50 values were improved by approximately 7-fold and 31-fold compared to the parent binding protein, respectively; and the monopolymerity as measured by analytical size exclusion chromatography>95%. In the second step, CD 70-specific binding protein +.>Protein #70 (SEQ ID NO: 109) and +.>Protein #71 (SEQ ID NO: 110) was subcloned into a derivative of pQE30 (Qiagen) expression vector pMPDV025, which contains an N-terminal His tag, and repeated protein expression in E.coli as a monovalent design ankyrin, purified and characterized according to standard protocols.

Example 2: pharmacokinetic analysis of recombinant proteins in female BALB/c mice

Pharmacokinetic analysis of CD3-specific ankyrin repeat proteins in female BALB/c mice

To determine whether the CD 3-specific ankyrin repeat domain of the invention can have an appropriate in vivo serum half-life that makes it useful for the development of therapeutic agents, analysis in miceProtein #1, < >>Protein #2,Protein #3 and->Protein #4 drugPharmacokinetic profile. To this end, the DARPin construct was subcloned and expressed as described above into a derivative of the pQE30 (Qiagen) expression vector containing an N-terminal His tag, an HSA binding ankyrin repeat domain for half-life extension, followed by one of the CD3 specific binding domains. For example, an expression vector encoding the following ankyrin repeat proteins is constructed:

protein #16 (SEQ ID NO:39 having a His tag fused to its N-terminus (SEQ ID NO: 33));

protein #17 (SEQ ID NO:40, having a His tag fused to its N-terminus (SEQ ID NO: 33));

protein #18 (SEQ ID NO:41, having a His tag fused to its N-terminus (SEQ ID NO: 33));

protein #19 (SEQ ID NO:42, having a His tag fused to its N-terminus (SEQ ID NO: 33));

In vivo administration and sample collection

For each ankyrin repeat fusion protein, human serum albumin specific ankyrin repeat domains were formattedProtein #16, < >>Protein #17, < >>Protein #18 and->Protein #19 was administered in a single intravenous bolus into the tail vein of 6 mice. The target dosage level was 1mg/kg, with an administration volume of 5mL/kg. Ankyrin repeat fusion proteins were formulated in Phosphate Buffered Saline (PBS) solution.

Mice were divided into 2 groups with the same number of animals. Four serum samples were collected from each mouse. Blood samples for pharmacokinetic studies were collected from saphenous vein 5 minutes, 4 hours, 24 hours, 48 hours, 76 hours, 96 hours and 168 hours after compound administration. The blood was kept at room temperature to allow clotting, followed by centrifugation and serum collection.

Biological analysis by ELISA to measure ankyrin repeat proteins in serum samples

100 μl of 10nM polyclonal goat anti-rabbit IgG antibody (Ab 18) in PBS per well was coated onto NUNC Maxisorb ELISA plates overnight at 4deg.C. After washing five times with 300 μl of PBST (PBS supplemented with 0.1% tween 20) per well, the wells were blocked with 300 μl of PBST (PBST-C) supplemented with 0.25% casein at Room Temperature (RT) on a Heidolph Titramax shaker (450 rpm) for 1 hour. The plates were washed as described above. Mu.l of 5nmol/L rabbit anti-in PBST-C was added Antibodies, and plates were incubated for 1 hour at room temperature (22 ℃) with orbital shaking (450 rpm). The plates were washed as described above.

100 μl of diluted serum samples (1:20-1:312500 in the 1:5 dilution step) or ankyrin repeat protein standard curve samples (0 nmol/L and 50nmol/L-0.0008nmol/L in the 1:3 dilution step) were applied with shaking at 450rpm for 2h at room temperature. The plates were washed as described above.

Wells were then incubated with 100 μl of murine anti-RGS-His-HRP IgG (Ab 06, 1:2000 in PBST-C) and incubated for 1 hour at 450rpm at room temperature. The plates were washed as described above. ELISA was developed 5 using 100. Mu.l/well TMB substrate solutionMinute, and by adding 100. Mu.l 1mol/L H ₂ SO ₄ And (5) terminating. The difference between the absorbance at 450nm and the absorbance at 620nm was calculated. Samples were measured in duplicate on two different plates. FIG. 1A showsProtein #16, < >>Protein #17, < >>Protein #18 and->Serum concentration as a function of time following a single intravenous administration of protein #19 to mice. The trace indicates a substantially single exponential elimination of the compound.

Pharmacokinetic analysis

Pharmacokinetic data analysis was performed at Molecular Partners using the WinNonlin program version 7.0 as part of Phoenix 64 (Pharsight, north Carolina). Calculation of pharmacokinetic parameters based on mean concentration-time data of animals dosed via intravenous bolus was performed by non-atrioventricular analysis (NCA models 200-202, IV bolus, linear trapezoidal linear interpolation). The following pharmacokinetic parameters were calculated:

AUCinf、AUClast、AUC_％extrapol、Cmax、Tmax、Cl_pred、Vss_pred、t1/2

The maximum serum concentration (Cmax) and time of occurrence (Tmax) were obtained directly from the serum concentration-time curve. The area under the serum concentration-time curve (AUCinf) was determined by a linear trapezoidal equation up to the last sampling point (Tlast) and extrapolated to infinity (assuming a single exponential drop at the end of the period). Extrapolation to infinity is performed using Clast/λz, where λz represents the endpoint rate constant estimated by log-linear regression and Clast represents the concentration estimated at Tlast by means of the endpoint log-linear regression. Total serum clearance (cl_pred) and apparent endThe terminal half-life is calculated as follows: cl_pred=i.v. dose/AUCinf, and t 1/2=ln2/λz. The steady state distribution volume Vss is determined by: vss=intravenous dose. AUMCinf/(AUCinf) 2.AUMCinf represents the total area under the first moment of the drug concentration-time curve extrapolated to infinity using the same extrapolation procedure as described in AUCinf calculation. In order to calculate PK parameters based on the concentration given in nmol/L, the dose values given in mg/kg were converted to nmol/kg by using the molecular weight of ankyrin repeat protein. Table 2a shows four ankyrin repeat proteins tested after a single intravenous administration of 1mg/kg Protein #16, < >>Protein #17,Protein #18 and->Summary of pharmacokinetic profile of protein # 19.

Table 2a: pharmacokinetic parameters of four exemplary HSA/CD3 specific ankyrin repeat proteins

B. Pharmacokinetic analysis of multispecific recombinant proteins in female BALB/c mice

To determine whether the multispecific recombinant binding proteins of the invention are capable of having an appropriate serum in vivo half-life that makes them useful for the development of therapeutics, assays in miceProtein #29, < >>Pharmacokinetic profile of protein #31 (in half-life extended form). To this end, DARPin constructs were subcloned into and expressed as described above in derivatives of pQE30 (Qiagen) expression vectors containing an N-terminal His tag, an ankyrin repeat domain for half-life extension (one or two as described in the previous examples), followed by a tumor-associated binding domain and CD3 binding domain, or an N-terminal His tag, a tumor-associated binding domain and CD3 binding domain, followed by an ankyrin repeat domain for half-life extension (one or two as described in the previous examples) binding to HSA. For example, an expression vector encoding the following ankyrin repeat proteins is constructed:

Protein #50 (SEQ ID NO:81 having a His tag fused to its N-terminus (SEQ ID NO: 33));

protein #53 (SEQ ID NO:84, having a His tag fused to its N-terminus (SEQ ID NO: 33));

protein #51 (SEQ ID NO:82, having a His tag fused to its N-terminus (SEQ ID NO: 33));

protein #54 (SEQ ID NO:85, having a His tag fused to its N-terminus (SEQ ID NO: 33));

protein #55 (SEQ ID NO:86 having a His tag fused to its N-terminus (SEQ ID NO: 33));

in vivo administration and sample collection

For each ankyrin repeat fusion protein, human serum albumin specific ankyrin repeat domains were formattedProtein #50, < >>Protein #53, < >>Protein #51, < >>Protein #54 and->Protein #55 was administered as a single intravenous bolus into the tail vein of 6 mice. Ankyrin repeat proteins were formulated in Phosphate Buffered Saline (PBS) at a dose of 1mg/kg and an administration volume of 100 μl.

100 μl of 10nM polyclonal goat anti-rabbit IgG antibody (Ab 18) in PBS per well was coated onto NUNC Maxisorb ELISA plates overnight at 4deg.C. After washing five times with 300 μl of PBST (PBS supplemented with 0.1% tween 20) per well, the wells were blocked with 300 μl of PBST (PBST-C) supplemented with 0.25% casein at Room Temperature (RT) on a Heidolph Titramax shaker (450 rpm) for 1 hour. The plate is as described aboveWashing is performed. Mu.l of 5nmol/L rabbit anti-in PBST-C was addedAntibodies, and plates were incubated for 1 hour at room temperature (22 ℃) with orbital shaking (450 rpm). The plates were washed as described above.

Wells were then incubated with 100 μl of murine anti-RGS-His-HRP IgG (Ab 06, 1:2000 in PBST-C) and incubated for 1 hour at 450rpm at room temperature. The plates were washed as described above. ELISA was developed for 5 min using 100. Mu.l/well TMB substrate solution and was performed by adding 100. Mu.l 1mol/L H ₂ SO ₄ And (5) terminating. The difference between the absorbance at 450nm and the absorbance at 620nm was calculated. Samples were measured in duplicate on two different plates. FIG. 1B showsProtein #50, < >>Protein #53, < >>Protein #51, < >>Protein #54 and->Serum concentration of protein #55 as a function of time following a single intravenous administration to mice. The trace indicates a substantially single exponential elimination of the compound.

Pharmacokinetic analysis

AUCinf, AUC_% extrapol, cmax, tmax, cl _pred, vss_pred, t1/2. The maximum serum concentration (Cmax) and time of occurrence (Tmax) were obtained directly from the serum concentration-time curve. The area under the serum concentration-time curve (AUCinf) was determined by a linear trapezoidal equation up to the last sampling point (Tlast) and extrapolated to infinity (assuming a single exponential drop at the end of the period). Extrapolation to infinity is performed using Clast/λz, where λz represents the endpoint rate constant estimated by log-linear regression and Clast represents the concentration estimated at Tlast by means of the endpoint log-linear regression. Total serum clearance (cl_pred) and apparent terminal half-life were calculated as follows: cl_pred=i.v. dose/AUCinf, and t 1/2=ln2/λz. The steady state distribution volume Vss is determined by: vss=intravenous dose. AUMCinf/(AUCinf) 2.AUMCinf represents the total area under the first moment of the drug concentration-time curve extrapolated to infinity using the same extrapolation procedure as described in AUCinf calculation. In order to calculate PK parameters based on the concentration given in nmol/L, the dose values given in mg/kg were converted to nmol/kg by using the molecular weight of ankyrin repeat protein. Table 2b shows five ankyrin repeat proteins tested after a single intravenous administration of 1mg/kg Protein #50, < >>Protein #53, < >>Protein #51,Protein #54 and->Summary of pharmacokinetic profile of protein # 55.

Table 2b: pharmacokinetic parameters of five exemplary multispecific recombinant proteins

As can be seen from table 2b above and figure 1,protein #51 shows the longest half-life, followed byProtein #50, < >>Protein #54 and->Protein #53, whereas half-life is prolonged at C-terminus +.>Protein #55 showed the shortest half-life.

In addition, according to the following formulaProtein #50, < >>Protein #53, < >>Protein #51, < >>Protein #54 and->Protein #55 the same experimental setup, analyzed in miceProtein #56 (SEQ ID NO: 95), ->Protein #57 (SEQ ID NO: 96), ->Protein #58 (SEQ ID NO: 97), ->Protein #59 (SEQ ID NO: 98), ->Protein #60 (SEQ ID NO: 99) and +.>Pharmacokinetic profile of protein #61 (SEQ ID NO: 100).

Table 2c shows six ankyrin repeat proteins tested after a single intravenous administration of 1mg/kgProtein #56, < >>Protein #57, < >>Protein #58, < >>Protein #59,Protein #60 and->Summary of pharmacokinetic profile of protein # 61.

Table 2c: pharmacokinetic parameters of six exemplary multispecific recombinant proteins

As can be seen from the above table 2c,protein #58 shows the longest half-life, followed by +.>Protein #60, < >>Protein #61, < >>Protein #56 and->Protein #59, anProtein #57 shows the shortest half-life.

_D Example 3:determination of dissociation constant (K) of Multi-specific recombinant ankyrin repeat proteins

A. Determination of multispecific re-specific for human CD3 by Surface Plasmon Resonance (SPR) analysis Dissociation constant (KD) of ankyrin repeat protein

The binding affinities of the four purified ankyrin repeat proteins to recombinant human CD3 target were analyzed using a ProteOn instrument (BioRad) and measured according to standard procedures known to the person skilled in the art. For this purpose, the invention isProtein #7, < >>Protein #8, < >>Protein #9 and->Protein #10 was subcloned into and expressed as described above in a derivative of the pQE30 (Qiagen) expression vector containing the N-terminal His tag, CD33 and CD123 binding ankyrin repeat domain, followed by one of four CD3 specific ankyrin repeat protein constructs.

Briefly, biotinylated human scCD3 εγ was diluted in PBST (PBS, pH 7.4, containing 0.005% Tween)) And coated on an NLC chip (BioRad) to a level of about 700-1400 Resonance Units (RU). Then 300. Mu.l of running buffer (PBS, pH 7.4, containing 0.005% Tween- >) The interaction of ankyrin repeat protein with human CD3 was then measured by injecting a running buffer stream at a constant flow rate of 60 μl/min for at least 10 minutes (dissociation rate measurement). Regeneration was performed using 30 μl of 10mM glycine pH 2. The signal (i.e., RU value) of the inter-spot and reference injections (i.e., injection of running buffer alone) was subtracted from the Resonance Unit (RU) trace obtained after injection of ankyrin repeat protein (double reference). Although toProtein #7 binds too weakly, but the binding parameters for CD3 were determined for affinity matured constructs (K _D Binding rate, dissociation rate). Binding Rate (k) _on ) And dissociation constant (K) _D ) Only given as an approximation, since the binding equilibrium is not properly reached even at the highest sample concentration, resulting in a non-best fit of the binding rate.

As a representative example, FIG. 2C shows formatting with engineered ankyrin repeat domains that bind CD33 and CD123SPR trace obtained for protein # 9. The dissociation constant (K is calculated from the estimated association and dissociation rates using standard procedures known to those skilled in the art _D ). Selected ankyrin repeat proteins bind to human CD3 and interact with each other _D Values were determined to be in the range of 6nM to 35nM (see table 3 a).

_D TABLE 3K value of ankyrin repeat protein-human CD3 interaction

B. Determination of the heavy Ends with binding specificity to human CD33 and CD123 by Surface Plasmon Resonance (SPR) analysis _D Dissociation constant (K) of ankyrin repeat protein。

Two purifications were analyzed using a Proteon instrument (BioRad)Binding affinities of ankyrin repeat proteins to recombinant human CD33 and CD123 targets and measured according to standard procedures known to the person skilled in the art. For this purpose, the invention isProtein #25 (binding specificity for CD33 and CD 3), ->Protein #26 (with binding specificity for CD123 and CD 33) was subcloned into and expressed in a derivative of the pQE30 (Qiagen) expression vector containing an N-terminal His tag, CD33 or CD123 binding ankyrin repeat domain, respectively, followed by one of four CD3 specific ankyrin repeat constructs as described above. The following steps were performed as described above in a. Briefly, the biotinylated extracellular domain of human CD33 (supplied by Speed BioSystems, catalogue #YCP2045) was used as target protein for evaluation +.>Protein #25 and accordingly the biotinylated extracellular domain of human (uniprot ID 26951) was used for evaluation +.>Protein #26.

K for binding interaction of selected ankyrin repeat proteins with human CD33 and CD123 _D The values are shown in the following table (see table 3 b).

_D TABLE 3K values for ankyrin repeat protein-human CD33 or human CD123 interactions

C. Determination of binding specificity for human CD33, CD123 and/or CD70 by Surface Plasmon Resonance (SPR) analysis _D Dissociation of the heterologous ankyrin repeat proteinNumber (K)

The binding affinities of 16 purified ankyrin repeat proteins to recombinant human CD33, CD123 and CD70 targets were analyzed using a ProteOn XPR36 instrument (BioRad) and measured according to standard procedures known to the person skilled in the art. For this purpose, it willProtein #27, < >>Protein #29, < >>Protein #47 andprotein #48 and->Protein #49, < >>Protein #50, < >>Protein #51, < >>Protein #52, < >>Protein #31, < >>Protein #53, < >>Protein #54, < >>Protein #55, < >>Protein #34, < >>Protein #33, < >>Protein #39 and->Protein #36 was subcloned into and expressed in a derivative of the pQE30 (Qiagen) expression vector as described above, which contains an N-terminal His tag, CD33, CD123 and/or CD70 binding ankyrin repeat domain, followed by a CD3 specific ankyrin repeat domain and for some constructs a human serum albumin specific ankyrin repeat domain.

Briefly, in PBST (PBS, pH 7.4, containing 0.005% Tween)) Biotinylated human CD70, CD123 and CD33 were diluted and coated on GLC chips (BioRad) to levels of about 200 Resonance Units (RU), 460RU and 450RU, respectively. HSA in NaOAc pH 5.0 was immobilized directly on GLC chips to a level of 800 RU. Bio.cd70, bio.cd33 and bio.cd123 require first coating with neutravidin to a level of 6000RU, and then biotinylated targets can be administered. Then by injection of 300. Mu.l running buffer (PBS, pH 7.4, containing 0.005% Tween->) The interaction of selected ankyrin repeat proteins with human CD70, CD123 and CD33 was measured with binding 120s and dissociation 1200s using a constant flow rate of 100. Mu.l/min, the running buffer containing ankyrin repeat protein serial dilutions covering a concentration range between 100nM and 2.6nM (100 nM, 40nM, 16nM, 6.4nM and 2.6nM, respectively)SPR measurements were performed on multiple traces. Regeneration was performed using 30 μl of 4mM glycine pH 2. The signal (i.e., RU value) of the inter-spot and reference injections (i.e., injection of running buffer alone) was subtracted from the Resonance Unit (RU) trace obtained after injection of ankyrin repeat protein (double reference).

K for binding interactions of selected ankyrin repeat proteins with human CD33, CD123 and CD70 _D The values are shown in the following table (see table 3 c).

_D TABLE 3K values for ankyrin repeat protein-human CD33, CD123 and CD70 interactions

/>

* nb: unbound material

Similarly, the binding affinity of two purified ankyrin repeat proteins to recombinant human CD3 target was analyzed as previously described. Briefly, the reaction was carried out at NaOAc pH 4.5Protein #53, < >>Protein #54 was coated on a GLC chip (BioRad) to a level of about 790-1500 Resonance Units (RU). The interaction of ankyrin repeat protein with human CD3 was then measured by: injection of the extract containing the extract in PBST (PBS, pH 7.4, containing 0.005% Tween->) In a serial dilution of human scCD3 εγ (56.4 uM) and covering a concentration range between 300nM and 19nM for a multi-trace SPR measurement of 300 μl running buffer (PBS, pH 7.4, containing 0.005% Tween)>) The buffer stream is then run for at least 10 minutes at a constant flow rate of 60 μl/min (dissociation rate measurement).

Regeneration was performed using 30 μl of 10mM glycine pH 2. The signal (i.e., RU value) of the inter-point and reference injections (i.e., injection of running buffer only) was subtracted from the Resonance Unit (RU) trace obtained after CD3 injection (double reference). Binding parameters (KD, binding rate, dissociation rate) for CD3 were determined by using a standard Langmuir (Langmuir) fitting model. As a representative example, fig. 2 (a-B) shows a schematic diagram for Protein #53 (A) and +.>SPR trace obtained for protein #54 (B).

_D TABLE 4K value of ankyrin repeat protein-human CD3 interaction

D. Determination of binding specificity to human CD33, CD123 and CD70 by Surface Plasmon Resonance (SPR) analysis _D Dissociation constant (K) of ankyrin repeat protein

Binding affinity of selected multi-specific ankyrin repeat proteins to recombinant targets was analyzed using ProteOn (BioRad) and Bruker Sierra SPR-32Pro instruments. The measurements were made according to standard procedures known to those skilled in the art. For this purpose, the invention isProtein #56 and->Protein #57 was subcloned into and expressed in a derivative of the pQE30 (Qiagen) expression vector as described above, which contains an N-terminal His tag, two human serum albumin specific binding domains and a CD70, CD123 and CD33 specific binding domain, followed by a CD3 specific binding domainBinding domain.

Briefly, human target CD70 (SEQ ID NO:89; hCD 70-Fc-trimer, ACRO Biosystems) was purified in PBST (PBS, pH7.4, containing 0.005% Tween)) Was diluted and captured to a level of about 36 Resonance Units (RU) on a PAGD200L protein a/G sensor chip (XanTec bioanalytics GmbH) using ProteOn (BioRad). The interaction of each of the ankyrin repeat proteins tested with human CD 70-Fc-trimer was then measured by: injection of 400. Mu.l of running buffer (PBS, pH7.4, containing 0.005% Tween- >) The buffer stream was then run at a constant flow rate of 100 μl/min for 2700 seconds (dissociation rate measurement). Regeneration was performed using 30 μl of 10mM glycine pH 2, and hCD 70-Fc-trimer was re-injected to prepare the surface for the next measurement. Each interaction was recorded in triplicate. The signal (i.e., RU value) of the inter-point and reference injections (i.e., injection of running buffer alone) was subtracted from the Resonance Unit (RU) trace obtained by injection of ankyrin repeat protein (double reference). Dissociation constants (KD) were calculated from the full-fit binding and dissociation rates using a standard 1:1-langmuir model.

Similarly, human target_hCD 123 (SEQ ID NO: 88) was purified in PBST (PBS, pH7.4, containing 0.005% Tween) And captured to a level of about 177 Resonance Units (RU) on a Bruker IgG capture sensor chip using Bruker Sierra SPR-32 pro. The interaction of each of the tested ankyrin repeat proteins with the target hCD123 was then measured by: 100 μl of running buffer (PBS, pH7.4, containing 0.005% Tween->) The buffer stream is then run at a constant flow rate of 25 μl/min for at least 1500 seconds (dissociation rate measurement). Regeneration was performed using 8.3 μl of 10mM glycine pH 2, and hCD123 was re-injected to prepare the surface for the next measurement. Each interaction was recorded in triplicate. The signal (i.e., RU value) of the empty surface and the reference injection (i.e., injection of running buffer alone) was subtracted from the Resonance Unit (RU) trace obtained by injection of ankyrin repeat protein (double reference). Dissociation constants (K _D )。

To measure the interaction with human CD33, one wouldProtein #56 and->Protein #57 was diluted in 10mM NaOAc pH 4.0 and immobilized on NHS/EDC activated HC200M sensor chip (XanTec bioanalytics GmbH) using ProteOn (BioRad) to a level of about 1000 Resonance Units (RU) (SMA 555) or 1400RUs (PSC 466). The surface was deactivated using ethanolamine. The interaction of the tested protein with human target hCD33 (SEQ ID NO: 87) was then measured by: injection of 400. Mu.l running buffer (PBS, pH7.4, containing 0.005% Tween->) The buffer stream was then run for 500 seconds at a constant flow rate of 100 μl/min (dissociation rate measurement). After each measurement, a 10min pause was introduced to allow complete dissociation of the analyte. Each interaction was recorded in triplicate. The signal (i.e. RU value) of the inter-point and reference injections (i.e. only running buffer) was subtracted from the Resonance Unit (RU) trace obtained by injecting human hCD33 (double reference). Dissociation constants (K _D ). Fig. 2D shows the case for +. >Protein #56 and->SPR trace obtained for protein # 57.

K for binding interactions of selected ankyrin repeat proteins with human CD33, CD123, CD70 and CD3 _D The values are shown in the following table (see table 4 b).

_D TABLE 4K values for ankyrin repeat protein-human CD33, CD123 and CD70 interactions

Example 4: determination of T cell or tumor cell binding。

Protein #11,/> Protein #12,/> Protein #13 Determination of CD3T cell binding of protein #14。

Determination of CD3 binding was performed on primary human T cells using Mirrorball laser scanning imaging cytometry. Thus, primary T cells were isolated from human Peripheral Blood Mononuclear Cells (PBMC) using pan-T cell purification kit (Miltenyi Biotec). Formatting into a trispecific form (including the ankyrin repeat domain that binds CD33 and CD 123)Protein #7, < >>Protein #8, and->Protein #9 and->Protein #10, formatted in tetraspecific form (including additional ankyrin repeat domains that specifically bind to human serum albumin)>Protein #11, < >>Protein #12, < >>Protein #13 and->The titres of protein #14UN were incubated with 50,000 pan-T cells/well in the presence of 600. Mu.M human serum albumin (to mimic physiological serum concentrations) for 30 minutes at 4 ℃. Two reference T cell conjugates (AMG 330 and Fu Tuozhu mab) were used as controls to target CD33 or CD123. After washing, CD3 binding was detected with anti-five-His Alexa Fluor 488 antibody (Qiagen) diluted 1:100. After incubation at 4 ℃ for 30 min, cells were washed and resuspended in Cytofix fixation buffer (BD Biosciences) and counterstained with 5 μm DRAQ5 (Abcam) for 15 min at room temperature. Median of mean fluorescence intensity of Alexa Fluor 488 bound to far red counterstained cells was measured by Mirrorball using Cellista software (SPT Labtech) and the data plotted using GraphPad Prism 8.

As shown in figures 20A and 20B,proteins show a broad range of affinities from +.>Protein 7 was undetectable to bind +.>Protein 10 binds as well as the reference molecule (known reference T cell conjugates: AMG330 with binding specificity for CD33 and Fu Tuozhu mab with binding specificity for CD 123). CD3 binding to T cells matches with CD3 affinity as measured by SPR. The presence of additional HSA binding domains (see fig. 3B) has only a minor effect on binding to T cells. Table 5a shows the CD3 binding affinities of four exemplary ankyrin repeat proteins, from their ECs ₅₀ The value represents.

TABLE 5a CD123-CD33-CD3 specificity ₅₀ EC value of protein

Protein #27,/> Protein #28, and/> protein #29 T cell binding of protein #30Fixing device。

Determination of T cell binding was performed on primary human T cells using Mirrorball laser scanning imaging cytometry. Thus, primary T cells were isolated from human Peripheral Blood Mononuclear Cells (PBMC) using pan-T cell purification kit (Miltenyi Biotec). Will beProtein #27, < >>Protein #28 and->Protein #29The titer of protein #30 was incubated with 50'000 pan-T cells/well in the presence of 600. Mu.M human serum albumin (to mimic physiological serum concentrations) for 30 minutes at 4 ℃. Two reference T cell conjugates (AMG 330 and Fu Tuozhu mab) were used as controls to target CD33 or CD123. After washing, CD3 binding was detected with anti-five-His Alexa Fluor 488 antibody (Qiagen) diluted 1:100. After incubation at 4 ℃ for 30 min, cells were washed and resuspended in Cytofix fixation buffer (BD Biosciences) and counterstained with 5 μm DRAQ5 (Abcam) for 15 min at room temperature. Median of mean fluorescence intensity of Alexa Fluor 488 bound to far red counterstained cells was measured by Mirrorball using Cellista software (SPT Labtech) and the data plotted using GraphPad Prism 8.

Table 5b shows the binding affinities of four exemplary ankyrin repeat proteins, from their ECs ₅₀ The value represents.

TABLE 5b multispecific EC of proteins ₅₀ Value of />

Determination of tumor cell binding of multispecific binding proteins。

Determination of binding of several multispecific proteins to tumor cells was performed by Fluorescence Activated Cell Sorting (FACS) flow cytometry. Thus, tumor cells (Molm-13 N1) were seeded at 100,000 cells/well in 96-well plates.Protein #56, < >>Protein #57, < >>Protein #58, < >>Protein #59, < >>Protein #60, < >>Protein #61 and->Protein #62 was titrated downward from 100nM at a dilution ratio of 1:5. With diluted +.>The tumor cells were resuspended in protein and incubated at 4℃for 60 minutes. The assay was performed in PBS containing 2% fetal bovine serum and 20 μm Human Serum Albumin (HSA). After washing twice with Phosphate Buffered Saline (PBS), unlabeled primary anti-rabbit +.2 ug/ml was added>Antibodies (anti-Rabbit 1-1-1 antibody, cePower) to detect +.>Protein-specific tumor cell binding. Followed by an incubation step at 4℃for at least 30 minutes. Thereafter, the cells were washed with PBS and 2ug/ml of a secondary anti-rabbit antibody labeled with Alexa Fluor 647 antibody (ThermoFisher) was added. The same incubation conditions were applied. Finally, the cells were washed twice and resuspended in Cytofix fixation buffer (BD Biosciences) for 15min at Room Temperature (RT). Alexa Fluor 647- >Median Fluorescence Intensity (MFI) of the labeled cells was measured by Attune NxT (Thermo Fisher), analyzed using FlowJo software and GraphPad Prism 8 was used to map the data. FIG. 20C shows targeting selected +.>Titration curves for proteins.

Table 5c shows the binding affinities of seven exemplary multispecific binding proteins, from their ECs ₅₀ The value represents.

TABLE 5c multispecific ₅₀ EC value of protein

Example 5: assessment of potency and specificity of multispecific recombinant proteins using in vitro short-term T cell activation assays。

A. Efficacy and specificity of multispecific recombinant proteins on Molm-13 target cells in Co-culture with human PanT cells Sex characteristics。

The specificity and potency of the aforementioned recombinant multi-specific ankyrin repeat proteins were assessed in an in vitro short term T cell activation assay by FACS measuring the CD25 activation markers on cd8+ T cells.

Thus, with the selectionSerial dilutions of protein or control reference molecule 50,000 purified pan-T effector cells and 50,000 Molm-13 target cells per well were co-incubated (E: T ratio 1:1) at 37 ℃ in duplicate in the presence of 600 μm human serum albumin for 24 hours. Cells were washed and stained with 1:1,000live/Dead Aqua (Thermo Fisher), 1:250 mouse anti-human CD8 Pacific Blue (BD) and 1:100 mouse anti-human CD25 PerCP-Cy5.5 (eBiosciences) antibodies for 30 minutes at 4 ℃. After washing and fixation, cells were analyzed on a FACS Canto II (BD) machine. T cell activation was assessed by measuring cd25+ cells on Live/Dead negative and cd8+ gated T cells. FACS data were analyzed using FlowJo software and plotted using GraphPad Prism 8.

As shown in FIG. 3, (A)Protein #27, (B)/(B)>Protein #28, (C)/(C)>Protein #29 and (D)/(D)>Protein #30 induced specific short-term T cell activation comparable to the reference molecule (known reference T cell conjugates: AMG330 with binding specificity for CD33 and Fu Tuozhu mab with binding specificity for CD 123). Furthermore, recombinant proteins when compared to a single targeting control +.>Protein #40 (PairCD33 has binding specificity),Protein #41 (with binding specificity for CD 123), ->Protein #42 (with binding specificity for CD 33)>Protein #43 (with binding specificity for CD 70), -A. About>Protein #44 (with binding specificity for CD 33)>Protein #45 (binding specificity for CD 70) and +.>Protein #46 (binding specificity for CD 123) when compared->Protein #27, < >>Protein #28,Protein #29 and->T cell activation of protein #30 showed a significant increase in avidity.

In addition, several multispecific recombinant proteins were tested in this similar assay with half-life extending binding domains, where the effect of the half-life extending on molecular potency was evaluated. (A)Protein #27 with half-life formatted +.A half-life with two tumor antigen specific binding domains (CD 123 and CD33, respectively) and one half-life extending domain at the N-terminus >Protein #47, a similar protein with two half-life extending domains located at the N-terminus (++>Protein # 48) and the like having one half-life extending domain at the C-terminus (/ -amino acid sequence)>Protein # 49); (B) And (2)>Protein #29 is half-life formatted with a half-life having two tumor antigen specific binding domains (CD 70 and CD33, respectively) and one half-life extending domain at the N-terminusProtein #50, and the like having two half-life extending domains at the N-terminus (++>Protein # 51) and the like having one half-life extending domain at the C-terminus (/ -amino acid sequence)>Protein # 52); />Half-life format of protein 31 with three tumor antigen specific binding domains (CD 70, CD123 and CD33, respectively) and one half-life extending domain at the N-terminusInnovative->Protein #53, and the like having two half-life extending domains at the N-terminus (++>Protein # 54) and the like having one half-life extending domain at the C-terminus (/ -amino acid sequence)>Protein # 55). As can be seen in fig. 4, the addition of half-life extending binding domains results in a half-life at +.>At least 20-fold loss of potency in the case of protein #27, at +. >At least 4-fold loss of potency in the case of protein #29 and at +.>Less potency loss than factor 10 in the case of protein # 31.

In addition, multi-specific ankyrin repeat proteins with binding specificity for CD3, CD33 and CD123 were evaluated in an in vitro short term T cell activation assay by FACS measuring the CD25 activation marker on cd8+ T cellsSpecificity and potency of protein # 8. Both specificity and potency were separately compared to known baseline T cell conjugates: AMG330 with binding specificity to CD33 and Fu Tuozhu mab with binding specificity to CD123 were compared and compared to recombinant proteins with binding specificity to CD3 and CD33, respectively->Protein #23 and a recombinant protein having binding specificity for CD3 and CD123>Protein #24 was compared.

Briefly, 50,000 purified pan-T effector cells and 50,000 MOLM-13 target cells per well were co-incubated (E: T ratio 1:1) at 37℃for 24 hours in duplicate with serial dilutions of recombinant protein or control reference molecules (Fu Tuozhu mab and AMG 330) in the presence of 600. Mu.M human serum albumin. Cells were washed and stained with 1:1'000live/Dead Aqua (Thermo Fisher), 1:250 mouse anti-human CD8 Pasific Blue (BD) and 1:100 mouse anti-human CD25 PerCP-Cy5.5 (eBiosciences) antibodies for 30 minutes at 4 ℃. After washing and fixation, cells were analyzed on a FACS Canto II (BD) machine. T cell activation was assessed by measuring cd25+ cells on Live/Dead negative and cd8+ gated T cells. FACS data were analyzed using FlowJo software and plotted using GraphPad Prism 8. As shown in FIG. 21, binding specificity for CD3, CD33 and CD123 and thus binding to two tumor specific targets on MOLM-13 Protein #8 induces specific short-term T-cell activation comparable to the reference molecule, whereas +.about.each binds to only one of the tumor specific targets (CD 33 or CD 123)>Protein #23 and->Protein #24 showed reduced efficacy to 1/10 to 1/100.

In a similar experimental set-up, the sample,protein #56, < >>Protein #57, < >>Protein #58, < >>Protein #59, < >>Protein #60 and->Protein #61 was also assessed by FACS measuring the CD25 activation marker on cd8+ T cells. Briefly, 80,000 purified pan-T effector cells and 20,000 Molm-13 or target cells per well were co-incubated (E: T ratio 4:1) in duplicate with serial dilutions of the selected test proteins in the presence of 20. Mu.M human serum albumin for 48 hours at 37 ℃. After 48 hours, cells were washed and stained with 1:1,000 live/read Green (Thermo Fisher), 1:400 mouse anti-human CD8 Pacific Blue (BD) and 1:100 mouse anti-human CD25 PerCP-Cy5.5 (eBiosciences) antibodies for 30min at 4 ℃. After washing and fixation, cells were analyzed on a FACS Canto II (BD) machine. T cell activation was assessed by measuring cd25+ cells on Live/Dead negative and cd8+ gated T cells. FACS data were analyzed using FlowJo software and plotted using GraphPad Prism 8 (3-PL-fit).

As shown in FIG. 51A, the test was performed with binding specificity for CD3, CD33, CD123 and CD70Protein: />Protein #56 and->Protein #57 induced potent T cell activation as expected (EC 50 on Molm-13 of 26.1pM and 10.4pM, respectively). The +.>Protein #58, < >>Protein #59, < >>Protein #60 and->Protein #61 showed approximately 5-fold differences from each other in terms of EC50 values as expected.

In addition, also testProtein #56 and->Comparison of protein #57 (in the same experimental setup) with non-binding designed ankyrin repeat protein used as negative control compound. Briefly, 100,000 purified pan-T effector cells and 20,000 Molm-13 target cells per well were co-incubated in duplicate with serial dilutions of the selected proteins in the presence of 20. Mu.M human serum albumin at 37℃for 48 hours (E: T ratio 5:1 for PanT cells). After 48 hours, cells were washed and stained with 1:1,000 live/read Green (Thermo Fisher), 1:400 mouse anti-human CD8 Pacific Blue (BD) and 1:100 mouse anti-human CD25PerCP-Cy5.5 (eBiosciences) antibodies for 30min at 4 ℃. After washing and fixation, cells were analyzed on a FACS Canto II (BD) machine. T cell activation was assessed by measuring cd25+ cells on Live/Dead negative and cd8+ gated T cells. FACS data were analyzed using FlowJo software and plotted using GraphPad Prism 8 (3-PL-fit).

As shown in FIG. 51B, the test was performed with binding specificity for CD3, CD33, CD123 and CD70Protein: />Protein #56 and->Protein #57 induced potent and specific T cell activation (23.3 pM EC50 on Molm-13, respectively) compared to their corresponding negative controls. Negative control proteins, each binding only to tumor specific targets or CD3, did not show up-regulation of the CD25 activation markers.

And also evaluateComparison of protein #56 (in the same experimental set-up) with the known reference molecule Fu Tuozhu mab analog and AMG330 analog. As shown in FIG. 51C, there is binding specificity for CD3, CD33, CD123 and CD70 +.>Protein #56 (thus binding to 3 tumor specific targets) induced potent and specific T cell activation.

B. Multi-specific recombinant proteins in Molm13 CRISPR Knockout (KO) target cells in co-culture with human PanT cells Efficacy and specificity on。

Assessment in an in vitro short term T cell activation assay by FACS measuring CD25 activation markers on CD8+ T cellsProtein #56 and->Specificity and potency of protein # 57; in this assay, pan T cells were co-cultured with tumor cells consisting of a combination of Molm-13 cells expressed from wild-type targets with CD33, CD123 and CD70 and various knockouts of the same targets (Molm 13 CRISPR Knockdown (KO) target cells) (fig. 51D and 51E).

To this end, 100,000 purified pan-T effector cells and 20,000 target cells per well were incubated in duplicate with serial dilutions of the selected proteins in the presence of 20. Mu.M human serum albumin (E: T ratio 5:1) for 48 hours at 37 ℃. After 48 hours, cells were washed and stained with 1:1,000 live/read Green (Thermo Fisher), 1:400 mouse anti-human CD8 Pacific Blue (BD) and 1:100 mouse anti-human CD25 PerCP-Cy5.5 (eBiosciences) antibodies for 30min at 4 ℃. After washing and fixation, cells were analyzed on a FACS Canto II (BD) machine. T cell activation was assessed by measuring cd25+ cells on Live/Dead negative and cd8+ gated T cells. FACS data were analyzed using FlowJo software and plotted using GraphPad Prism 8 (3-PL-fit).

In FIGS. 51D and 51E, specificity for CD33, CD123, CD70 and CD3 was shown in the presence of Molm-13 tumor cells and multiple target knockout combinationsProtein #56 and->Efficacy of protein #57 (T cell activation). The highest efficacy is achieved for both molecules shown if all three targets are co-expressed (curve 1), and additionally when two targets are co-expressed (curve 2 to curve 4). After co-culturing T cells with single target expressing tumor cells (such as cd33+, cd123+, cd70+) the efficacy was reduced by up to 1/10-1/100 (curve 5 to curve 7). / >Protein #56 shows similar EC50 values for CD33+ single expressing tumor cells, but is similar to +.>Protein #57, which achieved comparable efficacy for all knockout cell lines, was less potent.

Example 6: assessment of target specificity induced by recombinant multi-specific ankyrin repeat protein by LDH cytotoxicity assay Short-term tumor of different natureCell killing。

Multi-specific recombinant proteins with binding specificity for two or three different tumor-associated antigensProteins #7 to #14, ">Protein #27, < >>Protein #28, < >>Protein #29,The specificity and potency of protein #30 was assessed by LDH release by in vitro short-term cytotoxicity assays.

Effector cells and target cells were co-incubated in duplicate at a 5:1 E:T ratio in 96 well plates in the presence of 600. Mu.M human serum albumin (at a simulated physiological concentration). Non-contacted T cells were isolated from human PBMC using the pan-T cell isolation kit (Miltenyi). 100,000 purified pan-T cells (effector cells) and 20,000 Molm-13 cells (target cells) per well were incubated with the selected multispecific recombinant protein, control reference molecule, or serial dilutions of a control containing 1% Triton X-100 for 48 hours at 37 ℃. After 48 hours of incubation, the cells were centrifuged and 100. Mu.l of supernatant per well and 100. Mu.l of LDH reaction mixture per well (LDH detection kit; roche Applied Science) were incubated for 30 minutes. The absorbance at 492nm to 620nm was measured by a TECAN infinite M1000Pro reader. After background correction, OD values were plotted using GraphPad Prism 8.

As shown in FIG. 22A, for DARPin proteins without half-life extension, i.eProtein #8Protein #9 induces tumor cell killing comparable to a reference molecule (known reference T cell conjugate: AMG330 with binding specificity for CD 33), while +.>Protein #7 and->Protein #10 showed efficacy reduced to 1/10 to 1/100. In FIG. 22B, half-life is extended compared to the corresponding non-half-life extended moleculeProtein #10-14 showed efficacy reduced to about 1/4 to 1/70.

As shown in figure 5 of the drawings,protein #27, < >>Protein #28, < >>Protein #29Protein #30 showed target cell killing activity comparable to that of reference molecules AMG330 and Fu Tuozhu mab. Furthermore, recombinant proteins when compared to a single targeting control +.>Protein #40 (with binding specificity for CD 33),Protein #41 (with binding specificity for CD 123), ->Protein #42 (with binding specificity for CD 33)>Protein #43 (with binding specificity for CD 70), -A. About>Protein #44 (with binding specificity for CD 33)>Protein #45 (binding specificity for CD 70) and +.>Protein #46 (with binding specificity for CD 123) showed a significant increase in affinity when compared.

In addition, multispecific recombinant proteins were tested in this similar assay with half-life extending binding domains, where the effect of the half-life extending on molecular potency was evaluated. Will have two tumor antigen specific binding domains (CD 123 and CD33, respectively) and one half-life extending domain at the N-terminusProtein #47 is similar to protein with two half-life extending domains at the N-terminus (++>Protein # 48) and the like having one half-life extending domain at the C-terminus (/ -amino acid sequence)>Protein # 49) for comparison; will have two tumorsAntigen-specific binding domains (CD 70 and CD33, respectively) and a half-life extending domain at the N-terminus +.>Protein #50 and analogous protein having two half-life extending domains at the N-terminusProtein # 51) and proteins similar to those having a half-life extending domain at the C-terminusProtein # 52); will have three tumor antigen specific binding domains (CD 70, CD123 and CD33 respectively) and one half-life extending domain at the N-terminus ≡>Protein #53 is similar to protein with two half-life extending domains at the N-terminus (++ >Protein # 54) and the like having one half-life extending domain at the C-terminus (/ -amino acid sequence)>Protein # 55). As can be seen in fig. 6, the addition of half-life extending binding domains results in a half-life at +.>At least 20-fold loss of potency in the case of protein #27, atAt least 4-fold loss of potency in the case of protein #29 and at +.>ProteinsLess efficacy loss than factor 10 in case # 31.

In the same experimental setup, it was also evaluatedProtein #56, < >>Protein #57,Protein #58, < >>Protein #59, < >>Protein #60 and->Protein #61./>

As shown in FIG. 52A, proteins tested for binding specificity for CD3, CD33, CD123 and CD70Protein #56 and->Protein #57 induces effective tumor cell killing as expected (EC 50 on Molm-13 of 7.1pM and 1.6pM, respectively), while +_ shown in FIG. 51A>Protein #58, < >>Protein #59, < >>Protein #60 and->Protein #61 showed a 5-10 fold difference from each other in terms of EC50 values as expected.

And also testProtein #56 and->Comparison of protein #57 with non-binding designed ankyrin repeat protein used as negative control compound. As shown in FIG. 52B, the test protein +. >Protein #56 and->Protein #57 shows efficient and specific tumor cell killing in the presence of panT cells (IC 50 on Molm-13:. About.>Protein #56:4.6pM, & gt>Protein #57:1.3 pM). As expected, the negative control engineered ankyrin repeat protein bound only to tumor specific targets (or CD 3), resulting in little tumor cell killing at the maximum concentration used.

In addition, evaluateComparison of protein #56 with known reference molecule Fu Tuozhu mab analog and AMG330 analog. As shown in FIG. 52C, the +.>Protein #56 showed efficient and specific tumor cell killing in the presence of human PanT cells. Fu Tuozhu monoclonal antibodies and->The efficacy of protein #56 was comparable, whileThe EC50 value of protein #56 was lower than that of the votuzumab and AMG330 tested.

Assessment by measuring LDH release in an in vitro short term LDH cytotoxicity assaySpecificity and potency of protein # 56; in this assay, pan T cells were co-cultured with tumor cells consisting of a combination of Molm-13 cells expressed from wild-type targets with CD33, CD123 and CD70 and various knockouts of the same targets (Molm 13 CRISPR Knockdown (KO) target cells) (fig. 52D).

To this end, 100,000 purified pan-T cells (effector cells) and 20,000 Molm-13 KO cells (target cells) per well were incubated with the selected multispecific recombinant protein, control reference molecule or serial dilutions of a control containing 1% Triton X-100 for 48 hours at 37 ℃. After 48 hours of incubation, the cells were centrifuged and 100. Mu.l of supernatant per well and 100. Mu.l of LDH reaction mixture per well (LDH detection kit; roche Applied Science) were incubated for 30 minutes. The absorbance at 492nm to 620nm was measured by a TECAN infinite M1000Pro reader. After background correction, OD values were plotted using GraphPad Prism 8. As shown in fig. 52D, the highest efficacy was achieved for the test protein if all three targets were co-expressed (curve 1) and additionally when two targets were co-expressed (curve 2 to curve 4). After co-culturing T cells with single target expressing tumor cells (such as cd33+, cd123+, cd70+) the efficacy was reduced up to 10-100 fold (curves 5 toCurve 7). In addition, in the case of the optical fiber,the efficacy of protein #56 on cd33+ cells was also reduced.

Example 7: multispecific recombinant proteins targeting three tumor-associated antigens Swelling of protein #31 Assessment of tumor cell killing。

For analysis ofProtein #31 killing Activity against tumor cells effector cells (pan-T cells, 100,000 cells) and Molm13 tumor cells (20,000 cells) were seeded at an E:T ratio of 5:1 in 96 well plates. Pan-T cells were stained with CellTrace Violet (CTV) prior to seeding to enable differentiation from tumor cells during FACS analysis. Samples were analyzed for tumor cell killing by flow cytometry after co-cultivation in the presence of serial dilutions of the indicated molecules for 48 hours. Cells were washed and stained with 1:1,000live/Dead Aqua (Thermo Fisher) and incubated at 4℃for 30min. After washing and fixation, cells were analyzed on a FACS Canto II (BD) machine. Tumor cell killing was assessed by absolute count of remaining L/Dneg/CTVneg cells. FACS data were analyzed using FlowJo software and plotted using GraphPad Prism 8.

FIG. 7 shows a reference control molecule (known reference T cell conjugate: AMG330 with binding specificity for CD33 and Fu Tuozhu mab with binding specificity for CD 123) andprotein #31. The data show that in tumor cell killing assays, when compared to clinical baseline, the +.>Protein #31 exerts similar potency and efficacy.

Similar tests were also performed on Molm13 CRISPR Knockout (KO) cellsProtein #31 to investigate the functionality of this molecule. As can be seen in fig. 8, curve 7 represents the Molm13 parental cells expressing all three targets (CD 70, CD123 and CD 33). Here->Protein #31 showed full efficacy. Curves 4-6 represent a single KO cell, meaning that only two targets are still expressed. Here->Protein #31 remains active, indicating the potential to combat tumor heterogeneity. Curves 1-3 represent double KO cells, meaning that only one target is still expressed to mimic a healthy tissue compartment. Here the number of the elements is the number,protein #31 had significantly lower activity, meaning increased selectivity for healthy tissue. In summary, FIG. 7 shows +.>Protein #31 has the potential to counteract tumor heterogeneity and improve selectivity.

Example 8: effects of multispecific binding proteins on cytokine Release in human ex vivo Whole blood Loop models

Whole blood circuit systems are used to study interactions between blood and drug samples, including effects on cytokine release. The blood circuit system uniquely includes immune cells, immunoglobulins, and the complete complement and coagulation cascade system in the blood (Fletcher, E.A.K. et al, int Immunopharmacol, (2018) 54: pages 1-11). The system relies on fresh human whole blood that is kept spinning to avoid clotting, and it represents a model that mimics human blood circulation.

A. Recombinant binding proteins have been evaluated in the whole blood circuit system at four different concentrations#7、#8、/>#9 and->#10, and two known control reference T cell conjugates AMG330 and Futuzumab analogs (AMG 330 analog/Fu Tuozhu monoclonal antibody analog: 10nM, 0.1nM, 0.01nM and 0.001 nM;)>#7 to 10:10nM, 1nM, 0.1nM and 0.01 nM).

Cytokine release was analyzed at time points of 0 hours, 4 hours, 8 hours, 24 hours and 48 hours in the whole blood circuit system. In this study, PBS was used as a negative control, and AMG330 analog and Futuzumab analog were used as reference controls for cytokine release (T cell conjugates: AMG330 with binding specificity for CD33 and Fu Tuozhu mab with binding specificity for CD 123).

Drug samples were prepared to obtain the following concentrations in blood: AMG330 analog/Fu Tuozhu mab analog: 10nM, 0.1nM, 0.01nM and 0.001nM;#7 to 10:10nM, 1nM, 0.1nM and 0.01nM. Sterile PBS was used as vehicle and to dilute the test samples to obtain a final volume of 100 μl added to each loop. The remaining test sample solution was discarded.

All materials in the loop system are surface heparinized. To prevent clotting, blood was placed in a plastic tube for rotation, wherein the interior of the tube was pre-coated with a unique heparin conjugate prior to running. The coating allows blood circulation without adding large amounts of anticoagulant to the blood. Fresh whole blood was taken from two healthy volunteers (D1, D2). Immediately after blood collection, the blood was transferred into pre-coated plastic tubes to form a circuit, followed by administration of test items according to the following design:

1. vehicle (PBS)

AMG330 analog 0.001nM

AMG330 analog 0.01nM

AMG330 analog 0.1nM

AMG330 analog 10nM

6. Fu Tuozhu monoclonal antibody analog 0.001nM

7. Fu Tuozhu monoclonal antibody analog 0.01nM

8. Fu Tuozhu monoclonal antibody analog 0.1nM

9. Fu Tuozhu monoclonal antibody analog 10nM

10.#7 0.01nM

11.#7 0.1nM

12.#7 1nM

13.#7 10nM

14.#8 0.01nM

15.#8 0.1nM

16.#8 1nM

17.#8 10nM

18.#9 0.01nM

19.#9 0.1nM

20.#9 1nM

21.#9 10nM

22.#10 0.01nM

23.#10 0.1nM

24.#10 1nM

25.#10 10nM

Subsequently, the loops were set to rotate and for each donor, a set of 26 loops was divided onto two parallel rotating platforms.

Two healthy donors (men age. Gtoreq.18 years) were recruited (no acute infection and no intake of NSAIDs or any kind of corticosteroid within 7 days after blood donation).

Fresh blood from each donor (described as a time point 0 sample) stored directly after lancing is processed into plasma and included in the cytokine analysis. The circuit was sampled 4 hours, 8 hours, 24 hours and 48 hours after the addition of the test substance, and EDTA (concentration in blood: 10 mM) was added to each sample to stop the reaction at the sampling time point. The circuits were sampled at 4 hours, 8 hours, 24 hours and 48 hours for cytokine release and flow cytometry analysis. Plasma samples were prepared by centrifugation, aliquoted and stored at-60 ℃ until analysis.

Using a primer from Meso Scale Discovery MSDThe technique measures cytokines (ifnγ, tnfα) (fig. 11, fig. 12). Samples for cytokine analysis were collected at the 0, 4, 8, 24 and 48 hour time points. Blood samples were processed into plasma and stored at-60 ℃ until analysis, which in this study was performed within 22 days from sample collection. Samples were diluted 1:4, 1:8, 1:16, 1:32, or 1:100 and run in duplicate according to manufacturer's instructions.

The lower limit of detection (LLOD) was calculated by MSD software and defined as 2.5×sd above zero calibrator (standard-8). The upper limit of detection (ULOD) was calculated by MSD software from the signal value of standard-1. The lower and upper limits of quantitation (LLOQ and ULOQ) were verified by MSD and calculated from the percent recovery of the standard curve and diluent standards with an accuracy of 20% and an accuracy of 80% -120%. Three levels of ifnγ and tnfα from MSD multi-analyte controls (lot nos. a00C0640, a00C 0641) were used to evaluate the accuracy and precision between runs. Control and test samples were run in duplicate. A typical acceptance criterion for the control is CV <20%. In this study, all inter-run CV values were below 20%.

Cytokines were measured in the whole blood circuit system at the 0 (zero) hour, 4 hour, 8 hour, 24 hour and 48 hour time points. Responsive toThe concentrations of cytokines ifnγ and tnfα for #7 to #10 and controls are presented in fig. 27 and 28 as the mean of the two donors over time and the original mean concentration calculated by the MSD software for all values above 0 pg/ml. Cytokine production is described (arbitrarily classified) as: for values of 50pg/ml or less, low; at 50pg/ml <A value of between ∈500pg/ml, medium; at 500pg/ml<And a value of 5000pg/ml or less, is high.

IFNgamma levels

In the loop system, AMG330 analog and votuzumab analogs induced high levels of ifnγ at the two highest concentrations (10 nM and 0.1 nM), whereas at 0.01nM, the levels were medium to high for AMG330 analog and low to medium for votuzumab analog. For the two reference T cell conjugates at the lowest concentration (0.001 nM), ifnγ levels were low (fig. 27). IFNgamma level atGroup #7 was low and only the highest concentration (10 nM) was noted above vehicle, reaching a peak at 8 hours (average 34.8 pg/ml). In use->IFNγ levels in the #8-10 loop were high at 10nM and reached peak (++) after 4 hours>The average value of #8 was 6758.6pg/ml,the average value of #9 is 10988.6pg/ml, and +.>The average value of #10 was 18972.3 pg/ml), which was high at 1nM and reached peak (++) after 24 hours>Average value of #8 is 3914.4pg/ml，/>The average value of #9 is 6482.0pg/ml, and +.>The average value of #10 was 4511.3 pg/ml), low to high at 0.1nM, and peak (++A) after 24 hours>#8 has an average value of 222.4pg/ml, #8>The average value of #9 is 710.1pg/ml and +. >The average value of #10 was 326.4 pg/ml). At->At the lowest concentration of #7 to 10 (0.01 nM), ifnγ levels were low and comparable to vehicle at all four time points.

TNFalpha levels

Similar to ifnγ, high tnfα levels were observed in AMG330 analog and in the votuzumab analog at 10nM, with peak values after 24 hours for AMG330 analog (average 7680.9 pg/ml) and after 4 hours for Fu Tuozhu mab analog (average 4594.1 pg/ml) (fig. 28). At 0.1nM, the TNFα level is high and peaks after 24 hours (average 6159.6 pg/ml) for the AMG330 analog and 8 hours (average 4077.3 pg/ml) for the Futuzumab analog. At 0.01nM, the TNFα level was medium to high (peak after 24 hours, average 1813.9 pg/ml) for the AMG330 analog and low to medium (peak after 24 hours, 141.9 pg/ml) for the Futuzumab-mAb analog. T at 0.001nM in AMG330 and Futuzumab analoguesNfα levels were low and comparable to vehicle groups. At allTnfα levels in group #7 were low at all four time points, all comparable to vehicle, except that the highest concentration to peak after 48 hours was 18.5 pg/ml. In use- >TNFα levels were moderate to high at 10nM in the loop of #8-10 for +.>#8 (average value 2732.8 pg/ml) after 24 hours and for +.>#9-10 (average values 4671.9pg/ml and 6026.4 pg/ml) peaked after 8 hours, respectively. TNF alpha levels were moderate to high at 1nM, reaching a peak (+.>#8 has an average value of 2046.0pg/ml, #8>The average value of #9 is 3288.6pg/ml, and +.>The average value of #10 was 2522.7 pg/ml). At 0.1 nM->In #8-10, TNFα levels were low to moderate at the 4 hour and 8 hour time points, and moderate to high at the 24 hour to 48 hour levels (+.>Peak value of #8 is 88.3pg/ml +.48 hours later>Peak value of #9 is 800.7pg/ml, and 24 hours later +.>Peak value of #10 was 1382.8 pg/ml). At the lowest concentration (0.01 nM), allGroup #8-10 was comparable to the vehicle group at all four time points.

In general, in the known reference control group (at 10nM, 0.1nM and 0.01 nM) andan increase in the levels of all cytokines (ifnγ and tnfα) relative to vehicle was observed in groups #8-10 (at 10nM, 1nM and 0.1 nM) at all four time points. No response was observed>#7 (at all concentrations), AMG330 analogues and Futuzumab analogues (at 0.001 nM) and +. >Release of different cytokines from #8-10 (at 0.01 nM). This may suggest that ten times higher concentrations of +.>#8-10 to match cytokine release induced by the reference molecule AMG330 analog and the votuzumab analog. Furthermore, this indicates +.>The #8-10 compound has a more controlled cytokine release.

B. Recombinant binding proteins have been evaluated in the whole blood circuit system at three different concentrationsProtein #27, < >>Protein #29 and->Protein #31 and known control reference T cell conjugate Fu Tuozhu mab

Cytokine release was analyzed at time points of 0 hours, 2 hours, 4 hours, 8 hours and 24 hours in the whole blood circuit system. In this study, PBS was used as a negative control for cytokine release, fu Tuozhu mab was used as a baseline, positive control (Fu Tuozhu mab with binding specificity to CD 123).

Drug samples and positive controls were prepared to obtain the following concentrations in blood: 0.005nM, 0.1nM and 2nM. Sterile PBS was used as vehicle and to dilute the test samples to obtain a final volume of 100 μl added to each loop. The remaining test sample solution was discarded.

All materials in the loop system are surface heparinized. To prevent clotting, blood was placed in a plastic tube for rotation, wherein the interior of the tube was pre-coated with a unique heparin conjugate prior to running. The coating allows blood circulation without adding large amounts of anticoagulant to the blood. Fresh whole blood was taken from three healthy volunteers (D1-D3). Immediately after blood collection, the blood was transferred into pre-coated plastic tubes to form a circuit, followed by administration of test items according to the following design:

1. Vehicle (PBS)

2. Fu Tuozhu mab 0.005nM

3. Fu Tuozhu mab 0.1nM

4. Fu Tuozhu monoclonal antibody 2nM

5.Protein # 27.005 nM

6.Protein # 27.1nM

7.Protein #27 2nM

8.Protein # 29.005 nM

9.Protein # 29.1 nM->

10.Protein #29 nM

11.Protein # 31.005 nM

12.Protein # 31.1 nM

13.Protein #31 nM 2

Three healthy donors (two females and one male aged 18 years) were enrolled (no acute infection and no intake of NSAIDs or corticosteroids of any kind within 7 days after blood donation).

Fresh blood (described as a 0 time point sample) stored directly after lancing is used for hematology measurements. In addition, blood collected at time 0 was processed into plasma and included in cytokine analysis. Samples were extracted from each circuit 2 hours, 4 hours, 8 hours and 24 hours after the addition of the test substance, and EDTA (concentration in blood: 10 mM) was added to each sample to stop the reaction at the sampling time point. The circuits were sampled at 2 hours, 4 hours, 8 hours and 24 hours for hematology and cytokine release analysis. Plasma samples were prepared by centrifugation, aliquoted and stored at-60 ℃ until analysis.

Using a primer from Meso Scale Discovery (MSD)Techniques measure cytokines (IFNγ, IL-2, IL-6, IL-8, TNF. Alpha.). Cytokine levels are shown as averages over time (fig. 9, 10, 11, 12, 13). Samples for cytokine analysis were collected at 0, 2, 4, 8 and 24 hour time points. Blood samples were processed into plasma and stored at-60 ℃ until analysis, which in this study was performed within 8 days from sample collection. Samples were diluted 1:4 (or 1:100) and run in duplicate according to manufacturer's instructions.

The lower limit of detection (LLOD) was calculated by MSD software and defined as 2.5×sd above zero calibrator (standard-8). The upper limit of detection (ULOD) was calculated by MSD software from the signal value of standard-1. The lower and upper limits of quantitation (LLOQ and ULOQ) were verified by MSD and calculated from the percent recovery of the standard curve and diluent standards with an accuracy of 20% and an accuracy of 80% -120%. If the measured average concentration value of the diluted plasma sample is below LLOQ or above ULOQ, the data is defined as below LLOQ or above ULOQ. The data were further converted by dilution factor (4 x or 100 x) to obtain calculated average concentration values. Raw data corresponds to calculated average concentration values for all samples. Repeated acceptance criteria within a quantifiable range are set to Coefficient Values (CV). Ltoreq.20%. All samples had repeated CV values of 20% or less.

Cytokines were measured in the whole blood circuit system at 0 (zero) hour, 2 hours, 4 hours, 8 hours and 24 hours time points. The cytokines ifnγ, IL-2, IL-6, IL-8 and tnfα concentrations in response to the drug samples are presented in fig. 9, 10, 11, 12, 13 as the mean of the three donors over time. Cytokine production is described (arbitrarily classified) as: for values of 50pg/ml or less, low; a value between 50pg/ml < and ∈500pg/ml, medium; and a value between 500pg/ml < and 5000pg/ml, high.

IFNgamma levels

At high levels in samples with 2nM and 0.1nM of Fu Tuozhu mab (defined as>500 pg/ml) induced ifnγ, which increased in average from 1243.8pg/ml to 48937.8pg/ml (at 2 nM) and from 485.7pg/ml to 31335.2pg/ml (at 0.1 nM) over time, whereas in samples with 0.005nM of tuzumab, ifnγ levels were low to high, with average from 11.9pg/ml to 2064.8pg/ml over time (fig. 9).Protein #27 (at 2 nM) induced moderate to high levels of ifnγ, which increased in average from 233.5pg/ml to 6131.5pg/ml (at 2 to 24 hours) over time. At 0.1 nM->At protein #27, ifnγ levels increased relative to vehicle, from low to medium levels, with an average of 10.2pg/ml to 334.3pg/ml (at 2 to 24 hours). At 0.005 nM- >At protein #27, ifnγ levels were low and similar to vehicle in all donors and at all four time points. In use->In the sample of protein #29, ifnγ levels were low and similar to vehicle at 0.005nM and 0.1nM, while levels increased slightly relative to vehicle at 2nM at time points ranging from 2 to 24 hours, with averages from 3.1pg/ml to 55.2pg/ml. In the presence of 2 nM->In the sample of protein #29, IFNγ levels increased over time from an average value of 23.4pg/ml at 2 hours to 511.0pg/ml at 24 hours, and at 0.1nM and 0Ifnγ levels were low and similar to vehicle at 005nM and at all four time points.

IL-2 levels

IL-2 levels were low in samples with votuzumab (at 0.005 nM) at 2 and 4 hours, but increased gradually over time from 8 to 24 hours compared to vehicle, with an average peak at the 24 hour time point of 472.1pg/ml (FIG. 10). At 0.1nM and 2nM, the votuzumab ozogamicin induced low to high levels of IL-2, with average peaks at 1951.6pg/ml (at 0.1 nM) and 2756.6pg/ml (at 2 nM) at 8 hours time points. At 0.005At protein #27, IL-2 levels were low and similar to vehicle, but at 0.1nM, whereas in samples from D2 and D3, but not D1, increased relative to vehicle at 24 hours. At 2 nM- >IL-2 levels increased from low levels at 2 to 4 hours to high levels at 8 to 24 hours at protein #27, with an average peak at the 24 hour time point of 600.0pg/ml. In use->In the sample of protein #29, IL-2 levels were low at all concentrations and time points except at the 24 hour time point (where IL-2 levels increased relative to vehicle at 2nM in the samples from D1 and D3). In useIn the sample of protein #31, IL-2 levels were low at all concentrations and time points except for the highest concentration (2 nM), where IL-2 levels were increased relative to vehicle at three later time points, with an average peak of 99.9pg/ml at 24 hours.

IL-6 levels

IL-6 was induced at low to high levels in samples with 2nM and 0.1nM Fu Tuozhu mabAverage values increased over time from 3.5pg/ml to 432.2pg/ml (at 2 nM) and from 1.4pg/ml to 713.2pg/ml (at 0.1 nM), whereas in samples with 0.005nM valtuzumab, IL-6 levels were low and similar to vehicle at 2 to 4 hours and increased relative to vehicle at 8 to 24 hours with an average peak of 1744.4pg/ml at 24 hours (FIG. 11). In useIn the sample of protein #27 (at 2 nM), IL-6 levels increased relative to vehicle at three later time points (4 hours to 24 hours), while at 0.005nM and 0.1nM IL-6 levels were low or similar to vehicle except for D3 at the 24 hour time point where moderate levels (77.6 pg/ml) were observed. At all four time points, at +. >Protein #29 and->At all concentrations of protein #31, IL-6 levels were low (defined as<50 pg/ml) or similar to the vehicle.

IL-8 levels

In the samples with 2nM and 0.1nM of Fu Tuozhu mab, IL-8 levels increased from moderate to high over time, whereas at 0.005nM, at all four time points, the levels were similar to vehicle in D1 and increased relative to vehicle in samples from D2 (at 24 hours) and D3 (at 4 to 24 hours) (fig. 12). At 2nMIL-8 levels of all three donors increased relative to vehicle over time at protein #27, while IL-8 levels at 0.1nM increased relative to vehicle in samples from D2 (at 24 hours) and D3 (at 4 to 24 hours) instead of D1. At 0.005 nM->Protein #2IL-8 levels were similar to vehicle at all four time points at 7. In useIL-8 levels were low and similar to vehicle in all samples of protein #29, but +.>In the sample of protein #31, IL-8 levels increased slightly relative to vehicle at the highest concentration (2 nM) and at 4 to 24 hours.

TNFalpha levels

At moderate to high levels in samples with 2nM and 0.1nM Fu Tuozhu mab (defined as>500 pg/ml) induced tnfα, which increased in average from 412.1pg/ml to 7673.8pg/ml (at 2 nM) and from 184.2pg/ml to 4689.8pg/ml (at 0.1 nM) over time, whereas in samples with 0.005nM tuzumab, tnfα levels increased from low to high over time, with average from 3.9pg/ml to 458.9pg/ml (fig. 13). Protein #1 (at 2 nM) induced moderate to high levels of tnfα, which increased over time from an average of 57.8pg/ml to 1522.6pg/ml (at 2 to 24 hours). At 0.1 nM->At protein #27, tnfα levels increased relative to vehicle, from low to medium levels, with an average value of 4.0pg/ml to 95.4pg/ml (at 2 to 24 hours). At 0.005nMAt protein #27, tnfα levels were low and similar to vehicle in all donors and at all four time points. In use->In the sample of protein #29, at all three concentrations and at all four timesAt the point, tnfα levels were low and similar to vehicle. In the presence of 2 nM->In the sample of protein #31, tnfα levels increased over time from low to medium levels, with average values increasing from 6.5pg/ml at 2 hours to 98.6pg/ml at 24 hours, while ifnγ levels were low and similar to vehicle at 0.1nM and 0.005nM and at all four time points.

In summary, ifnγ, IL-2, IL-6 and tnfα levels recorded in the vehicle group at all four time points (2 hours, 4 hours, 8 hours and 24 hours) were generally similar to those recorded in the 0 time point group. In contrast, for IL-8, a difference was observed between time point 0 and vehicle, and this may indicate that in a system with an active cascade system, a background level of cytokines was released from the donor in response to the vehicle. In conclusion, fu Tuozhu mab (0.005 nM, 0.1nM and 2 nM), Protein #27 (0.1 nM and 2 nM) and +.>Protein #31 (at 2 nM) induced cytokine release increased over time, on the order of Fu Tuozhu mab>/>Protein #27>/>Protein #31. On the contrary, the->Protein #29 did not induce cytokines or induced low levels of cytokines at all three concentrations and at all four time points.

C. Recombinant binding proteins targeting CD70, CD123 and CD33 have been evaluated in the whole blood circuit system at three different concentrationsProtein #58, < >>Protein #59, < >>Protein #57, < >>Protein #60, < >>Protein #61 and->Protein #56 and associating them with a half-life extended non-TAA (Ni 2C) in the presence of a non-TAA inhibitor>Protein #74 was compared to the votuzumab analogue as a reference molecule.

Cytokine release was analyzed at time points of 0 hours, 2 hours, 4 hours, 8 hours and 24 hours in the whole blood circuit system. In this study, PBS was used as a negative control for cytokine release, and a Fu Tuozhu mab analog was used as a baseline, positive control (Fu Tuozhu mab with binding specificity to CD 123).

TABLE 9a

Using a primer from Meso Scale Discovery (MSD)Techniques measure cytokines (IFNγ, IL-2, IL-6, IL-8, TNF. Alpha.). Cytokine levels are shown as averages over time (fig. 44, 45, 46, 47, 48). Samples for cytokine analysis were collected at 0, 2, 4, 8 and 24 hour time points. Blood sample is takenPlasma was processed and stored at-60 ℃ until analysis, which in this study was performed within 8 days from sample collection. Samples were diluted 1:4 (or 1:100) and run in duplicate according to manufacturer's instructions.

Cytokine production is described (arbitrarily classified) as: for values of 50pg/ml or less, low; a value between 50pg/ml < and ∈500pg/ml, medium; and a value between 500pg/ml < and 5000pg/ml, high.

Overall, the lowest concentration (0.005) compared to vehicleProtein #56, < >>Protein #57 did not induce increased IL-2, IL-8 or TNF. Alpha. Furthermore, the lowest concentration and the medium concentration +.>Protein #56 and->The time point for induction of IL-2 and IL-6 by protein #60 was later than all other test proteins at the same concentration. At 8h and 24hIntermediate, highest concentration +.>Protein #60 induced tnfα levels similar to most of the test items. In contrast, the highest concentration +.>Protein #56 induced tnfα values were lower than all other test items. As expected, the CD3 and CD123 binding properties of Fu Tuozhu mab induced cell depletion and cytokine release, however +_ compared to vehicle>Protein #74 did not significantly alter cell depletion and immune response.

Example 9: multi-specific binding proteins for T cell activation, cell viability, blood in human ex vivo whole blood circuit models Effects of platelet count and white blood cell count

A. Effect of multispecific binding proteins on T cell activation and cell viability

As in example 8, in the whole blood circuit system, the evaluation was responsive to AMG330 analog/Fu Tuozhu mab analog and at the 0 hour (pre-dosing), 4 hour, 8 hour, 24 hour, and 48 hour time pointsT cell activation and cell viability of #7 to 10. All sample preparations were performed as described in example 10. Blood cells were stained with fluorescent-labeled monoclonal antibodies (supplied by Biolegend) for detection: bone marrow cells (CD33+ and CD123+ -antibodies used: CD33-PE/Cy7, CD 123-PerCP/Cy5.5), T cells (CD 3-antibody used: CD3-BV 510), activation markers (CD 69, CD 25-antibodies used: CD69-BV421, CD 25-APC) and vital dyes (fixable vital dyes eFluor 780) to stain dead cells. A cytometry bead was added to calculate the relative cell count over time for each loop. Will be used for flow cytometryThe analyzed sample was incubated with the fluorescently labeled antibody, and the erythrocytes were lysed and washed. Samples were run on a CytoFlex flow cytometer (Beckman Coulter) and the data was analyzed by FlowJo v10 to present a percentage of cell types positive for activation markers and vital dyes (dead cell%). In addition, zero samples of both donors were analyzed for CD33 and CD123 expression by T cells. In addition to analyzing blood samples collected from the circuit, time 0 samples of each donor were incubated with antibodies specific for whole blood cells and without activation markers. All controls were as expected. To compensate for spectral overlap of fluorophores during flow cytometry, compensation is performed. Beads (catalog No. B22804, beckman Coulter) were stained with antibodies having the same fluorophores as those contained in the study, and the auto-compensation calculation was performed by software (cytexert, beckman Coulter). The compensation settings were evaluated using single stained cells and optimized if necessary.

Flow cytometry analysis was performed on samples collected at 4, 8, 24, and 48 hour time points. In addition, time point 0 samples were analyzed to directly determine cell activation status after blood collection. T cells and bone marrow cells were detected using surface markers (CD 3, cd33+ and cd123+). Cell activation and viability were measured by activation markers CD25, CD69 (activated) and viability dye binding to dead cells. In addition, counting beads were included to analyze how the cell count of cd3+, cd33+ and cd123+ cells varied over time (fig. 29-30).

T cell activation

The percentage of cd25+ T cells was similar to vehicle in all groups (average 9.3% positive cells at 4 hours and 7.0% positive cells at 8 hours) (fig. 29). In the group of AMG330 analog/Fu Tuozhu mab analog (at 10nM and 0.1 nM) at 24 hours and 48 hours, the percentage of cd25+ T cells was increased relative to vehicle ranging from 15.4% -28.4% positive T cells, while the two lowest concentrations of AMG330 analog/Fu Tuozhu mab analog were similar to vehicle at 24 hours and 48 hours time points (average 13.5% at 24 hours, average 1% at 48 hours)3.9%). At the position of The percentage of cd25+ T cells in group #7 was similar to vehicle at four time points.#8-10 was similar to vehicle at 4 hours and 8 hours. At 24 hours and 48 hours, the percentage of CD25% T cells has been increased relative to vehicle, ranging from 11.9% -23.2% (for +.># 8), 12.3% -26.2% (for# 9) and 12.4% -26.5% (for +.># 10). At the two lowest +.>The percentage of CD25 positive T cells at concentrations #8-10 (0.1 nM and 0.01 nM) was similar to vehicle at all four time points.

AMG330 analogs activated T cells expressed CD69 at 10nM (28.9% positive T cells on average peak at 4 hours), 0.1nM (30.0% positive T cells on average peak at 8 hours), and 0.01nM (22.0% positive T cells on average peak at 24 hours) (fig. 30). The AMG330 analog group was similar to vehicle (average 2.5% at 4 hours, average 3.0% at 8 hours, average 5.6% at 24 hours, and average 7.9% at 48 hours) at 0.001 nM. Fu Tuozhu mAb analogs induced CD69 expression at 10nM (52.2% positive T cells on average peak at 8 hours), 0.1nM (39.7% positive T cells on average peak at 8 hours), and 0.01nM (39.7% positive T cells on average peak at 24 hours). CD69 expression in group #7 was found in allFour time points were similar to vehicle. At->CD69 expression was for +.10 in group #8-10 at 10nM>#10 peaked at 10nM at 4 hours (average 41.4% positive T cells) and was for +.>#8-9 peaked after 8 hours at 10nM (for +.>The average value of #8 is 26.4%, for +.>The average value of #9 is 33.1%). CD69 is expressed in +.1 nM and 0.1nM>The peak was reached in group #8-10 at 24 hours to 48 hours, with the mean value of positive T cells ranging from 14.4% -30.6% (fig. 30). At 0.01 nM->Group #8-10 was similar to vehicle at all four time points.

Cell viability

In addition to cell type markers and activation markers, viability dyes are included in the staining to assess viability of target cells and effector cells (presented as% dead cells). In addition, since vital dye staining was dependent on the expression of cell type markers, cell counts were reported to give a comprehensive picture of viability and cell count over time (fig. 31-32).

For all test molecules, the viability of dye-positive T cells (i.e., dead cells%)<11.0% (FIG. 31). Except forExcept for #7 (average 20.8% decrease at 1nM and 19.2% decrease at 10 nM), the number of T cells in all molecules was similar to vehicle at 4 hours (defined as compared to vehicle group) <20% drop). At 8 hours, T cell numbers decreased compared to vehicle at 10nM (33.3% average for AMG330 analog, 26.3% average for Fu Tuozhu mab analog, 26.3% average for->#9 drop 19.8% on average and for +.>#10 dropped 63.6% on average), while all other groups were similar to vehicle at the 8 hour time point. After 24 hours, the T cell count in the vehicle group decreased and was except +.about.0.01 nM>Except for #8 (average 43.6% drop compared to vehicle), all groups were similar to vehicle. After 48 hours, T cell counts decreased with increasing molecular concentration (average decrease in the range of 15.5% -86.5% for AMG330 analog, 45.4% -83.0% for Fu Tuozhu mab analog, and>the average drop range of #7 is 48.7% -65.2% for +.>The average drop range of #9 is 27.9% -62.4% for +.>The average drop in #10 ranged from 20.7% to 58.9%), except for#8，The maximum drop was at the two lowest concentrations compared to vehicle, at 0.1nM (average drop of 63.9%) and 0.01nM (average drop of 23.5%).

Cd33+ bone marrow cells were stained as viable dye positive (i.e., dead cell%) at 10nM (56.4% viable dye positive cells on average at 8 hours, 56.1% viable dye positive cells on average at 24 hours, and 52.8% viable dye positive cells on average at 48 hours), 0.1nM (15.3% viable dye positive cells on average at 8 hours, 58.0% viable dye positive cells on average at 24 hours, and 51.1% viable dye positive cells on average at 48 hours) and 0.01nM (19.8% viable dye positive cells on average at 24 hours, and 31.5% viable dye positive cells) in the AMG330 analog group (fig. 32). Fu Tuozhu monoclonal antibody analogs at 10nM (average 53.2% viable dye-positive cells at 4 hours, and 8 to 48 hours >98.0% vital dye-positive cells) and 0.1nM (average 76.3% vital dye-positive cells at 24 hours, and average 80.1% vital dye-positive cells at 48 hours). At lower molecular concentrations (0.001 nM for AMG330 analog/Fu Tuozhu monoclonal antibody analog, for#8-10 was 0.01nM for#7 is 0.1 nM), activity was similar to vehicle at all four time points<10% viable dye positive cells). At 4 hours, at +.>Group #7 (average 13.6% vital dye positive cells at 10nM, and average 11.2% vital dye positive cells at 1 nM),>group #8 (average 19.7% vital dye positive cells at 0.1 nM) and +.>In group #10 (average 14.0% vital dye positive cells at 10nM, average 13.9% vital dye positive cells at 1nM, and average 14.2% vital dye positive cells at 0.01 nM), the percentage of vital dye positive cells was increased compared to vehicle. At 10nM for 8-48 hours, compared to vehicle>Group #7 to 10 increased the percentage of viable dye positive cells (for +.>The average value of #7 ranges from 10.5% to 30.2%, forThe average value of #8 ranges from 73.1% to 69.1% for +.>The average value of #9 ranges from 90.9% to 92.6% and for +. >The average value of #10 ranges from 93.7% to 91.9%). At 1nM->#8 induced viable positive cells (average range of 39.6% -42.1%) from 24 hours to 48 hours, ">#9 induced viable positive cells (average range 13.7% -60.7%) from 8 hours to 48 hours,>#10 induced viable positive cells at 8-24 hours (average range 13% -47.1%). In addition, a->#9 and->#10 for +.>#9 increased the percentage of viable positive cells at 48 hours (mean 33.9%) and for v10 at 24 hours to 48 hours (mean 22.9% and 14.7%, respectively).

Similarly, in addition toIn all the test groups except #7, cell counts of CD33+ cells decreased as compared to vehicle at the highest molecular concentration (10 nM) at the 4-8 hour time point>30% difference) (fig. 35). The second highest concentration (0.1 nM for AMG330 analog/Fu Tuozhu monoclonal antibody analog, and for all test groups)#7 to 10 at 1 nM) resulted in a decrease in CD33+ cells at the 8-24 hour time point. At lower concentrations (0.01 nM-0.001nM for AMG330 analog/Fu Tuozhu monoclonal antibody analog, and +.>#7 to 10 was 0.1nM-0.01 nM), cd33+ cells were similar to vehicle at the earlier time point and declined at the later time point for some groups.

The percent of viable dye positive cd123+ cells (i.e., dead cells%) was similar to vehicle and averaged over 4 hours for all test molecules<8.0% and average over 8 hours<20.0%. At 24 hours, at the highest concentration of AMG330 analog,#8、/>#9 and->The percentage of viable positive cd123+ cells at #10 (mean range 10.2% -29.2% viable dye positive cells) was increased compared to vehicle. At 48 hours, the percentage of viable dye positive cells ranged from 3.0% to 50.3% in all groups, some of which were larger than the vehicle group and some of which were smaller than the vehicle group (average 13.9%) (fig. 31-33).

Cd123+ cell numbers in AMG330 analog group were decreased over time at 10nM (average decrease in 4 hours of 54.6%, average decrease in 8 hours of 85.1%, average decrease in 24 hours of 89.5%, and average decrease in 48 hours of 78.6%), at 0.1nM (average decrease in 8 hours of 40.3%, average decrease in 24 hours of 81.5%, and average decrease in 48 hours of 86.6%) and at 0.01nM (average decrease in 24 hours of 49.3%, and average decrease in 48 hours of 72.4%) compared to vehicle (fig. 36). Fu Tuozhu mAb analogs induced a decrease in CD123+ cells at 10nM (average decrease in 8 hours 60.2% at 10nM, average decrease in 65.2% at 24 hours, and average decrease in 42.4% at 48 hours) and at 0.1nM (average decrease in 79.0% at 24 hours, and average decrease in 63.4% at 24 hours). At lower concentrations of AMG330 analog (at 0.001 nM) and Fu Tuozhu mab analog (0.01 nM and 0.001 nM), counts were similar to vehicle at all four time points. At the position of In group #7, cd123+ cells% were similar to vehicle except that a 31.2% decrease in the 1nM group compared to vehicle was noted at the 4 hour time point. As the molecular concentration and time point increased, compared to vehicle group,/was->#8-10 reduced cd123+ cell numbers. />#8 decreased 50.4% on average at 8 hours, 47.7% on average at 24 hours and 72% on average at 48 hours at 10 nM.9%) and at 1nM (average drop 38.1% at 24 hours and 68.1% at 48 hours). For->#9, cd123+ cells decreased at 10nM (58.1% average decrease at 8 hours, 67.9% average decrease at 24 hours, and 79.1% average decrease at 48 hours), at 1nM (29.6% average decrease at 24 hours, and 76.2% average decrease at 48 hours), and at 0.1nM (34.4% average decrease at 24 hours, and 48.6% average decrease at 48 hours) compared to vehicle. />#10 decreased at 10nM (average 31.3% decrease at 4 hours, average 72.3% decrease at 8 hours, average 81.3% decrease at 24 hours and average 76.3% decrease at 48 hours), at 1nM (average 74.4% decrease at 24 hours and average 72.7% decrease at 48 hours) and at 0.1nM (average 57.2% decrease at 24 hours and 62.9%) resulted in CD123+ cell decrease compared to vehicle. At a lower concentration +. >#8 (0.1 nM and 0.01 nM),>#9 (0.01 nM) and +.>The counts were similar to vehicle at all four time points at #10 (0.01 nM).

B. Effect of multispecific binding proteins on platelet count

As in example 8, response to recombinant binding proteins was evaluated at the 0 hour (pre-dosing), 2 hour, 4 hour, 8 hour and 24 hour time points in the whole blood circuit systemProtein #27, < >>Protein #29Platelet count (PLT (109/L)) for protein # 31. All samples were prepared as described in example x and measured by a hematology analyzer Sysmex XN-L350. The technical function and reagent system of the instrument was monitored with XN-CHECK level 1 and level 2 prior to each run. XN-CHECK is a control blood specifically designed for an analyzer that allows for efficient and reliable internal and external quality control of the instrument. XN-CHECK level 2 covers the normal range of hematological parameters, while XN-CHECK level 1 is used for the abnormally low range of hematological parameters. The PLT count is measured as a means of checking that no micro-clot is formed, which may be a marker of cell/blood activation. In addition, the presence of blood clots was visually assessed. If noted, the hemolysis is visually classified.

Platelet count

The donor exhibited a normal PLT range (i.e., a range of values between 150-400 x 109 cells/L) at time point 0 (fig. 14). PLT count reduction at time 0>20% are classified as platelet aggregation (threshold determined based on measurement reproducibility at the 4 hour time point under the same conditions. Threshold at time points exceeding 4 hours has not yet been determined). Macroscopic blood clots (. Gtoreq.1 mm) were observed in the loop incubated with 0.005nM (at 24 hours for D1 and D3), 0.1nM (at 8 hours for D1-D2, and at 24 hours for D1-D3) and 2nM (at 24 hours for D1-D3) of Fu Tuozhu monoclonal antibody. In 2nMIn the sample of protein #27 (24 hours for D1-D3), however at +.>Protein #29No macroscopic clot was observed in the protein #31 sample at any time point. Small micro-clots were observed in vehicle samples from D1 at 8 hours. PLT levels in the vehicle group were similar to the 0 time point at 2 hours and 4 hours (defined as level +.20% difference), whereas at 8 hours the PLT count for D1 was reduced by 35.0%, and at 24 hours the PLT for D1 was reduced by 37.9%, the PLT for D2 was reduced by 24.9%. Typically, fu Tuozhu mab induced a decrease in PLT counts with increasing concentration at 8 hours and 24 hours. +.f at the two highest concentrations (0.1 nM and 2 nM) >Protein #27 decreased PLT counts at two later time points, while in use +.>Protein #29 and->PLT counts in samples of protein #31 were similar to vehicle at all concentrations and time points.

In response to Fu Tuozhu mab, PLT counts were reduced by +.20% at 0.005nM, except for D1 and D2 at 24 hours (65.1% and 85.8% reduction compared to vehicle, respectively). At 0.1nM, PLT counts per donor were within 20% of vehicle samples at 2 and 4 hours, while PLT counts were reduced for all three donors at 8 and 24 hours, by 83.7% and 88.4%, respectively, on average. At 2nM, PLT counts were reduced in blood from all donors at two later time points>20% vehicle (average 75.1% decrease at 8 hours, and 88.9% decrease at 24 hours). The highest concentration of 2nM in blood from D1 (85.4% decrease at 8 hours, and 97.1% decrease at 24 hours), D2 (94.7% decrease at 24 hours), and D3 (90.4% decrease at 24 hours), at all three concentrationsVehicle-like with PLT count decrease of 20% or less at protein #27The product is obtained. At->Protein #29 and->At all three concentrations of protein #31, PLT counts for all three donors were within 20% of the vehicle samples at all four time points.

C. Effects of multispecific binding proteins on platelet count and leukocyte count

As in example 8, in the whole blood circuit system, the evaluation was responsive to Fu Tuozhu mab analogue at the 0 hour (pre-dose), 2 hour, 4 hour, 8 hour and 24 hour time points,Protein #58, < >>Protein #59,Protein #57, < >>Protein #60, < >>Protein #61 and->Platelet (PLT) and White Blood Cell (WBC) counts of protein #56 (10 ⁹ /L). All samples were prepared as described in example 8 and measured by a hematology analyzer Sysmex XN-L350. The technical function and reagent system of the instrument was monitored with XN-CHECK level 1 and level 2 prior to each run. XN-CHECK is a control blood specifically designed for an analyzer that allows for efficient and reliable internal and external quality control of the instrument. X is XN-CHECK level 2 covers the normal range of hematological parameters, while XN-CHECK level 1 is used for the abnormally low range of hematological parameters. The PLT count is measured as a means of checking that no micro-clot is formed, which may be a marker of cell/blood activation. In addition, the presence of blood clots was visually assessed.

Platelets in time point 0 and vehicle group

All donors exhibited a normal PLT range at time 0 (i.e.values in the range 150-400X 10 ⁹ Range between individual cells/L) (fig. 49). PLT levels in vehicle groups were similar to time 0 samples of all donors (defined as levels<20% difference). No macroscopic blood clots were observed in the fresh blood incubated with vehicle, but were detected in several test items at 8 hours and 24 hours (table 9B).

TABLE 9b extent of macroscopic blood clot

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Empty = no clot observed, 1 = small clot (< 2 mm), 2 = medium size clot (2 mm-5 mm), 3 = large clot (> 5 mm). At 2h, 4h and 8h, only a portion of the blood was sampled and clotting was possible even if not observed.

All donors exhibited a normal PLT range (i.e. a range of values between 150-400 x 109 cells/L) at time 0 (fig. 49). PLT levels in the vehicle group were similar to the 0 time point samples of all donors (defined as level <20% difference). No macroscopic blood clots were observed in fresh blood incubated with vehicle (PBS), but were detected in several test items at 8 hours and 24 hours (table 9 b).

All donorsIs within the normal range at time 0 (i.e., values of 4.0-11.0X10) ⁹ Within a range between individual cells/L). The change of + -10% of the sample at the 4 hour time point relative to the 0 time point was judged normal (fig. 50).

In summary, at all three concentrations (0.005 nM, 0.1nM and 2nM of test protein), PLT and WBC counts were similar to vehicle groups up to the 4h time point. After 4 hours, a concentration-dependent decrease in PLT and WBC counts over time was detected, typically compared to vehicle, for all test proteins in all donors. However, 0.005nM and 0.1nMProtein #56 and->Protein #60 did not induce a significant decrease in WBC counts. For some test proteins, blood clots were first detected at 8h, and all test proteins showed blood clots at different concentrations at 24 h. However, no blood clot was observed in the vehicle sample, indicating that clot formation is the effect of immune stimulation. For->No blood clot was detected by protein #74, which showed limited immune activity in the protein by this control.

Example 10: determination of the combined affinity of the multispecific binding proteins for two surface ligands (CD 33 and CD 123)

Designed ankyrin repeat proteins for the determination of exemplary multispecificProtein #20Protein #27 (binding specificity for CD3, CD33 and CD 123) and its corresponding single TAA target control (binding specificity for CD3 and CD123 +.>Protein #21 and +.sup.f with binding specificity for CD3 and CD33>Protein #22; a binding specificity for CD3 and CD123->Protein #41 and +.sup.f with binding specificity for CD3 and CD33>Affinity and avidity of protein # 40), one use was made ofResearch on the technology (Dynamic Biosensors). />The technique allows high sensitivity both in the detection affinity constant (with a detection limit of 10 fM) and in the binding kinetics (with detection limits of 1E3-1E8 1/Ms and 1E-6-1E 0/s for the binding rate and dissociation rate constants, respectively). (Langer et al; 2013.Protein analysis by time-resolved measurements with an electro-switchable DNA chip. Nat Commun 4:2099).

This technique makes it possible to monitor two independent signals from two interactions simultaneously and on the same sensor spot, using two different fluorophores. Thereby measuring affinity and avidity kinetic multispecific binders simultaneously for two target molecules.

Materials and methods

Designed ankyrin repeat proteins were tested at concentrations of 0.16nM and 64nM (in buffer PE140 containing 10mM Na2HPO4/NaH2PO4, 140mM NaCl, 0.05% Tween20, 50. Mu.M EDTA, 50. Mu.M EGTA):protein #20, < >>Protein #21, < >>Protein #22, < >>Protein #27, < >>Protein #40 and->Binding of protein #41 to two ligands hCD33 and hCD123 immobilized simultaneously to the surface of DNA nanoles at a predetermined ratio (2:1, CD33: CD 123) and 10% density (100:50) on an ADP-48-1-0 chip, wherein 100% density ≡1000 receptors/. Mu.m2 (pre-incubated R1-B48-CD123 and G1-A48-CD33 (100 nM) diluted in cNL-48 (dark)). Briefly, the following steps are performed: />

-conjugation. Two ligands (CD 33 and CD 123) were coupled to DNA nanoever (A48, B48 respectively) amine and were found to be at 25 ^℃ Is used at the placeThe conjugate was purified from the solution (Dynamic Biosensors GmbH).

-evaluating all combined affinities and avidities of all different proteins for both ligands attached to the ADP chip surface using a drx+ instrument (dynamic BIOSENSORS).

Statistical analysis (repeated 2 to 5 times) comprising regeneration and long dissociation times of the chip surface for each analysis concentration, and fitting the data using a double-exponential global fit.

TABLE 6

Results and conclusions

Multi-specific recombinant proteinsProtein #20 shows biphasic dissociation, representing two interactions on the chip: 1E ^-2 s ^-1 Affinity of the order (faster dissociation rate) and 1E ^-3 s ^-1 Affinity of the stage (slower off-rate, where the analyte interacts with both ligands). In addition, with CD33 (1E ⁶ M ^-1 s ^-1 ) In contrast, the binding rate to CD123 seems to be slightly higher (1E ⁷ M ^-1 s ^-1 ). For->Protein #21 and->Protein #22 controls both, only binding interactions with one of the targets were observed. For example, as expected, +.>Protein #21 interacts only with CD123 because it does not have a CD33 subunit. Thus, it shows k _off 6E of ^-2 s ^-1 Is a single phase combination and dissociation of (a). No observation was madeInteraction between protein #21 and CD 33. Similarly, as expected, +.>Protein #22 interacted only with CD33 because it did not have a CD123 subunit. Thus, it shows k _off 1E of ^-2 s ^-1 Is a single phase combination and dissociation of (a). No +.>Interaction between protein #22 and CD 123.

In summary, designed ankyrin repeat proteins (αcd33- αcd 123) comprising both a CD33 specific binding domain and a CD123 specific binding domain are shown to bind simultaneously to two targets attached to the chip surface and they show affinity and avidity, whereas designed ankyrin repeat proteins with CD33 specific binding domain or CD123 specific binding domain show only affinity for CD33 and affinity for CD123, respectively. In fact, only for Protein #20, a biphasic dissociation was observed representing two interactions (populations) on the chip: affinity (2E) ^-2 s ^-1 Faster dissociation rate) and affinity (1E ^-3 s ^-1 Slower dissociation rate).

As can be seen in fig. 38, the CD123 binding domain shows a faster binding rate to its target than the CD33 binding domain, fig. 37 (for CD123, k _on For 7E ⁶ M ^-1 s ^-1 Which is the k of CD33 _on (equal to 1.3E ⁶ M ^-1 s ^-1 ) About 5 times). For the followingProtein #20, two dissociation rates were observed: rapid (3E) ^-2 s ^-1 ) Which is associated with affinity binding, a slower (1.5E ^-3 s ^-1 ) It has an affinity action. Thus, +.>Two K of protein #20 _D ：K _D,1 It is higher and associated with affinity binding, about 17.6nM for CD33 and 4.9nM for CD 123. K is as follows _D,2 Which is low and associated with affinity (binding both CD33 and CD 123), in the pM range (fig. 39 and fig. 40).

Multi-specific recombinant proteinsProtein #27 shows biphasic dissociation, representing two interactions on the chip: 1E ^-2 s ^-1 Affinity of the order (faster dissociation rate) and 1E ^-3 s ^-1 Affinity of the stage (slower off-rate, where the analyte interacts with both ligands). In addition, with CD33 (1E ⁶ M ^-1 s ^-1 ) In contrast, the binding rate to CD123 seems to be slightly higher (1E ⁷ M ^-1 s ^-1 ). For->Protein #40 and->Protein #41 controls both, only binding interactions with one of the targets were observed. For example, as expected, +.>Protein #15 interacted only with CD123 because it did not have a CD33 subunit. Thus, it shows k _off 6E of ^-2 s ^-1 Is a single phase combination and dissociation of (a). No observation was madeInteraction between protein #41 and CD 33. Similarly, as expected, +.>Protein #40 interacted only with CD33 because it did not have a CD123 subunit. Thus, it shows k _off 1E of ^-2 s ^-1 Is a single phase combination and dissociation of (a). No +.>Between protein #40 and CD123Is described in (a) and (b) interact with each other. (FIGS. 15 and 16)

In summary, it is shown that both a CD33 specific binding domain and a CD123 specific binding domain are comprisedProtein #27 (αcd33- αcd 123) can bind to both targets attached to the chip surface simultaneously and they show affinity and avidity, whereas the engineered ankyrin repeat proteins with CD 33-specific binding domains or CD 123-specific binding domains show only affinity for CD33 or affinity for CD123, respectively. Thus, it can be determined thatTwo K of protein #27 _D ：K _D,1 It is higher and associated with affinity binding, about 20nM for CD33 and 4.5nM for CD 123. And K was determined in the range of 0.2nM to 0.3nM _D,2 Which is low and associated with affinity (binding to both CD33 and CD 123) (fig. 17 and 18), is at least 1/14 of the measured affinity.

Example 11: target-specific short-term T cell activation and ifnγ secretion were assessed.

The specificity and efficacy of the aforementioned recombinant multi-specific ankyrin repeat proteins were assessed in an in vitro short-term T cell activation assay by FACS measuring the CD25 activation marker on cd8+ T cells and by ELISA measuring ifnγ secretion.

Thus, with the selectionSerial dilutions of protein or control reference molecule 100,000 purified pan-T effector cells and 100,000 Molm-13 target cells per well were co-incubated (E: T ratio 1:1) at 37 ℃ in duplicate in the presence of 600 μm human serum albumin for 24 hours. After 24 hours, 100 μl of supernatant per well was transferred to measure ifnγ secretion by the human ifnγ standard ABTS ELISA development kit (PeproTech) according to the manufacturer's protocol. Cells were washed and anti-mouse with 1:1'000live/Dead Aqua (Thermo Fisher), 1:250Human CD8 Pasific Blue (BD) and 1:100 mouse anti-human CD25 PerCP-Cy5.5 (eBiosciences) antibodies were stained at 4℃for 30 minutes. After washing and fixation, cells were analyzed on a FACS Canto II (BD) machine. T cell activation was assessed by measuring cd25+ cells on Live/Dead negative and cd8+ gated T cells. FACS data were analyzed using FlowJo software and plotted using GraphPad Prism 8.

As shown in fig. 23A and 24A, for the case without half-life extensionProteins, #8 and #9 induced specific short-term T-cell activation comparable to the reference molecule (known reference T-cell conjugate: AMG330 with binding specificity for CD 33), however>Protein #7 and->Protein #10 showed efficacy reduced to 1/10 to 1/100. Half-life extended +.>Proteins 11-14 (see FIGS. 23B and 24B) showed efficacy reduced to about 1/3 to 1/100.

Example 12: evaluation of target-specific long-term tumor cell killing by intucyte

The specificity and efficacy of the recombinant multi-specific ankyrin repeat proteins described above were assessed by in vitro long term killing assay using the IncuCyte S3 platform.

The Molm-13 cells were first transduced with NucLight Red (NLR) lentiviral particles (Sartorius) and Red fluorescent cells were selected by 0.7 μg/ml puromycin and/or FACS sorting. Long term tumor cell killing was then assessed using the IncuCyte S3 system (Sartorius). Effector cells and target cells were co-incubated in duplicate on 0.01% poly-L-ornithine coated 96-well plates at a 5:1 E:1T ratio in the presence of 1:200 annexin V green (Sartorius) and 600. Mu.M human serum albumin (at simulated physiological concentrations). Serial dilutions of ankyrin repeat proteins or control reference molecules selected from 50,000 purified (Miltenyi) pan-T cells per well (isolated from healthy donor PBMC) and 10,000 Molm-13 NLR cells were used for up to 6 days at 37 ℃. Images were taken every 2 hours to assess cell proliferation (red fluorescence from NLR) and cell death (green fluorescence from annexin V). Total cell proliferation and tumor cell killing were analyzed by calculating the area under the curve after 6 days of co-culture using GraphPad Prism 8.

As shown in fig. 25, testedProteins #8, #9, #10 showed potent and specific tumor cell killing comparable to the reference molecules (known reference T cell conjugates: AMG330 with binding specificity to CD33 and Fu Tuozhu mab with binding specificity to CD 123), independent of half-life extension. With only low affinityProtein #7 showed a decrease in killing efficacy.

Example 13: assessment of target-specific long-term T cell activation and proliferation by FACS

The specificity and potency of recombinant multi-specific ankyrin repeat proteins were assessed by FACS-based in vitro long-term T cell activation assays (described in examples 5, 6, 7 and 8).

To assess drug-specific and target-specific T cell activation and proliferation, effector and target cells were co-incubated in duplicate at a 1:1 ratio of E to T in the presence of serial dilutions of the selected molecules. Purified (Miltenyi) pan-T cells (isolated from healthy donor PBMC) were first labeled with 5 μ M CellTrace Violet (CTV) (Thermo Fischer) for 20min at 37 ℃. 50,000 CTV-labeled pan-T cells and 50,000 Molm-13 cells per well were then incubated with serial dilutions of selected CD 3-specific ankyrin repeat proteins or control reference molecules TCE1 and TCE2 in the presence of 600. Mu.M human serum albumin (at a simulated physiological concentration) at 37 ℃. After 5 days, cells were washed with PBS and stained with 1:5'000 live/read Green (Thermo Fisher), 1:100 mouse anti-human CD8 PE (BD) and 1:100 mouse anti-human CD25 PerCP-Cy5.5 (eBiosciences) antibodies for 30 minutes at 4 ℃. After washing 2 times with PBS, cells were fixed for 20min at 4 ℃ using CellFIX (BD), and finally buffer was replaced with PBS. Stained cells were analyzed on a FACS Canto II (BD) machine. T cell activation was assessed by measuring cd25+ cells on Live/Dead negative and cd8+ gated T cells. T cell proliferation was assessed by gating Live/Dead negative and CTV positive cells. FACS data was analyzed using FlowJo software; data were plotted using GraphPad Prism 8.

As shown in fig. 26 and 27, for the case without half-life extension#8 and #9 induced long-term T cell activation and proliferation comparable to the reference molecule (known reference T cell conjugates: AMG330 with binding specificity for CD33 and Fu Tuozhu mab with binding specificity for CD 123), however->Proteins #7 and #10 showed efficacy reduced to 1/10 to 1/100. Half-life extended +.>Protein #11-14 showed efficacy reduced to about 1/5 to 1/30.

Example 14: exemplary multispecific binding proteins in PBMC humanized mice and MOLM-13 tumor models Evaluation of in vivo efficacy

Experiment A four different designed ankyrin repeat proteins with binding specificity for CD33, CD123 and CD3 were tested in a Peripheral Blood Mononuclear Cell (PBMC) humanized mouse model carrying the tumor cell line MOLM-13 as a solid subcutaneous tumorProtein #8, < >>Protein #12, < >>Protein #13 and->Protein #14 and comparing them to the known CD 33-targeted T cell conjugate AMG330 currently tested in clinical trials.

In addition, in the case of the optical fiber,protein #12, < >>Protein #13 and->Protein #14 also included as half-life extending moieties a designed ankyrin repeat domain with binding specificity for human serum albumin. Protein #8 is->A non-half-life extended form of protein # 12.

Materials and methods

Animals:60 female NOG mice, 65 days old (provider Taconic Biosciences) at the beginning of the study

Test and control molecules: proteins#8、/>Protein #12, < >>Protein #13 and->Protein #14 was produced at a concentration of 5mg/ml as previously described; control AMG330 0.59mg/ml: provided by Evitria AG

Treatment group:60 mice were enrolled in this study. All animals were randomly assigned to 8 different study groups. The date of tumor cell inoculation is indicated as day 0.

The method comprises the following steps:body weights were recorded two days before the start of the experiment, and mice were randomized to have equal average weights and similar standard deviations in each group. The average weight was 19.6g.

On day-2, 5X 10 was used ⁶ Mice were injected intraperitoneally with PBMCs.

On day 0, 10 is applied to the right flank ⁶ Mice were subcutaneously injected with MOLM-13 cells.

On day 5, treatment was started with intraperitoneal injection according to table 8. The last treatment was on day 16.

Tumor measurements and weighing were performed on days 7, 10. Tumor measurements and weighing were performed on day 12, day 14, day 17, day 19. Tumor volumes were calculated according to the following formula: [ Length× (width) ² ×π]/6。

Tumor volume data were analyzed by Anova comparison of growth curves and following the nonparametric Kruskal-Wallis test (Dunn test) corrected for multiple comparisons.

The tumor volume of the treated group was compared to the volume of the control group. Data from two different PBMC donors were analyzed together and separately.

Table 7: treatment group assignment and treatment regimen

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Results

Tumor growth inhibition

Tumor growth curves after exposure to the tested multispecific binding proteins and known reference T cell conjugates are summarized in fig. 25A-25C, including two donors together and separately. Fig. 26A-26L show individual growth curves for all mice.

Table 8 (two donors), table 9 (donor a) and table 10 (donor B): statistics of tumor growth

+=p <0.1; * = p <0.05; ++ + =p <. 0-01; ++ + +=p <0.001; ns = insignificant

PBMCs from donor B resulted in lower amounts of CD8 positive lymphocytes in the mouse blood. Generally, an antitumor effect was observed in all test molecules. In contrast to this, the process is performed,protein #12 and AMG330 showed effects starting 5 days after the start of treatment, however +.>Protein #13 and->The effect of protein #14 was significant and relevant starting 7 days after the start of treatment. When only subgroup a was compared, a statistically significant effect was observed for all test molecules starting from day 5 after the start of treatment. / >Protein #12, < >>Protein #13 and->Protein #14 has a stronger effect than AMG330 and +.>Effect of protein # 8. When comparing subgroup B, for +.>A significant anti-tumor effect was observed for protein #13 and baseline AMG 330.

The results obtained with the benchmarks verify the model used. At the same time, the less pronounced effect of the baseline observed in PBMC humanized mice from donor B suggests that mice belonging to this subgroup are less sensitive. This is also reflected by the significantly lower amount of human CD8 positive lymphocytes found at the end of the experiment in PBMC humanized mice from donor B.

Thus, more relevance is due to the data generated with PBMC humanized mice from donor a.

The high variability in tumor size is due to the fact that randomization and range selection is not performed in mice, as treatment is initiated before tumors can be identified in all mice. Despite this high variability, statistical analysis can be performed without the need to normalize the initial tumor volume.

We demonstrate that all the multi-specific binding proteins tested show anti-tumor activity and that half-life extended molecules Protein #12, < >>Protein #13 and->Protein #14 is ++increased over non-half-life at the dose and administration regimen used>Protein #8 has greater antitumor activity. Without wishing to be bound by theory, it is believed that due to the shorter half-life of this molecule, the +_s>The exposure of protein #8 may be lower.

In summary, all four of the tested multispecific binding proteins showed in vivo antitumor activity comparable to or greater than the baseline AMG 330T cell conjugate.

Experiment B in a similar experimental setup and procedure as experiment A, additional designed ankyrin repeat proteins with binding specificity for CD70, CD33, CD123 and CD3 were tested in a Peripheral Blood Mononuclear Cell (PBMC) humanized mouse model carrying the tumor cell line MOLM-13 as a solid subcutaneous tumor, i.eProtein #56 and compared to AMG 330.

Briefly, in vivo experiments were performed in six 9 week old female immunodeficiency NXG mice (supplied by Janvier Labs). Mice were maintained in standard rodent mini-spacer cages (20+/-1 ℃ room temperature, 50+/-10% relative humidity and 12 hours light dark cycle) under standard environmental conditions. Mice received irradiated food and bedding and 0.22um filtered drinking water. All experiments were performed in accordance with swiss animal protection laws (Swiss Animal Protection Law) and were authorized by state and federal veterinary authorities.

Mice were injected intraperitoneally with hPBMC and then xenografted with cancer cells as in experiment a. MOLM-13 cells were xenografted subcutaneously (s.c.) into mice in the right flank region. Two hPBMC donors were used. Intravenous injection (i.v.) treatment was started four days after cancer cell implantation. The treatment was administered as follows:

-will beProtein #56 was administered by intravenous injection three times per week at 0.5mg/kg for 2 weeks

AMG330 was administered at 0.5mg/kg by daily intravenous injection for 2 weeks

Tumor size was assessed by caliper measurements. Tumor volume was calculated using the formula tumor volume (mm ³ ) =0.5 x length x width ² 。

As can be seen in fig. 58 (a-B), during the whole experimental time (fig. 58A) and 17 days after the first injection (fig. 58B),protein #56 shows good efficacy in inhibiting tumor growth and tumor volume.

Experiment C two designed ankyrin repeat proteins with binding specificity for CD70, CD33, CD123 and CD3 were tested in a Peripheral Blood Mononuclear Cell (PBMC) humanized mouse model carrying the tumor cell line MOLM-13 as a solid subcutaneous tumor in an experimental setup and procedure similar to experiment A and experiment B, namelyProtein #56Protein #57 and monoclonal antibodies to AMG330 and Fu Tuozhu as positive controls and as negative controls Protein #74 (CD 3 specific binding protein) and +.>Protein #75 (CD 33/CD70/CD123 specific binding protein) was compared.

As in experiment B, NXG mice were injected intraperitoneally with hPBMC and then xenografted with cancer cells. MOLM-13 cells were xenografted subcutaneously (s.c.) in the right flank region into NXG mice. Two to six hPBMC donors were used depending on the treatment. Intravenous injection (i.v.) treatment (therapeutic treatment) was started 8 days after cancer cell implantation. All tumors were established at this time point. The average tumor volume was about 150mm ³ 。

Figure 65 shows the effect of test proteins on tumor volume over time after treatment. It can be seen that the two tested multispecific proteinsProtein #56 and->Protein #57 provides effective inhibition of tumor growth. FIG. 66 shows the +.>Effect of protein #56 (0.02 mg/kg to 2 mg/kg) on tumor growth. Tumor growth inhibition with the administration +.>An increase in the amount of protein #56 becomes more potent.

The treatment was administered as follows:

intravenous administration three times per weekProtein #56, < >>Protein #74, < >>Protein #75 and->Protein #57, for 2 weeks. All molecules were administered at 0.5 mg/kg. />Protein #56 was also tested at several other doses (0.02 mg/kg, 0.2mg/kg and 2 mg/kg).

Fu Tuozhu mab analogue and AMG analogue were administered intravenously daily at 0.5mg/kg for 2 weeks.

In order to correlate in vivo efficacy with the mode of action of the multi-specific proteins tested, ex vivo assays were performed in parallel with in vivo models. To this end, NXG mice were injected intraperitoneally with hPBMC from the same donor and treated as described above. More specifically, T cell activation was assessed by FACS and by Luminex, respectively ^TM And Meso Scale Discovery (MSD) assays to determine cytokine/chemokine release in mouse serum and mouse tumor supernatants. Briefly, for T cell activation assessment, a portion of the mice were sacrificed three days after the first injection of test protein. By genetleMACS according to vendor's protocol ^TM Tumors were dissociated and FACS measurements were performed on cell suspensions. FIG. 67 shows the frequency of human immune cells (hCD45+) and activated T cells (CD4+/CD25+/CD69+ and CD8+/CD25+/CD69+) in dissociated MOLM-13 tumors. Data are expressed as mean and SEM (standard error of mean).

To evaluate cytokine and chemokine release in serum, luminex was performed in mouse serum from mouse blood ^TM The blood of the mice was collected four hours after the first injection of test protein. Luminex ^TM The assay is available from R&D Systems' human customized multiplex 4 bead array assay. More specifically, luminex is used according to manufacturer's recommendations ^TM MAGPIX instruments measure IFN-gamma, IL-6, IL-2 and TNF-alpha levels. All test samples were thawed on ice, centrifuged at 2000rpm for 3 minutes, and then diluted 1:2 in calibrator diluent. Each test sample was assayed in duplicate. In addition, four QC (quality control) samples were diluted 1:2 in duplicate in calibrator dilutions from different cytokine standard samples (S2, S3, S5, S6, i.e., IFN-gamma, IL-6, IL-2, and TNF-alpha) according to manufacturer' S instructions. Cytokine standards provided by the manufacturer were assayed in duplicate and used to calculate the concentrations of the test samples as well as QC samples. Cytokine levels were measured in serum samples taken 4 hours after the 1 st injection. FIG. 68 shows INFγ, IL-6, IL-2 and TNFα levels in mouse serum. Data are presented as mean and SEM.

In addition, three days after the first injection of test protein, mice were sacrificed and tumors were harvested. Using genetleMACS according to the vendor's protocol ^TM Dissociating the tumor. Cytokine levels in tumor supernatants were measured using the VPlex pro-inflammatory panel 1 (human) kit (K151A 9H-4) tailored for IFNγ, IL-2, IL-6 and TNFα of the Meso Scale Discovery (MSD) Multi-Spot assay system. The subsequent assay protocols are described in more detail in examples 18 and 19. FIG. 69 shows IFNγ and IL-6 levels in mouse tumor supernatants as examples.

In this experimental set-up of experiment C,protein #74 and->Protein #75 did not show any efficacy in vivo (fig. 65). These data indicate that both a T cell-engaging binding domain and a tumor-associated antigen-specific binding domain are present in the same molecule (e.g., at +.>Protein #56 and->Protein # 57) is necessary for induction of antitumor efficacy in vivo. Two tested T cell conjugates DARRin protein (/ -A)>Protein #56 and->Protein # 57) showed good antitumor efficacy in vivo (fig. 65). This antitumor efficacy was concentration-dependent (fig. 66). The in vivo antitumor activity of the TCE proteins of the present invention is believed to be due to the increased number of immune cells and T cell activation in tumor tissue (fig. 67). The TCE proteins of the present invention trigger cytokine/chemokine release in tumor tissue (fig. 69), but are much less or not significant in serum (fig. 68), consistent with the beneficial safety profile.

Example 15: target specific short term autologous and allogeneic in co-cultures with BMMC tumor cells from AML patients Assessment of allogeneic T cell activation and tumor cell killing。

Assessment in an in vitro short term T cell activation assay by FACS measuring CD25 activation markers on CD8+ T cells in the presence of AML patient cells and in an in vitro tumor cell killing assay by FACSSpecificity and potency of protein # 56.

Thus, 120,000 purified pan-T effector cells were labeled with CellTrace Violet (CTV) according to the manufacturer's protocol (Thermofisher) and they were used with 30,000 AML tumor cells (BMMC, M type 4) per well (E: T ratio 4:1) in the presence of 200. Mu.M human serum albuminSerial dilutions of protein #56 were co-cultured in duplicate at 37 ℃ for 48 hours. After 48 hours, cells were washed and washed with 1:3,000 live/read Green (Thermo Fisher), 1:400 mouse anti-human CD8 PacThe ific Blue (BD) and 1:100 mouse anti-human CD25PerCP-Cy5.5 (eBiosciences) antibodies were stained at 4℃for 30min. After washing and fixation, cells were analyzed on a FACS Canto II (BD) machine. Allogeneic T cell activation was assessed by measuring cd25+ cells positive for CTV on Live/read negative and cd8+ gated T cells. Autologous T cell activation was assessed by measuring cd25+ cells negative for CTV Violet staining on Live/read negative and cd8+ gated T cells. FACS data were analyzed using FlowJo software and plotted using GraphPad Prism 8 (3-PL-fit).

As shown in FIG. 53A, the test was for binding specificity to CD3, CD33, CD123 and CD70Protein #56 (thus binding to 3 tumor specific targets) induced potent and specific allogeneic T cell activation. In addition, up-regulation of CD25 markers on autologous T cells may be shown.

To assess drug-specific and target-specific tumor cell killing, inProtein #56, negative control->Effector cells and target cells were co-incubated in duplicate at a 4:1 E:T ratio in the presence of serial dilutions of protein #74, votuzumab and AMG330 analogs. Purified (Miltenyi) pan-T cells (isolated from healthy donor PBMC) were first labeled with Celltrace Violet (Thermo Fischer) according to the manufacturer's protocol. 120,000 CTV-labeled pan-T cells and 30,000 AML tumor cells (BMMC, type M4) per well were then incubated with serial dilutions of selected CD 3-specific ankyrin repeat proteins or control molecules in the presence of 200 μm human serum albumin (at a simulated physiological concentration) at 37 ℃. After 48 hours, cells were washed with PBS and stained with 1:3,000 live/read Green (Thermo Fisher) for 20min at 4 ℃. After washing and fixation, cells were analyzed on a FACS Canto II (BD) machine. By being negative for CTV, single, live Gating was performed on/head negative cells to assess tumor cell killing. FACS data was analyzed using FlowJo software; data were normalized to co-cultured-only background and plotted using GraphPad Prism 8 (3 PL-Fit).

As shown in figure 53B of the drawings,protein #56 showed potent and specific tumor cell killing of AML patient-derived cells with efficacy between the comparative AMG330 analog and the votuzumab analog. The negative control resulted in little tumor cell killing.

Example 16: effect of multispecific binding proteins in autologous killing of ex vivo AML cells

Effect of selected multispecific binding proteins in autologous killing of ex vivo AML cells ex vivo personalized drug testing in a native environment provided by Vivia Biotech uses proprietary automated flow cytometry-based techniques (Pharmaflow ^TM ) To evaluate for complete pharmacological characterization of the drug in an ex vivo patient sample. Furthermore, vivia can simplify clinical development by: generating predictive in vitro natural environment assays and identifying reliable and robust predictive biomarkers in parallel with drug profiling and characterization provides predictive data for high levels of reliable, clinically viable compounds, and compound activity.

The purpose of this study was:

1. cell surface expression of targets of molecules (CD 123, CD33 and CD 70) was assessed at baseline by flow cytometry.

2. The in vitro pharmacological activity of the different test proteins with Fu Tuozhu mab as a single agent was performed in a natural environmental immunooncology assay of Vivia.

3. The supernatant, conditions and time points of each well were saved for post-assay measurement of soluble cytokines.

4. Assessment of different cytokine levels in the stored supernatants was performed.

The experimental design was performed as follows:

bead quantification of molecular target expression (CD 123, CD33 and CD 70) on pathological cells in triplicate at baseline.

-simultaneously measuring depletion and activation on pathological cells and T cells by a dose response curve:

8 different concentrations (50 nM, 10nM, 2nM, 0.4nM, 0.08nM, 0.016nM, 0.0032nM and 0.00064 nM) of test proteinProtein #56 (SEQ ID NO: 95), ->Protein #57 (SEQ ID NO: 96),Protein #58 (SEQ ID NO: 97), ->Protein #59 (SEQ ID NO: 98), ->Protein #60 (SEQ ID NO: 99) and +.>Protein #61 (SEQ ID NO: 100)

-8 different concentrations of one reference molecule (Fu Tuozhu mab (MPEXT 118)) in monotherapy (as described above).

1 high concentration (in triplicate)Protein #74 served as a negative control.

1 negative control (in triplicate) (negative control means no compound, the wells contain only solvent).

-2 incubation time points (72 h and 120 h).

The supernatant is saved for future measurement of soluble cytokines:

after each incubation time (72 h and 120 h), the supernatants from all experimental conditions were stored at-80 ℃.

-measuring different cytokine levels in the supernatant.

Sample collection

Vivia 5 frozen bone marrow samples from adult AML patients co-expressing CD123 and CD33 were used for this program (sample ID:13043, 13045, 13271, 13272, 15131). Samples were taken at the corresponding hospital center after clinical practice at the center to Vacutainer containing heparin as anticoagulant within the patient's routine treatment regimen ^TM In the tube. A portion of the samples were sent to the Vivia Biotech laboratory under the ethical committee approved study protocol and signed patient informed consent.

A small portion of the sample was stained with a specific monoclonal antibody (MoAb) to identify pathological cells and cell viability. Once the number of living cells was established and considered sufficient for analysis, a second staining was performed in a different portion of the sample to determine target protein (CD 123, CD33, CD 70) expression compared to isotype control. The expression of the target was quantified using the QuantiBRITE PE kit (Becton Dickinson) following the manufacturer's recommendations. All 5 samples showed the expression of CD33 and CD123, CD33 being the strongest TAA expressed. CD70 expression was below the detection limit of the kit.

For the killing assay, each sample was diluted to a final volume of 60 μl per well in an appropriate volume of IMDM/L-glutamine 4mM supplemented with 20% (v/v) FBS and 1% antibiotic ("Vivia medium"). The mixture was dispensed into 96-well plates containing the compounds previously prepared using an automated Echo 550 liquid processor. The plates containing the samples were incubated in humid air containing 5% CO2 at 37℃for 72h and 120h. At the final endpoint, cells were stained with mAb plus annexin V to better differentiate pathological cells from baseline characterization to measure cell death, and with CD25 to measure T cell activation. Finally, the plates were analyzed on the PharmaFlow platform of Vivia. The killing fit curve is shown on figure 54B as the median of the single donor fits. T cell activation is shown in% of CD25 positive T cells in figure 54A. Both killing and T cell activation data showed that all the tested proteins could induce killing and T cell activation of AML cells in an autologous environment.

Example 17: effect of multispecific binding proteins on killing of CD34+ sorted LSCs and HSCs

To test the efficacy and selectivity of selected multispecific binding proteins in killing Leukemia Stem Cells (LSCs) and Hematopoietic Stem Cells (HSCs), FACS sorting of cd34+ cells was performed on frozen AML (peripheral blood and/or bone marrow) and healthy donor bone marrow samples.

At the beginning of the experiment (day 0), a portion of cd34+ sorted cells were stained (CD 70PE, CD123 BV421 and CD33 APC) and measured by flow cytometry (BD LSR Forttessa Analyzer) to quantify target expression by calculating delta Median Fluorescence Intensity (MFI) relative to isotype control. Figure 55 shows median expression, expressed as Δmfi, in different LSC and HSC samples.

For the LSC/HSC killing assay, sorted cd34+ AML LSCs or healthy donor HSCs were first combined with allogeneic T cells (isolated from buffy coats of healthy donors) at a 1:1 ratio in 96-well U-bottom plates at 100pM, 10pM, or 1pMProtein #56, < >>Protein #57, < >>Protein #58, < >>Protein #59, < >>Protein #60 and->Protein #61 or reference molecule (Fu Tuozhu mab analog and AMG330 analog) were co-cultured for 4 days. The StemSpan medium used also contained a cytokine mixture (SCF, IL-6, IL-3, flt 3) and 10. Mu.M Human Serum Albumin (HSA). After 4 days of incubation, cells were resuspended and transferred to semi-solid methylcellulose medium to check colony forming ability after an additional 2 weeks of incubation. Finally colony counts were normalized to untreated control (=100), i.e. no killing. FIG. 56 shows the use of +. >Protein #56Protein #57 preferentially kills LSCs (solid bars), confirming that there is a window of opportunity between LSCs and HSCs. Furthermore, two reference compounds (Fu Tuozhu mab analog and AMG330 analog) were included to show the difference in specificity of the selected DARPin protein compared to the two molecules. Figure 56 shows that both reference molecules are effective against LSC and that trastuzumab kills HSCs equally well. AMG330 also shows some windows, but is effective at killing HSCs at low pM concentrations. Will->Protein #76, a non-TAA-bound but CD 3-bound protein, was used as a negative control. Will->Protein #78, a CD70-CD3 specific binding protein, was used as a positive control, also demonstrating that killing by targeting only CD70 was possible (and specific for LSC) even though CD70 expression was very low.

Example 18: shadow of multispecific binding proteins on killing AML Molm-13 cells and inducing cytokine release Sound box

Evaluation by FACS-based in vitro tumor cell killing assay in the presence of AML cell line Molm-13Protein #56, < >>Protein #57, < >>Protein #59 and->Specificity and potency of protein #62 and reference molecules (Fu Tuozhu mab analogs and AMG330 analogs). To assess drug-specific and target-specific tumor cell killing, effector cells and target cells were co-incubated in duplicate at a 5:1 E:T ratio in the presence of serial dilutions of the selected molecules. Purified (Miltenyi) pan-T cells (isolated from healthy donor PBMC) were first labeled with CellTrace Violet (Thermo Fischer) according to the manufacturer's protocol. 100,000 CTV-labeled pan-T cells and 20,000 Molm-13 cells per well were then incubated with serial dilutions of the selected multispecific protein or control molecule in the presence of 20 μm human serum albumin (to provide saturation of the HSA binding domain) at 37 ℃. After 48h, the supernatant was collected and stored at-80 ℃ for cytokine measurement, and the cells were washed with PBS and stained with 1:3,000 live/read Green (Thermo Fisher) for 20min at 4 ℃. After washing and fixation, cells were analyzed on a FACS Canto II (BD) machine. Tumor cell killing was assessed by gating CTV negative, single, live/read negative cells. FACS data was analyzed using FlowJo software; data were normalized to co-cultured-only background and plotted using GraphPad Prism 8 (3 PL-Fit).

Cytokine levels in supernatants were measured using the VPlex pro-inflammatory panel 1 (human) kit (K151A 9H-4) tailored for IFNγ, IL-2, IL-6 and TNFα of the Meso Scale Discovery (MSD) Multi-Spot assay system. Using detection antibodies labeled with MSD Sulfo-TAG, the MSD instrument uses the Electrochemiluminescent (ECL) signal generated in the wells of the MSD plate to provide a quantitative measurement of the analytes present in each sample. The signal is reported as the actual photon count from each measured 'spot' layered on the working electrode. Experiments were performed according to the manufacturer's instructions. Briefly, all supernatants were thawed at Room Temperature (RT) and diluted 5-fold in diluent 2 before addition to the MSD plates. The lyophilized calibrator blends were reconstituted in diluent 2 (4 times higher than standard protocol) and a series of 5-fold dilutions were performed to obtain 7 calibrators and 8 th zero calibrator (diluent 2). The SulfoTag antibodies for each of the 4 analytes were combined by 50-fold dilution in diluent 3. The 2X read buffer was prepared by diluting 4X read buffer T2-fold with deionized water. The wash buffer used to wash the MSD plates was PBS+0.05% Tween-20. The plate was washed 3 times with 150 μl/well of wash buffer, followed by 50 μl of sample/calibrator per well, and incubated at room temperature for 2h on an orbital shaker at 700 rpm. Followed by washing 3 times with 150. Mu.L/well of wash buffer, followed by addition of 25. Mu.L/well of detection antibody solution. The antibody was incubated for 2h at room temperature on an orbital shaker at 700rpm, followed by washing 3 times with 150. Mu.L/well of wash buffer. After the last wash, 150 μl/well of 2X read buffer T was added and the plate was read using MSD MESO QuickPlex SQ. All data files were analyzed using MSD discovery bench software by assigning assay analytes, plate layout and dilutions to each plate measured. The software provides the calculated concentration in pg/mL by reverse fitting to a 4-parameter logistic fit of the calibration curve for all 4 analytes from each plate (batch specific). The output values were plotted as a non-linear regression plot using GraphPad Prism.

As shown in FIG. 57 (A-H), the proteins were testedProtein #56, < >>Protein #57,Protein #59/>Protein #62 showed effective killing of Molm-13 cells (EC 50Protein # 56.4 pM,/i>Protein # 57.0 pM,/l>Protein # 59.1.4pM,Protein # 62.9 pM, votuzumab 0.4pM, AMG330 0.5 pM). Quantification of IFNγ in the supernatant also shows +.>Protein #56 showed the best safety profile, inducing the lowest ifnγ release.

Example 19: multispecific binding proteins are useful for killing patient-derived AML cells and inducing cytokine release Influence of。

Evaluation by FACS-based ex vivo tumor cell killing assay in the presence of AML donor patient cellsProtein #56 and->Specificity and potency of protein #57 as a reference molecule for positive control (Fu Tuozhu mab analogue and AMG330 analogue). To assess drug-specific and target-specific tumor cell killing, AML patient-derived cells were incubated in duplicate and in the presence of serial dilutions of selected molecules alone (autologous setting) or with Pan-T cells from healthy donorsCo-incubations (allogeneic setting) were performed together. In an allogeneic setting, an effector cell (E) (i.e., T cell) to target cell (T) (i.e., AML cell) ratio of about 4:1 is achieved. In autologous settings, the E:T ratio is much lower due to the limited number of T cells in the AML donor patient cell sample.

For autologous setting, 25,000 AML patient cells per well were incubated with serial dilutions of selected CD 3-specific DARPin compounds or control molecules in the presence of 20 μm human serum albumin (to provide saturation of the HSA binding domain) and 40ng/ml of each cytokine (IL-6, GM-CSF, SCF, TPO, flt3L, G-CSF) at 37 ℃. After 48h and 120h, supernatants were collected and stored at-80℃for cytokine measurement, and cells were washed with PBS and stained with 1:3000 live/read Green (Thermo Fisher) for 20min at 4 ℃. After washing and fixation, cells were analyzed on a FACS Canto II (BD) machine. Tumor cell killing was assessed by gating on individual Live/read negative cells. FACS data was analyzed using FlowJo software; data were normalized to co-cultured-only background and plotted using GraphPad Prism 8 (3 PL-Fit).

For allogeneic setting, purified (Miltenyi) pan-T cells (isolated from healthy donor PBMC) were first labeled with CellTrace Violet (available from Thermo Fischer) according to the manufacturer's protocol. Then, 100,000 CTV-labeled pan-T cells and 25,000 AML patient cells per well were incubated with serial dilutions of selected CD 3-specific DARPin compounds or control molecules in the presence of 20 μm human serum albumin (to provide saturation of the HSA binding domain) and 40ng/ml of each cytokine (IL-6, GM-CSF, SCF, TPO, flt3L, G-CSF) at 37 ℃. After 48h, the supernatant was collected and stored at-80 ℃ for cytokine measurement, and the cells were washed with PBS and stained with 1:3000 live/read Green (available from Thermo Fisher) for 20min at 4 ℃. After washing and fixation, cells were analyzed on a FACS Canto II (BD) machine. Tumor cell killing was assessed by gating CTV negative, single, live/read negative cells. FACS data was analyzed using FlowJo software; data were normalized to co-cultured-only background and plotted using GraphPad Prism 8 (3 PL-Fit).

Cytokine levels in supernatants were measured using the VPlex pro-inflammatory panel 1 (human) kit (K151A 9H-4) tailored for IFNγ, IL-2 and TNF α of the Meso Scale Discovery (MSD) Multi-Spot assay system. Using detection antibodies labeled with MSD Sulfo-TAG, the MSD instrument uses the Electrochemiluminescent (ECL) signal generated in the wells of the MSD plate to provide a quantitative measurement of the analytes present in each sample. The signal is reported as the actual photon count from each measured 'spot' layered on the working electrode. Experiments were performed according to the manufacturer's instructions. Briefly, all supernatants were thawed at Room Temperature (RT) and diluted 5-fold in diluent 2 before addition to the MSD plates. The lyophilized calibrator blends were reconstituted in diluent 2 (4 times higher than standard protocol) and a series of 5-fold dilutions were performed to obtain 7 calibrators and 8 th zero calibrator (diluent 2). The SulfoTag antibodies for each of the 4 analytes were combined by 50-fold dilution in diluent 3. The 2X read buffer was prepared by diluting 4X read buffer T2-fold with deionized water. The wash buffer used to wash the MSD plates was PBS+0.05% Tween-20. The plate was washed 3 times with 150 μl/well of wash buffer, followed by 50 μl of sample/calibrator per well, and incubated at room temperature for 2h on an orbital shaker at 700 rpm. Followed by washing 3 times with 150. Mu.L/well of wash buffer, followed by addition of 25. Mu.L/well of detection antibody solution. The antibody was incubated for 2h at room temperature on an orbital shaker at 700rpm, followed by washing 3 times with 150. Mu.L/well of wash buffer. After the last wash, 150 μl/well of 2X read buffer T was added and the plate was read using MSD MESO QuickPlex SQ. All data files were analyzed using MSD discovery bench software by assigning assay analytes, plate layout and dilutions to each plate measured. The software provides the calculated concentration in pg/mL by reverse fitting to a 4-parameter logistic fit of the calibration curve for all 4 analytes from each plate (batch specific). The output values were plotted as a non-linear regression plot using GraphPad Prism.

As shown in FIGS. 59 (A-L) and 60 (A-L), the proteins were testedProtein #56/>Protein #57 showed effective autogenous killing of AML patient cells in a concentration-dependent manner within 5 days (EC 50 value:protein #56:33 pM->Protein #57:2.9pM, fu Tuozhu mab analogue: 0.4pm, amg330 analog: 4 pM). Comparison of cytokine release induction with patient AML cell killing data indicates +.>Protein #56 is potentially the best safety profile at effective concentrations because it induces the lowest level of cytokines. Also, the process of the present invention is,protein #57 induced less cytokine than baseline votuzumab analogues.

Similarly, as can be seen in FIG. 61 (A-L), the proteins were testedProtein #56 and->Protein #57 also showed effective killing of tumor cells in the allogeneic setting (EC 50:/->Protein #56:103 pM->Protein #57:22.7pM, fu Tuozhu mab analogue: 0.6pm, amg330 analog: 4.1 pM). Also in this experimental setup, +.>Protein #56 and->Protein #57 induced less cytokine than baseline votuzumab analogues.

Example 20: cytokinin after incorporation of Molm-13 cells in whole blood in the presence of a multispecific binding protein Effect of sub-Release 。

Assessment using whole blood spiked with Molm13 cells from two healthy donorsProtein #56Cytokine release by protein # 57. 400,000 Molm13 cells were incubated with whole blood of healthy donors in the presence of serial dilutions of selected molecules. The number of Molm13 cells was selected to achieve an E:T ratio of 1:2 and to obtain a tumor burden of about 20% -25% in each well (since the average white blood cell count in 180 μl blood is close to 1,300,000 white blood cells, for a total of about 200,000T cells). Molm13 cells were incubated with whole blood of the healthy donor and molecules at 37℃for 30 minutes and 6 hours. Supernatants from each donor plate were collected at 2 different time points (30 min and 6 h) and stored at-80 ℃ until ready for cytokine measurement assays. No significant cytokine release was observed after 30min incubation time.

Cytokine levels in supernatants were measured using a VPlex pro-inflammatory panel 1 (human) kit (K151 A9H-4) tailored for ifnγ, IL-2, IL-6 and tnfα of the Meso Scale Discovery (MSD) Multi-Spot assay system following the same protocol as described in example 19.

As shown in FIG. 62 (A-H), quantitative analysis of IFNγ, IL-2, IL-6 and TNF. Alpha. In the supernatant revealed Protein #56 induced minimal release of the four tested cytokines in both donors, indicating +.>Protein #56 may have the best safety profile in the test molecule. Likewise, a->Protein #57 showed lower cytokine induction than the baseline molecule population.

Example 21: determination by Surface Plasmon Resonance (SPR) Protein #56 and CD33, CD123, Simultaneous binding of CD70, CD3 and serum albumin。

Evaluation by Surface Plasmon Resonance (SPR)Protein #56 binds to human CD33, human CD123, human CD70 and human CD3 targets simultaneously with human serum albumin.

In SierraSPR measurements were performed on an instrument (Bruker). PBS pH7.4 containing 0.005% Tween 20 was used as running buffer. 2600RU of 300nM Human Serum Albumin (HSA) was immobilized on an HCA sensor chip. Continuous administration of 1. Mu.M +.>Protein #56 (bound for 60s, dissociated for 0 s), 200nm hcd70 (bound for 40s, dissociated for 0 s) and 500nm hcd123 (bound for 120s, dissociated for 0 s) were used as independent analyte injection steps. Immediately thereafter, a double injection step was performed, injecting 150nM hCD33 (binding 150s, dissociation 0 s), followed by 1.35. Mu.M scCD3 (binding 150s, dissociation 180 s). More specifically, the- >Protein #56 injection resulted in a 750RU response. Then, hCD70 of 300RU was combined with +.>Protein #56 binds. Subsequently, hCD123 was injected, and can be shown to be associated with an increase of 700RUBinding of protein # 56. Using double injection, hCD33 binding produced another 800RU response, followed by subsequent binding of scCD3 at the cessation of hCD33 injection, further increasing by 150RU. Notably, a decrease in RU during the hCD3 injection phase indicates that hCD33 is dissociating during the scCD3 injection phase.

Only whenThis arrangement allows binding of hCD70, hCD123, hCD33 and scCD3 when protein #56 has bound to HSA. The requirement of the arrangement is->Protein #56 binds HSA with high affinity and hCD70/hCD123 with high affinity to prevent rapid loss of signal before the final target is applied. To overcome the faster off-rates of hCD33 and scCD3, dual injections were used for these targets to shorten the time between the cessation of hCD33 injection and the onset of scCD3 injection. Briefly, dual injections are performed by separating two target solutions within one syringe by air bubbles. Furthermore, as described in the injection protocol in table 11, a single injection control run was performed by injecting no target (PBST) or only one of the targets (analyte 2-analyte 5). The signal is referenced to an empty control point on the same channel. All steps were performed at a flow rate of 10. Mu.l/min, except for a double injection at 20. Mu.l/min.

FIG. 63 showsSPR trace of simultaneous binding of protein #56 to CD70, CD123, CD33 and CD 3.Simultaneous binding of protein #56 to CD70, CD123, CD33 and CD3 occurs at +.>Protein # 56. These findings indicate +.>Protein #56 is able to bind all five of its targets simultaneously.

Table 11: injection protocol for SPR measurements

In addition, a theoretical sensorgram of simultaneous binding was calculated by summing the values of a single injection of all targets (by subtracting three timesProtein #56 single injection to achieve extra +.>Control of protein #56 addition). Superposition of the "measured" and "calculated" sensor patterns confirms +.>Protein #56 can bind all five of its targets simultaneously (fig. 64). FIG. 64 shows a comparison of "measured" and "calculated" SPR traces.

The present specification is to be understood most thoroughly in light of the teachings of the references cited within the present specification. Aspects within this specification provide an illustration of aspects of the invention and should not be construed as limiting the scope of the invention. The skilled artisan will readily recognize that the invention encompasses many other aspects. All publications, patents, and GenBank sequences cited in this disclosure are incorporated by reference in their entirety. To the extent that the material incorporated by reference contradicts or is inconsistent with the present specification, the present specification will supersede any such material. Citation of any reference herein is not an admission that such reference is prior art to the present invention.

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

Sequence listing

/>

Sequence listing

<110> MOLECULAR PARTNERS AG

<120> novel multispecific T cell conjugates based on DARPin

<130> P031

<160> 124

<170> BiSSAP 1.3.6

<210> 1

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> DARPin protein #1

<400> 1

Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Ser Gln Gly Trp Thr Pro Leu His Thr Ala Ala Gln Thr Gly His Leu

35 40 45

Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Asp Lys Gly Val Thr Pro Leu His Leu Ala Ala Ala Leu Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120

<210> 2

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> DARPin protein #2

<400> 2

Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asn

20 25 30

Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala Gln Thr Gly His Leu

35 40 45

Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Asp Lys Gly Val Thr Pro Leu His Leu Ala Ala Ala Leu Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120

<210> 3

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> DARPin protein #3

<400> 3

Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asn

20 25 30

Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala Gln Thr Gly His Leu

35 40 45

Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asn Asp Lys Arg Val Thr Pro Leu His Leu Ala Ala Ala Leu Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Arg Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly

100 105 110

His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120

<210> 4

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> DARPin protein #4

<400> 4

Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asn

20 25 30

Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala Gln Thr Gly His Leu

35 40 45

Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Thr Asn Lys Arg Val Thr Pro Leu His Leu Ala Ala Ala Leu Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Arg Asp Thr Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly

100 105 110

His Arg Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120

<210> 5

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> DARPin protein #5

<400> 5

Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn Ala Lys Asn

20 25 30

Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala Gln Thr Gly His Leu

35 40 45

Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asn Asp Lys Arg Val Thr Pro Leu His Leu Ala Ala Ala Leu Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Arg Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly

100 105 110

His Gly Asp Ile Ala Glu Val Leu Gln Lys Leu Asn

115 120

<210> 6

<211> 125

<212> PRT

<213> artificial sequence

<220>

<223> DARPin protein #6

<400> 6

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Leu Asp

20 25 30

Trp Leu Gly His Thr Pro Leu His Leu Ala Ala Tyr Glu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Ile

50 55 60

Asp Asp Asn Asn Gly Phe Thr Pro Leu His Leu Ala Ala Ile Asp Gly

65 70 75 80

His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn

85 90 95

Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn

100 105 110

Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala

115 120 125

<210> 7

<211> 421

<212> PRT

<213> artificial sequence

<220>

<223> DARPin protein #7

<400> 7

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Leu Asp

20 25 30

Trp Leu Gly His Thr Pro Leu His Leu Ala Ala Tyr Glu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Ile

50 55 60

Asp Asp Asn Asn Gly Phe Thr Pro Leu His Leu Ala Ala Ile Asp Gly

65 70 75 80

His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn

85 90 95

Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn

100 105 110

Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala Gly Ser Pro

115 120 125

Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

130 135 140

Thr Pro Thr Gly Ser Asp Leu Gly Asp Lys Leu Leu Leu Ala Ala Thr

145 150 155 160

Ser Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp

165 170 175

Val Asn Ala Lys Asp Tyr Asp Gly Asp Thr Pro Leu His Leu Ala Ala

180 185 190

Asp Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

195 200 205

Asp Val Asn Ala Lys Asp Tyr Ser Gly Ser Thr Pro Leu His Ala Ala

210 215 220

Ala Ala Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly

225 230 235 240

Ala Asp Val Asn Ala Gln Asp Val Phe Gly Tyr Thr Pro Ala Asp Leu

245 250 255

Ala Ala Tyr Val Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala

260 265 270

Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr

275 280 285

Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu

290 295 300

Glu Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys

305 310 315 320

Ala Gly Ala Asp Val Asn Ala Lys Asp Ser Gln Gly Trp Thr Pro Leu

325 330 335

His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu

340 345 350

Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Asp Lys Gly Val Thr Pro

355 360 365

Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val Glu Val Leu

370 375 380

Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ser Trp Gly Thr Thr

385 390 395 400

Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Glu Asp Ile Ala Glu Val

405 410 415

Leu Gln Lys Ala Ala

420

<210> 8

<211> 421

<212> PRT

<213> artificial sequence

<220>

<223> DARPin protein #8

<400> 8

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Leu Asp

20 25 30

Trp Leu Gly His Thr Pro Leu His Leu Ala Ala Tyr Glu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Ile

50 55 60

Asp Asp Asn Asn Gly Phe Thr Pro Leu His Leu Ala Ala Ile Asp Gly

65 70 75 80

His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn

85 90 95

Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn

100 105 110

Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala Gly Ser Pro

115 120 125

Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

130 135 140

Thr Pro Thr Gly Ser Asp Leu Gly Asp Lys Leu Leu Leu Ala Ala Thr

145 150 155 160

Ser Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp

165 170 175

Val Asn Ala Lys Asp Tyr Asp Gly Asp Thr Pro Leu His Leu Ala Ala

180 185 190

Asp Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

195 200 205

Asp Val Asn Ala Lys Asp Tyr Ser Gly Ser Thr Pro Leu His Ala Ala

210 215 220

Ala Ala Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly

225 230 235 240

Ala Asp Val Asn Ala Gln Asp Val Phe Gly Tyr Thr Pro Ala Asp Leu

245 250 255

Ala Ala Tyr Val Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala

260 265 270

Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr

275 280 285

Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu

290 295 300

Glu Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys

305 310 315 320

Ala Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu

325 330 335

His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu

340 345 350

Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Asp Lys Gly Val Thr Pro

355 360 365

Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val Glu Val Leu

370 375 380

Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ser Trp Gly Thr Thr

385 390 395 400

Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Glu Asp Ile Ala Glu Val

405 410 415

Leu Gln Lys Ala Ala

420

<210> 9

<211> 421

<212> PRT

<213> artificial sequence

<220>

<223> DARPin protein #9

<400> 9

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Leu Asp

20 25 30

Trp Leu Gly His Thr Pro Leu His Leu Ala Ala Tyr Glu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Ile

50 55 60

Asp Asp Asn Asn Gly Phe Thr Pro Leu His Leu Ala Ala Ile Asp Gly

65 70 75 80

His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn

85 90 95

Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn

100 105 110

Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala Gly Ser Pro

115 120 125

Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

130 135 140

Thr Pro Thr Gly Ser Asp Leu Gly Asp Lys Leu Leu Leu Ala Ala Thr

145 150 155 160

Ser Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp

165 170 175

Val Asn Ala Lys Asp Tyr Asp Gly Asp Thr Pro Leu His Leu Ala Ala

180 185 190

Asp Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

195 200 205

Asp Val Asn Ala Lys Asp Tyr Ser Gly Ser Thr Pro Leu His Ala Ala

210 215 220

Ala Ala Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly

225 230 235 240

Ala Asp Val Asn Ala Gln Asp Val Phe Gly Tyr Thr Pro Ala Asp Leu

245 250 255

Ala Ala Tyr Val Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala

260 265 270

Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr

275 280 285

Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu

290 295 300

Glu Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys

305 310 315 320

Ala Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu

325 330 335

His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu

340 345 350

Lys Ala Gly Ala Asp Val Asn Ala Lys Asn Asp Lys Arg Val Thr Pro

355 360 365

Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val Glu Val Leu

370 375 380

Leu Lys Ala Gly Ala Asp Val Asn Ala Arg Asp Ser Trp Gly Thr Thr

385 390 395 400

Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Gln Asp Ile Ala Glu Val

405 410 415

Leu Gln Lys Ala Ala

420

<210> 10

<211> 421

<212> PRT

<213> artificial sequence

<220>

<223> DARPin protein #10

<400> 10

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Leu Asp

20 25 30

Trp Leu Gly His Thr Pro Leu His Leu Ala Ala Tyr Glu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Ile

50 55 60

Asp Asp Asn Asn Gly Phe Thr Pro Leu His Leu Ala Ala Ile Asp Gly

65 70 75 80

His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn

85 90 95

Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn

100 105 110

Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala Gly Ser Pro

115 120 125

Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

130 135 140

Thr Pro Thr Gly Ser Asp Leu Gly Asp Lys Leu Leu Leu Ala Ala Thr

145 150 155 160

Ser Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp

165 170 175

Val Asn Ala Lys Asp Tyr Asp Gly Asp Thr Pro Leu His Leu Ala Ala

180 185 190

Asp Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

195 200 205

Asp Val Asn Ala Lys Asp Tyr Ser Gly Ser Thr Pro Leu His Ala Ala

210 215 220

Ala Ala Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly

225 230 235 240

Ala Asp Val Asn Ala Gln Asp Val Phe Gly Tyr Thr Pro Ala Asp Leu

245 250 255

Ala Ala Tyr Val Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala

260 265 270

Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr

275 280 285

Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu

290 295 300

Glu Ala Ala Trp Ala Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys

305 310 315 320

Ala Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu

325 330 335

His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu

340 345 350

Lys Ala Gly Ala Asp Val Asn Ala Lys Thr Asn Lys Arg Val Thr Pro

355 360 365

Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val Glu Val Leu

370 375 380

Leu Lys Ala Gly Ala Asp Val Asn Ala Arg Asp Thr Trp Gly Thr Thr

385 390 395 400

Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Arg Asp Ile Ala Glu Val

405 410 415

Leu Gln Lys Ala Ala

420

<210> 11

<211> 569

<212> PRT

<213> artificial sequence

<220>

<223> DARPin protein #11

<400> 11

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val

165 170 175

Asn Ala Leu Asp Trp Leu Gly His Thr Pro Leu His Leu Ala Ala Tyr

180 185 190

Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp

195 200 205

Val Asn Ala Ile Asp Asp Asn Asn Gly Phe Thr Pro Leu His Leu Ala

210 215 220

Ala Ile Asp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly

225 230 235 240

Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile

245 250 255

Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala

260 265 270

Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr

275 280 285

Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Asp Lys Leu Leu

290 295 300

Leu Ala Ala Thr Ser Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Ala

305 310 315 320

Ala Gly Ala Asp Val Asn Ala Lys Asp Tyr Asp Gly Asp Thr Pro Leu

325 330 335

His Leu Ala Ala Asp Glu Gly His Leu Glu Ile Val Glu Val Leu Leu

340 345 350

Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Tyr Ser Gly Ser Thr Pro

355 360 365

Leu His Ala Ala Ala Ala Tyr Gly His Leu Glu Ile Val Glu Val Leu

370 375 380

Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Val Phe Gly Tyr Thr

385 390 395 400

Pro Ala Asp Leu Ala Ala Tyr Val Gly His Glu Asp Ile Ala Glu Val

405 410 415

Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr

420 425 430

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly

435 440 445

Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg

450 455 460

Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Ser Gln Gly

465 470 475 480

Trp Thr Pro Leu His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe

485 490 495

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Asp Lys

500 505 510

Gly Val Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile

515 520 525

Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ser

530 535 540

Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Glu Asp

545 550 555 560

Ile Ala Glu Val Leu Gln Lys Ala Ala

565

<210> 12

<211> 569

<212> PRT

<213> artificial sequence

<220>

<223> DARPin protein #12

<400> 12

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val

165 170 175

Asn Ala Leu Asp Trp Leu Gly His Thr Pro Leu His Leu Ala Ala Tyr

180 185 190

Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp

195 200 205

Val Asn Ala Ile Asp Asp Asn Asn Gly Phe Thr Pro Leu His Leu Ala

210 215 220

Ala Ile Asp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly

225 230 235 240

Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile

245 250 255

Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala

260 265 270

Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr

275 280 285

Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Asp Lys Leu Leu

290 295 300

Leu Ala Ala Thr Ser Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Ala

305 310 315 320

Ala Gly Ala Asp Val Asn Ala Lys Asp Tyr Asp Gly Asp Thr Pro Leu

325 330 335

His Leu Ala Ala Asp Glu Gly His Leu Glu Ile Val Glu Val Leu Leu

340 345 350

Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Tyr Ser Gly Ser Thr Pro

355 360 365

Leu His Ala Ala Ala Ala Tyr Gly His Leu Glu Ile Val Glu Val Leu

370 375 380

Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Val Phe Gly Tyr Thr

385 390 395 400

Pro Ala Asp Leu Ala Ala Tyr Val Gly His Glu Asp Ile Ala Glu Val

405 410 415

Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr

420 425 430

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly

435 440 445

Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg

450 455 460

Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly

465 470 475 480

Trp Thr Pro Leu His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe

485 490 495

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Asp Lys

500 505 510

Gly Val Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile

515 520 525

Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ser

530 535 540

Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Glu Asp

545 550 555 560

Ile Ala Glu Val Leu Gln Lys Ala Ala

565

<210> 13

<211> 569

<212> PRT

<213> artificial sequence

<220>

<223> DARPin protein #13

<400> 13

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val

165 170 175

Asn Ala Leu Asp Trp Leu Gly His Thr Pro Leu His Leu Ala Ala Tyr

180 185 190

Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp

195 200 205

Val Asn Ala Ile Asp Asp Asn Asn Gly Phe Thr Pro Leu His Leu Ala

210 215 220

Ala Ile Asp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly

225 230 235 240

Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile

245 250 255

Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala

260 265 270

Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr

275 280 285

Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Asp Lys Leu Leu

290 295 300

Leu Ala Ala Thr Ser Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Ala

305 310 315 320

Ala Gly Ala Asp Val Asn Ala Lys Asp Tyr Asp Gly Asp Thr Pro Leu

325 330 335

His Leu Ala Ala Asp Glu Gly His Leu Glu Ile Val Glu Val Leu Leu

340 345 350

Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Tyr Ser Gly Ser Thr Pro

355 360 365

Leu His Ala Ala Ala Ala Tyr Gly His Leu Glu Ile Val Glu Val Leu

370 375 380

Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Val Phe Gly Tyr Thr

385 390 395 400

Pro Ala Asp Leu Ala Ala Tyr Val Gly His Glu Asp Ile Ala Glu Val

405 410 415

Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr

420 425 430

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly

435 440 445

Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg

450 455 460

Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly

465 470 475 480

Trp Thr Pro Leu His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe

485 490 495

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asn Asp Lys

500 505 510

Arg Val Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile

515 520 525

Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Arg Asp Ser

530 535 540

Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Gln Asp

545 550 555 560

Ile Ala Glu Val Leu Gln Lys Ala Ala

565

<210> 14

<211> 569

<212> PRT

<213> artificial sequence

<220>

<223> DARPin protein #14

<400> 14

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val

165 170 175

Asn Ala Leu Asp Trp Leu Gly His Thr Pro Leu His Leu Ala Ala Tyr

180 185 190

Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp

195 200 205

Val Asn Ala Ile Asp Asp Asn Asn Gly Phe Thr Pro Leu His Leu Ala

210 215 220

Ala Ile Asp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly

225 230 235 240

Ala Asp Val Asn Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile

245 250 255

Ser Ile Asp Asn Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala

260 265 270

Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr

275 280 285

Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Asp Lys Leu Leu

290 295 300

Leu Ala Ala Thr Ser Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Ala

305 310 315 320

Ala Gly Ala Asp Val Asn Ala Lys Asp Tyr Asp Gly Asp Thr Pro Leu

325 330 335

His Leu Ala Ala Asp Glu Gly His Leu Glu Ile Val Glu Val Leu Leu

340 345 350

Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Tyr Ser Gly Ser Thr Pro

355 360 365

Leu His Ala Ala Ala Ala Tyr Gly His Leu Glu Ile Val Glu Val Leu

370 375 380

Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Val Phe Gly Tyr Thr

385 390 395 400

Pro Ala Asp Leu Ala Ala Tyr Val Gly His Glu Asp Ile Ala Glu Val

405 410 415

Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr

420 425 430

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly

435 440 445

Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Leu Asp Glu Val Arg

450 455 460

Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly

465 470 475 480

Trp Thr Pro Leu His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe

485 490 495

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Thr Asn Lys

500 505 510

Arg Val Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile

515 520 525

Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Arg Asp Thr

530 535 540

Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Arg Asp

545 550 555 560

Ile Ala Glu Val Leu Gln Lys Ala Ala

565

<210> 15

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> DARPin protein #15

<400> 15

Asp Leu Gly Asp Lys Leu Leu Leu Ala Ala Thr Ser Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Asp Gly Asp Thr Pro Leu His Leu Ala Ala Asp Glu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Tyr Ser Gly Ser Thr Pro Leu His Ala Ala Ala Ala Tyr Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Val Phe Gly Tyr Thr Pro Ala Asp Leu Ala Ala Tyr Val Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120

<210> 16

<211> 30

<212> PRT

<213> artificial sequence

<220>

<223> N-terminal capping Module

<400> 16

Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

20 25 30

<210> 17

<211> 30

<212> PRT

<213> artificial sequence

<220>

<223> N-terminal capping Module

<400> 17

Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

20 25 30

<210> 18

<211> 30

<212> PRT

<213> artificial sequence

<220>

<223> N-terminal capping Module

<400> 18

Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

20 25 30

<210> 19

<211> 30

<212> PRT

<213> artificial sequence

<220>

<223> N-terminal capping Module

<400> 19

Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn Ala

20 25 30

<210> 20

<211> 30

<212> PRT

<213> artificial sequence

<220>

<223> N-terminal capping Module

<400> 20

Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

20 25 30

<210> 21

<211> 30

<212> PRT

<213> artificial sequence

<220>

<223> N-terminal capping Module

<400> 21

Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn Ala

20 25 30

<210> 22

<211> 30

<212> PRT

<213> artificial sequence

<220>

<223> N-terminal capping Module

<220>

<221> variant

<222> 4

<223> Xaa can be any naturally occurring amino acid

<220>

<221> variant

<222> 5

<223> Xaa can be any naturally occurring amino acid

<220>

<221> variant

<222> 11

<223> Xaa can be any naturally occurring amino acid

<220>

<221> variant

<222> 12

<223> Xaa can be any naturally occurring amino acid

<220>

<221> variant

<222> 17

<223> Xaa can be any naturally occurring amino acid

Xaa can be any naturally occurring amino acid

<220>

<221> variant

<222> 20

<223> Xaa can be any naturally occurring amino acid

<220>

<221> variant

<222> 22

<223> Xaa can be any naturally occurring amino acid

<220>

<221> variant

<222> 23

<223> Xaa can be any naturally occurring amino acid

<220>

<221> variant

<222> 24

<223> Xaa can be any naturally occurring amino acid

<400> 22

Asp Leu Gly Xaa Xaa Leu Leu Gln Ala Ala Xaa Xaa Gly Gln Leu Asp

1 5 10 15

Xaa Val Arg Xaa Leu Xaa Xaa Xaa Gly Ala Asp Val Asn Ala

20 25 30

<210> 23

<211> 28

<212> PRT

<213> artificial sequence

<220>

<223> C-terminal end capping Module

<400> 23

Gln Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly

1 5 10 15

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

20 25

<210> 24

<211> 28

<212> PRT

<213> artificial sequence

<220>

<223> C-terminal end capping Module

<400> 24

Arg Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly

1 5 10 15

His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

20 25

<210> 25

<211> 28

<212> PRT

<213> artificial sequence

<220>

<223> C-terminal end capping Module

<400> 25

Arg Asp Thr Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly

1 5 10 15

His Arg Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

20 25

<210> 26

<211> 28

<212> PRT

<213> artificial sequence

<220>

<223> C-terminal end capping Module

<400> 26

Gln Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly

1 5 10 15

His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Ala

20 25

<210> 27

<211> 28

<212> PRT

<213> artificial sequence

<220>

<223> C-terminal end capping Module

<400> 27

Gln Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly

1 5 10 15

His Glu Asp Ile Ala Glu Val Leu Gln Lys Leu Asn

20 25

<210> 28

<211> 28

<212> PRT

<213> artificial sequence

<220>

<223> C-terminal end capping Module

<400> 28

Arg Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly

1 5 10 15

His Gln Asp Ile Ala Glu Val Leu Gln Lys Leu Ala

20 25

<210> 29

<211> 28

<212> PRT

<213> artificial sequence

<220>

<223> C-terminal end capping Module

<400> 29

Arg Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly

1 5 10 15

His Gln Asp Ile Ala Glu Val Leu Gln Lys Leu Asn

20 25

<210> 30

<211> 28

<212> PRT

<213> artificial sequence

<220>

<223> C-terminal end capping Module

<400> 30

Arg Asp Thr Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly

1 5 10 15

His Arg Asp Ile Ala Glu Val Leu Gln Lys Leu Ala

20 25

<210> 31

<211> 28

<212> PRT

<213> artificial sequence

<220>

<223> C-terminal end capping Module

<400> 31

Arg Asp Thr Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly

1 5 10 15

His Arg Asp Ile Ala Glu Val Leu Gln Lys Leu Asn

20 25

<210> 32

<211> 28

<212> PRT

<213> artificial sequence

<220>

<223> C-terminal end capping Module

<220>

<221> variant

<222> 1

<223> Xaa is any naturally occurring amino acid

<220>

<221> variant

<222> 3

<223> Xaa is any naturally occurring amino acid

<220>

<221> variant

<222> 4

<223> Xaa is any naturally occurring amino acid

<220>

<221> variant

<222> 6

<223> Xaa is any naturally occurring amino acid

<220>

<221> variant

<222> 11

<223> Xaa is any naturally occurring amino acid

<220>

<221> variant

<222> 15

<223> Xaa is any naturally occurring amino acid

<220>

<221> variant

<222> 19

<223> Xaa is any naturally occurring amino acid

<220>

<221> variant

<222> 22

<223> Xaa is any naturally occurring amino acid

<220>

<221> variant

<222> 26

<223> Xaa is any naturally occurring amino acid

<400> 32

Xaa Asp Xaa Xaa Gly Xaa Thr Pro Ala Asp Xaa Ala Ala Arg Xaa Gly

1 5 10 15

His Gln Xaa Ile Ala Xaa Val Leu Gln Xaa Ala Ala

20 25

<210> 33

<211> 10

<212> PRT

<213> artificial sequence

<220>

<223> His tag

<400> 33

Met Arg Gly Ser His His His His His His

1 5 10

<210> 34

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> ankyrin repeat Domain specific for human serum albumin

<400> 34

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asn Glu Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120

<210> 35

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> ankyrin repeat Domain specific for human serum albumin

<400> 35

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120

<210> 36

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> ankyrin repeat Domain specific for human serum albumin

<400> 36

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Asp Ala Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120

<210> 37

<211> 22

<212> PRT

<213> artificial sequence

<220>

<223> PT connector

<400> 37

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

1 5 10 15

Pro Thr Pro Thr Pro Thr

20

<210> 38

<211> 222

<212> PRT

<213> artificial sequence

<220>

<223> cCD3εγ_Avi-Bio

<400> 38

Met Gln Asp Gly Asn Glu Glu Met Gly Gly Ile Thr Gln Thr Pro Tyr

1 5 10 15

Lys Val Ser Ile Ser Gly Thr Thr Val Ile Leu Thr Cys Pro Gln Tyr

20 25 30

Pro Gly Ser Glu Ile Leu Trp Gln His Asn Asp Lys Asn Ile Gly Gly

35 40 45

Asp Glu Asp Asp Lys Asn Ile Gly Ser Asp Glu Asp His Leu Ser Leu

50 55 60

Lys Glu Phe Ser Glu Leu Glu Gln Ser Gly Tyr Tyr Val Cys Tyr Pro

65 70 75 80

Arg Gly Ser Lys Pro Glu Asp Ala Asn Phe Tyr Leu Tyr Leu Arg Ala

85 90 95

Arg Val Gly Ser Ala Asp Asp Ala Lys Lys Asp Ala Ala Lys Lys Asp

100 105 110

Asp Ala Lys Lys Asp Asp Ala Lys Lys Asp Gly Ser Gln Ser Ile Lys

115 120 125

Gly Asn His Leu Val Lys Val Tyr Asp Tyr Gln Glu Asp Gly Ser Val

130 135 140

Leu Leu Thr Cys Asp Ala Glu Ala Lys Asn Ile Thr Trp Phe Lys Asp

145 150 155 160

Gly Lys Met Ile Gly Phe Leu Thr Glu Asp Lys Lys Lys Trp Asn Leu

165 170 175

Gly Ser Asn Ala Lys Asp Pro Arg Gly Met Tyr Gln Cys Lys Gly Ser

180 185 190

Gln Asn Lys Ser Lys Pro Leu Gln Val Tyr Tyr Arg Met Gly Gly Gly

195 200 205

Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu

210 215 220

<210> 39

<211> 272

<212> PRT

<213> artificial sequence

<220>

<223> DARPin protein #16

<400> 39

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Ser Gln Gly Trp Thr Pro Leu His Thr Ala Ala Gln

180 185 190

Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Asp Lys Gly Val Thr Pro Leu His Leu Ala Ala

210 215 220

Ala Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala

245 250 255

Ala Lys Tyr Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

<210> 40

<211> 272

<212> PRT

<213> artificial sequence

<220>

<223> DARPin protein #17

<400> 40

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala Gln

180 185 190

Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Asp Lys Gly Val Thr Pro Leu His Leu Ala Ala

210 215 220

Ala Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala

245 250 255

Ala Lys Tyr Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

<210> 41

<211> 272

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #18

<400> 41

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala Gln

180 185 190

Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asn Asp Lys Arg Val Thr Pro Leu His Leu Ala Ala

210 215 220

Ala Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Arg Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala

245 250 255

Ala Lys Tyr Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

<210> 42

<211> 272

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #19

<400> 42

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala

145 150 155 160

Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala Gln

180 185 190

Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Thr Asn Lys Arg Val Thr Pro Leu His Leu Ala Ala

210 215 220

Ala Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Arg Asp Thr Trp Gly Thr Thr Pro Ala Asp Leu Ala

245 250 255

Ala Lys Tyr Gly His Arg Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

<210> 43

<211> 417

<212> DNA

<213> artificial sequence

<220>

<223> nucleic acid encoding the domain of SEQ ID NO 1

<400> 43

atgagaggat cgcatcacca tcaccatcac ggctcagatc tgggtcaaaa gttattggag 60

gcagcttggg ctggacaaga tgacgaagta cgtgaattgt taaaagcggg agcagatgta 120

aacgcaaaag atagtcaagg ttggactccg ttacacacag cagcgcaaac cggccacctg 180

gaaattttcg aggtgttatt gaaggctgga gcagatgtga atgcaaaaga cgacaaaggg 240

gtgactccgc tgcatctggc agcggcgttg gggcacttgg aaatcgttga ggtccttctg 300

aaagcaggcg ctgatgtgaa tgcgcaagac tcctggggaa ccacaccagc ggacctggcg 360

gctaagtacg gccacgaaga tattgctgaa gttctgcaga aggcagcata atgatag 417

<210> 44

<211> 417

<212> DNA

<213> artificial sequence

<220>

<223> nucleic acid encoding the domain of SEQ ID NO 2

<400> 44

atgagaggat cgcatcacca tcaccatcac ggctcagatc tgggtcaaaa gttgttggaa 60

gctgcctggg cgggacagga tgatgaggtg cgcgaattac ttaaggcggg agcagacgtg 120

aatgcgaaaa actctcgtgg ctggacacca ctgcacacgg ccgcgcaaac tggtcacctt 180

gaaattttcg aagtgcttct gaaggcaggc gcagatgtaa acgccaagga tgacaaaggg 240

gtaacaccgc ttcatctggc tgctgcactg ggacatcttg agattgtcga agtactgctt 300

aaggcaggtg ctgacgtaaa cgctcaggat tcatggggga ccacaccggc ggacctggcg 360

gctaaatacg gacatgaaga tattgctgaa gttctgcaga aggcagcata atgatag 417

<210> 45

<211> 417

<212> DNA

<213> artificial sequence

<220>

<223> nucleic acid encoding the domain of SEQ ID NO 3

<400> 45

atgagaggat cgcatcacca tcaccatcac ggctcagatc tgggtcaaaa gctgttggaa 60

gccgcgtggg cgggtcagga cgatgaagtc cgtgagctgc ttaaagcagg agccgacgtg 120

aacgcgaaga actcacgcgg gtggacgcca cttcacacgg ccgcgcagac aggtcacctt 180

gaaatctttg aggttcttct gaaggcagga gcagacgtta acgccaaaaa cgacaagcgc 240

gtgactccgt tgcaccttgc cgcagctctg gggcatttgg agatcgttga ggtactgttg 300

aaagcgggag cagatgttaa tgctcgcgac agttggggga cgacaccagc agacctggcc 360

gcaaaatacg gacaccaaga cattgctgaa gttctgcaga aggcagcata atgatag 417

<210> 46

<211> 417

<212> DNA

<213> artificial sequence

<220>

<223> nucleic acid encoding the domain of SEQ ID NO 4

<400> 46

atgagaggat cgcatcacca tcaccatcac ggctcagatc tgggtcaaaa attgttagag 60

gcagcctggg cgggacagtt agacgaggtt cgtattcttt tgaaagctgg tgcggatgtg 120

aacgcaaaga attctcgtgg atggactccg ttgcacaccg ccgcacagac tgggcacttg 180

gaaatctttg aggttctgtt aaaagcaggg gcagatgtta acgctaaaac taataaacgt 240

gtcacccccc ttcacctggc tgcggcttta ggccatttag aaatcgtgga agtattactt 300

aaagccgggg ctgacgttaa cgcccgtgac acttggggga caacccctgc ggatctggcc 360

gccaaatatg gtcaccgcga cattgctgaa gttctgcaga aggcagcata atgatag 417

<210> 47

<211> 421

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #20

<400> 47

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Leu Asp

20 25 30

Trp Leu Gly His Thr Pro Leu His Leu Ala Ala Tyr Glu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Ile

50 55 60

Asp Asp Asn Asn Gly Phe Thr Pro Leu His Leu Ala Ala Ile Asp Gly

65 70 75 80

His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn

85 90 95

Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn

100 105 110

Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala Gly Ser Pro

115 120 125

Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

130 135 140

Thr Pro Thr Gly Ser Asp Leu Gly Asp Lys Leu Leu Leu Ala Ala Thr

145 150 155 160

Ser Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp

165 170 175

Val Asn Ala Lys Asp Tyr Asp Gly Asp Thr Pro Leu His Leu Ala Ala

180 185 190

Asp Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

195 200 205

Asp Val Asn Ala Lys Asp Tyr Ser Gly Ser Thr Pro Leu His Ala Ala

210 215 220

Ala Ala Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly

225 230 235 240

Ala Asp Val Asn Ala Gln Asp Val Phe Gly Tyr Thr Pro Ala Asp Leu

245 250 255

Ala Ala Tyr Val Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala

260 265 270

Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr

275 280 285

Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu

290 295 300

Glu Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Ala

305 310 315 320

Ala Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu

325 330 335

His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu

340 345 350

Lys Ala Gly Ala Asp Val Asn Ala Lys Asn Asp Lys Arg Val Thr Pro

355 360 365

Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val Glu Val Leu

370 375 380

Leu Lys Ala Gly Ala Asp Val Asn Ala Arg Asp Ser Trp Gly Thr Thr

385 390 395 400

Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Gly Asp Ile Ala Glu Val

405 410 415

Leu Gln Lys Leu Ala

420

<210> 48

<211> 421

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #21

<400> 48

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Leu Asp

20 25 30

Trp Leu Gly His Thr Pro Leu His Leu Ala Ala Tyr Glu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Ile

50 55 60

Asp Asp Asn Asn Gly Phe Thr Pro Leu His Leu Ala Ala Ile Asp Gly

65 70 75 80

His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn

85 90 95

Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn

100 105 110

Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala Gly Ser Pro

115 120 125

Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

130 135 140

Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg

145 150 155 160

Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp

165 170 175

Val Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala

180 185 190

Arg Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

195 200 205

Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala

210 215 220

Ala Arg Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly

225 230 235 240

Ala Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu

245 250 255

Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala

260 265 270

Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr

275 280 285

Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu

290 295 300

Glu Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Ala

305 310 315 320

Ala Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu

325 330 335

His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu

340 345 350

Lys Ala Gly Ala Asp Val Asn Ala Lys Asn Asp Lys Arg Val Thr Pro

355 360 365

Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val Glu Val Leu

370 375 380

Leu Lys Ala Gly Ala Asp Val Asn Ala Arg Asp Ser Trp Gly Thr Thr

385 390 395 400

Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Gly Asp Ile Ala Glu Val

405 410 415

Leu Gln Lys Leu Ala

420

<210> 49

<211> 420

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #22

<400> 49

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg Glu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg Glu Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Asp Lys Leu Leu Leu Ala Ala Thr Ser

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Tyr Asp Gly Asp Thr Pro Leu His Leu Ala Ala Asp

180 185 190

Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Tyr Ser Gly Ser Thr Pro Leu His Ala Ala Ala

210 215 220

Ala Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Val Phe Gly Tyr Thr Pro Ala Asp Leu Ala

245 250 255

Ala Tyr Val Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

275 280 285

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu Glu

290 295 300

Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Ala Ala

305 310 315 320

Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu His

325 330 335

Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu Lys

340 345 350

Ala Gly Ala Asp Val Asn Ala Lys Asn Asp Lys Arg Val Thr Pro Leu

355 360 365

His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val Glu Val Leu Leu

370 375 380

Lys Ala Gly Ala Asp Val Asn Ala Arg Asp Ser Trp Gly Thr Thr Pro

385 390 395 400

Ala Asp Leu Ala Ala Lys Tyr Gly His Gly Asp Ile Ala Glu Val Leu

405 410 415

Gln Lys Leu Ala

420

<210> 50

<211> 272

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #23

<400> 50

Asp Leu Gly Asp Lys Leu Leu Leu Ala Ala Thr Ser Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Asp Gly Asp Thr Pro Leu His Leu Ala Ala Asp Glu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Tyr Ser Gly Ser Thr Pro Leu His Ala Ala Ala Ala Tyr Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Val Phe Gly Tyr Thr Pro Ala Asp Leu Ala Ala Tyr Val Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala Gln

180 185 190

Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Asp Lys Gly Val Thr Pro Leu His Leu Ala Ala

210 215 220

Ala Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala

245 250 255

Ala Lys Tyr Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

<210> 51

<211> 273

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #24

<400> 51

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Leu Asp

20 25 30

Trp Leu Gly His Thr Pro Leu His Leu Ala Ala Tyr Glu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Ile

50 55 60

Asp Asp Asn Asn Gly Phe Thr Pro Leu His Leu Ala Ala Ile Asp Gly

65 70 75 80

His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn

85 90 95

Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn

100 105 110

Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala Gly Ser Pro

115 120 125

Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

130 135 140

Thr Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp

145 150 155 160

Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp

165 170 175

Val Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala

180 185 190

Gln Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala

195 200 205

Asp Val Asn Ala Lys Asp Asp Lys Gly Val Thr Pro Leu His Leu Ala

210 215 220

Ala Ala Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly

225 230 235 240

Ala Asp Val Asn Ala Gln Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu

245 250 255

Ala Ala Lys Tyr Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala

260 265 270

Ala

<210> 52

<211> 1308

<212> DNA

<213> artificial sequence

<220>

<223> nucleic acid encoding the protein of SEQ ID NO 7

<400> 52

atgagaggat cgcatcacca tcaccatcac ggatccgacc tgggtaaaaa gctgcttgag 60

gctgcccgtg caggtcaaga tgacgaagtc cgcatcctta tggcaaatgg tgccgatgta 120

aatgcactgg actggcttgg ccacacaccc cttcatctgg cagcctacga ggggcacttg 180

gagattgtcg aagttttgtt aaaaaacggc gcggatgtaa acgcgattga tgacaacaac 240

ggatttactc cacttcactt ggcggctatc gacggtcact tagaaattgt agaggtgttg 300

ttgaagaacg gggcagacgt taatgcacaa gataagttcg gcaaaacggc attcgatatc 360

tccattgata atggtaatga agatttagct gaaatcctgc agaaggcagc aggctcgccg 420

actccgaccc cgaccacccc aactccaaca ccgaccaccc cgacccctac cccaacagga 480

tccgacctgg gtgacaaact gctgctggct gctacttctg gtcaggacga cgaagttcgt 540

atcctgctgg ctgctggcgc cgacgttaat gctaaagact acgacggtga cactccgctg 600

cacctggctg ctgacgaagg tcacctggaa atcgttgaag ttctgctgaa ggctggtgct 660

gacgttaatg ctaaagacta ctctggttct actccgctgc acgctgctgc tgcttacggt 720

cacctggaaa tcgttgaagt tctgctgaag gctggtgctg acgttaacgc tcaggacgtt 780

ttcggttaca ctccggctga tctggctgct tacgttggtc acgaggatat cgctgaagtt 840

ctgcagaagg ctgcggggag tccaaccccg acgccaacca cacccactcc tacgcctaca 900

actccaactc cgacgcctac cggatcagat ctgggtcaaa agttattgga ggcagcttgg 960

gctggacaag atgacgaagt acgtgaattg ttaaaagcgg gagcagatgt aaacgcaaaa 1020

gatagtcaag gttggactcc gttacacaca gcagcgcaaa ccggccacct ggaaattttc 1080

gaggtgttat tgaaggctgg agcagatgtg aatgcaaaag acgacaaagg ggtgactccg 1140

ctgcatctgg cagcggcgtt ggggcacttg gaaatcgttg aggtccttct gaaagcaggc 1200

gctgatgtga atgcgcaaga ctcctgggga accacaccag cggacctggc ggctaagtac 1260

ggccacgaag atattgctga agttctgcag aaggcagcat aatgatag 1308

<210> 53

<211> 1308

<212> DNA

<213> artificial sequence

<220>

<223> nucleic acid encoding the protein of SEQ ID NO 8

<400> 53

atgagaggat cgcatcacca tcaccatcac ggatccgacc tgggtaaaaa gctgcttgag 60

gctgcccgtg caggtcaaga tgacgaagtc cgcatcctta tggcaaatgg tgccgatgta 120

aatgcactgg actggcttgg ccacacaccc cttcatctgg cagcctacga ggggcacttg 180

gagattgtcg aagttttgtt aaaaaacggc gcggatgtaa acgcgattga tgacaacaac 240

ggatttactc cacttcactt ggcggctatc gacggtcact tagaaattgt agaggtgttg 300

ttgaagaacg gggcagacgt taatgcacaa gataagttcg gcaaaacggc attcgatatc 360

tccattgata atggtaatga agatttagct gaaatcctgc agaaggcagc aggctcgccg 420

actccgaccc cgaccacccc aactccaaca ccgaccaccc cgacccctac cccaacagga 480

tccgacctgg gtgacaaact gctgctggct gctacttctg gtcaggacga cgaagttcgt 540

atcctgctgg ctgctggcgc cgacgttaat gctaaagact acgacggtga cactccgctg 600

cacctggctg ctgacgaagg tcacctggaa atcgttgaag ttctgctgaa ggctggtgct 660

gacgttaatg ctaaagacta ctctggttct actccgctgc acgctgctgc tgcttacggt 720

cacctggaaa tcgttgaagt tctgctgaag gctggtgctg acgttaacgc tcaggacgtt 780

ttcggttaca ctccggctga tctggctgct tacgttggtc acgaggatat cgctgaagtt 840

ctgcagaagg ctgcggggag tccaaccccg acgccaacca cacccactcc tacgcctaca 900

actccaactc cgacgcctac cggatcagat ctgggtcaaa agttgttgga agctgcctgg 960

gcgggacagg atgatgaggt gcgcgaatta cttaaggcgg gagcagacgt gaatgcgaaa 1020

aactctcgtg gctggacacc actgcacacg gccgcgcaaa ctggtcacct tgaaattttc 1080

gaagtgcttc tgaaggcagg cgcagatgta aacgccaagg atgacaaagg ggtaacaccg 1140

cttcatctgg ctgctgcact gggacatctt gagattgtcg aagtactgct taaggcaggt 1200

gctgacgtaa acgctcagga ttcatggggg accacaccgg cggacctggc ggctaaatac 1260

ggacatgaag atattgctga agttctgcag aaggcagcat aatgatag 1308

<210> 54

<211> 1308

<212> DNA

<213> artificial sequence

<220>

<223> nucleic acid encoding the protein of SEQ ID NO 9

<400> 54

atgagaggat cgcatcacca tcaccatcac ggatccgacc tgggtaaaaa gctgcttgag 60

gctgcccgtg caggtcaaga tgacgaagtc cgcatcctta tggcaaatgg tgccgatgta 120

aatgcactgg actggcttgg ccacacaccc cttcatctgg cagcctacga ggggcacttg 180

gagattgtcg aagttttgtt aaaaaacggc gcggatgtaa acgcgattga tgacaacaac 240

ggatttactc cacttcactt ggcggctatc gacggtcact tagaaattgt agaggtgttg 300

ttgaagaacg gggcagacgt taatgcacaa gataagttcg gcaaaacggc attcgatatc 360

tccattgata atggtaatga agatttagct gaaatcctgc agaaggcagc aggctcgccg 420

actccgaccc cgaccacccc aactccaaca ccgaccaccc cgacccctac cccaacagga 480

tccgacctgg gtgacaaact gctgctggct gctacttctg gtcaggacga cgaagttcgt 540

atcctgctgg ctgctggcgc cgacgttaat gctaaagact acgacggtga cactccgctg 600

cacctggctg ctgacgaagg tcacctggaa atcgttgaag ttctgctgaa ggctggtgct 660

gacgttaatg ctaaagacta ctctggttct actccgctgc acgctgctgc tgcttacggt 720

cacctggaaa tcgttgaagt tctgctgaag gctggtgctg acgttaacgc tcaggacgtt 780

ttcggttaca ctccggctga tctggctgct tacgttggtc acgaggatat cgctgaagtt 840

ctgcagaagg ctgcggggag tccaaccccg acgccaacca cacccactcc tacgcctaca 900

actccaactc cgacgcctac cggatcagat ctgggtcaaa agctgttgga agccgcgtgg 960

gcgggtcagg acgatgaagt ccgtgagctg cttaaagcag gagccgacgt gaacgcgaag 1020

aactcacgcg ggtggacgcc acttcacacg gccgcgcaga caggtcacct tgaaatcttt 1080

gaggttcttc tgaaggcagg agcagacgtt aacgccaaaa acgacaagcg cgtgactccg 1140

ttgcaccttg ccgcagctct ggggcatttg gagatcgttg aggtactgtt gaaagcggga 1200

gcagatgtta atgctcgcga cagttggggg acgacaccag cagacctggc cgcaaaatac 1260

ggacaccaag acattgctga agttctgcag aaggcagcat aatgatag 1308

<210> 55

<211> 1308

<212> DNA

<213> artificial sequence

<220>

<223> nucleic acid encoding the protein of SEQ ID NO 10

<400> 55

atgagaggat cgcatcacca tcaccatcac ggatccgacc tgggtaaaaa gctgcttgag 60

gctgcccgtg caggtcaaga tgacgaagtc cgcatcctta tggcaaatgg tgccgatgta 120

aatgcactgg actggcttgg ccacacaccc cttcatctgg cagcctacga ggggcacttg 180

gagattgtcg aagttttgtt aaaaaacggc gcggatgtaa acgcgattga tgacaacaac 240

ggatttactc cacttcactt ggcggctatc gacggtcact tagaaattgt agaggtgttg 300

ttgaagaacg gggcagacgt taatgcacaa gataagttcg gcaaaacggc attcgatatc 360

tccattgata atggtaatga agatttagct gaaatcctgc agaaggcagc aggctcgccg 420

actccgaccc cgaccacccc aactccaaca ccgaccaccc cgacccctac cccaacagga 480

tccgacctgg gtgacaaact gctgctggct gctacttctg gtcaggacga cgaagttcgt 540

atcctgctgg ctgctggcgc cgacgttaat gctaaagact acgacggtga cactccgctg 600

cacctggctg ctgacgaagg tcacctggaa atcgttgaag ttctgctgaa ggctggtgct 660

gacgttaatg ctaaagacta ctctggttct actccgctgc acgctgctgc tgcttacggt 720

cacctggaaa tcgttgaagt tctgctgaag gctggtgctg acgttaacgc tcaggacgtt 780

ttcggttaca ctccggctga tctggctgct tacgttggtc acgaggatat cgctgaagtt 840

ctgcagaagg ctgcggggag tccaaccccg acgccaacca cacccactcc tacgcctaca 900

actccaactc cgacgcctac cggatcagat ctgggtcaaa aattgttaga ggcagcctgg 960

gcgggacagt tagacgaggt tcgtattctt ttgaaagctg gtgcggatgt gaacgcaaag 1020

aattctcgtg gatggactcc gttgcacacc gccgcacaga ctgggcactt ggaaatcttt 1080

gaggttctgt taaaagcagg ggcagatgtt aacgctaaaa ctaataaacg tgtcaccccc 1140

cttcacctgg ctgcggcttt aggccattta gaaatcgtgg aagtattact taaagccggg 1200

gctgacgtta acgcccgtga cacttggggg acaacccctg cggatctggc cgccaaatat 1260

ggtcaccgcg acattgctga agttctgcag aaggcagcat aatgatag 1308

<210> 56

<211> 272

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #25

<400> 56

Asp Leu Gly Asp Lys Leu Leu Leu Ala Ala Thr Ser Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Asp Gly Asp Thr Pro Leu His Leu Ala Ala Asp Glu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Tyr Ser Gly Ser Thr Pro Leu His Ala Ala Ala Ala Tyr Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Val Phe Gly Tyr Thr Pro Ala Asp Leu Ala Ala Tyr Val Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala Gln

180 185 190

Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asn Asp Lys Arg Val Thr Pro Leu His Leu Ala Ala

210 215 220

Ala Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Arg Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala

245 250 255

Ala Lys Tyr Gly His Gly Asp Ile Ala Glu Val Leu Gln Lys Leu Asn

260 265 270

<210> 57

<211> 273

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #26

<400> 57

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Ile Leu Met Ala Asn Gly Ala Asp Val Asn Ala Leu Asp

20 25 30

Trp Leu Gly His Thr Pro Leu His Leu Ala Ala Tyr Glu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn Ala Ile

50 55 60

Asp Asp Asn Asn Gly Phe Thr Pro Leu His Leu Ala Ala Ile Asp Gly

65 70 75 80

His Leu Glu Ile Val Glu Val Leu Leu Lys Asn Gly Ala Asp Val Asn

85 90 95

Ala Gln Asp Lys Phe Gly Lys Thr Ala Phe Asp Ile Ser Ile Asp Asn

100 105 110

Gly Asn Glu Asp Leu Ala Glu Ile Leu Gln Lys Ala Ala Gly Ser Pro

115 120 125

Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

130 135 140

Thr Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp

145 150 155 160

Ala Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp

165 170 175

Val Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala

180 185 190

Gln Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala

195 200 205

Asp Val Asn Ala Lys Asn Asp Lys Arg Val Thr Pro Leu His Leu Ala

210 215 220

Ala Ala Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly

225 230 235 240

Ala Asp Val Asn Ala Arg Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu

245 250 255

Ala Ala Lys Tyr Gly His Gly Asp Ile Ala Glu Val Leu Gln Lys Leu

260 265 270

Asn

<210> 58

<211> 421

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #27

<400> 58

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Leu Glu Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Gln Glu Gly Tyr Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Ser Ile Gly Arg Thr Pro Leu His Leu Ala Ala Tyr Lys Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Leu Leu Gly Glu Thr Pro Ala Asp Leu Ala Ala Glu Gln Gly

100 105 110

His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Val Lys Leu Leu Leu Ala Ala Ser Arg

145 150 155 160

Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Ile Asp Glu Gly Tyr Thr Pro Leu His Ile Ala Ala

180 185 190

Tyr Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

195 200 205

Asp Val Asn Ala Lys Asp Arg Tyr Gly Lys Thr Pro Leu His Leu Ala

210 215 220

Ala Ile Ser Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly

225 230 235 240

Ala Asp Val Asn Ala Gln Asp Asp Lys Gly Asp Thr Pro Ala Asp Leu

245 250 255

Ala Ala Asp Tyr Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala

260 265 270

Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr

275 280 285

Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu

290 295 300

Glu Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys

305 310 315 320

Ala Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu

325 330 335

His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu

340 345 350

Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Asp Lys Gly Val Thr Pro

355 360 365

Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val Glu Val Leu

370 375 380

Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ser Trp Gly Thr Thr

385 390 395 400

Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Glu Asp Ile Ala Glu Val

405 410 415

Leu Gln Lys Ala Ala

420

<210> 59

<211> 421

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #28

<400> 59

Asp Leu Gly Tyr Lys Leu Leu Gln Ala Ala Tyr Asp Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Ser Arg Gly Gln Thr Pro Leu His Tyr Ala Ala Ser Ile Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Asp His Gly Trp Thr Pro Leu His Leu Ala Ala Trp Ser Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Gln Glu Gly Thr Thr Pro Ala Asp Leu Ala Ala Val Gln Gly

100 105 110

His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Trp Lys Leu Leu Leu Ala Ala Ser Arg

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Ile Asp Glu Gly Tyr Thr Pro Leu His Ile Ala Ala

180 185 190

Tyr Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

195 200 205

Asp Val Asn Ala Lys Asp Arg Tyr Gly Lys Thr Pro Leu His Leu Ala

210 215 220

Ala Ile Ser Gly His Glu Asp Ile Ala Glu Val Leu Leu Lys Ala Gly

225 230 235 240

Ala Asp Val Asn Ala Gln Asp Asp Lys Gly Asp Thr Pro Ala Asp Leu

245 250 255

Ala Ala Asp Tyr Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala

260 265 270

Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr

275 280 285

Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu

290 295 300

Glu Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys

305 310 315 320

Ala Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu

325 330 335

His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu

340 345 350

Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Asp Lys Gly Val Thr Pro

355 360 365

Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val Glu Val Leu

370 375 380

Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ser Trp Gly Thr Thr

385 390 395 400

Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Glu Asp Ile Ala Glu Val

405 410 415

Leu Gln Lys Ala Ala

420

<210> 60

<211> 421

<212> PRT

<213> artificial sequence

<220>

<223> DARPin protein #29

<400> 60

Asp Leu Gly Tyr Lys Leu Leu Gln Ala Ala Tyr Asp Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Ser Arg Gly Gln Thr Pro Leu His Tyr Ala Ala Ser Ile Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Asp His Gly Trp Thr Pro Leu His Leu Ala Ala Trp Ser Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Gln Glu Gly Thr Thr Pro Ala Asp Leu Ala Ala Val Gln Gly

100 105 110

His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Val Lys Leu Leu Leu Ala Ala Ser Arg

145 150 155 160

Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Ile Asp Glu Gly Tyr Thr Pro Leu His Ile Ala Ala

180 185 190

Tyr Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

195 200 205

Asp Val Asn Ala Lys Asp Arg Tyr Gly Lys Thr Pro Leu His Leu Ala

210 215 220

Ala Ile Ser Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly

225 230 235 240

Ala Asp Val Asn Ala Gln Asp Asp Lys Gly Asp Thr Pro Ala Asp Leu

245 250 255

Ala Ala Asp Tyr Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala

260 265 270

Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr

275 280 285

Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu

290 295 300

Glu Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys

305 310 315 320

Ala Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu

325 330 335

His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu

340 345 350

Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Asp Lys Gly Val Thr Pro

355 360 365

Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val Glu Val Leu

370 375 380

Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ser Trp Gly Thr Thr

385 390 395 400

Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Glu Asp Ile Ala Glu Val

405 410 415

Leu Gln Lys Ala Ala

420

<210> 61

<211> 568

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #30

<400> 61

Asp Leu Gly Tyr Lys Leu Leu Gln Ala Ala Tyr Asp Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Ser Arg Gly Gln Thr Pro Leu His Tyr Ala Ala Ser Ile Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Asp His Gly Trp Thr Pro Leu His Leu Ala Ala Trp Ser Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Gln Glu Gly Thr Thr Pro Ala Asp Leu Ala Ala Val Gln Gly

100 105 110

His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Leu Glu

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Gln Glu Gly Tyr Thr Pro Leu His Leu Ala Ala Ala

180 185 190

Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Ser Ile Gly Arg Thr Pro Leu His Leu Ala Ala

210 215 220

Tyr Lys Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Leu Leu Gly Glu Thr Pro Ala Asp Leu Ala

245 250 255

Ala Glu Gln Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

275 280 285

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Val Lys Leu Leu Arg

290 295 300

Ala Ala Phe His Gly Gln Asp Asp Glu Val Arg Ile Leu Leu Ala Ala

305 310 315 320

Gly Ala Asp Val Asn Ala Lys Asp Thr Asp Gly Glu Thr Pro Leu His

325 330 335

Tyr Ala Ala Gln Phe Gly His Leu Glu Ile Val Glu Val Leu Leu Lys

340 345 350

Ala Gly Ala Asp Val Asn Ala Lys Asp Ala Tyr Gly Ala Thr Pro Leu

355 360 365

His Trp Ala Ala Trp His Gly His Leu Glu Ile Val Glu Val Leu Leu

370 375 380

Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Val Ser Gly Ala Thr Pro

385 390 395 400

Ala Asp Leu Ala Ala Lys Val Gly His Glu Asp Ile Ala Glu Val Leu

405 410 415

Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

420 425 430

Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Gln

435 440 445

Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg Glu

450 455 460

Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly Trp

465 470 475 480

Thr Pro Leu His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe Glu

485 490 495

Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Asp Lys Gly

500 505 510

Val Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val

515 520 525

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ser Trp

530 535 540

Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Glu Asp Ile

545 550 555 560

Ala Glu Val Leu Gln Lys Ala Ala

565

<210> 62

<211> 568

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #31

<400> 62

Asp Leu Gly Tyr Lys Leu Leu Gln Ala Ala Tyr Asp Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Ser Arg Gly Gln Thr Pro Leu His Tyr Ala Ala Ser Ile Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Asp His Gly Trp Thr Pro Leu His Leu Ala Ala Trp Ser Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Gln Glu Gly Thr Thr Pro Ala Asp Leu Ala Ala Val Gln Gly

100 105 110

His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Leu Glu

145 150 155 160

Gly Gln Leu Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Gln Glu Gly Tyr Thr Pro Leu His Leu Ala Ala Ala

180 185 190

Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Ser Ile Gly Arg Thr Pro Leu His Leu Ala Ala

210 215 220

Tyr Lys Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Leu Leu Gly Glu Thr Pro Ala Asp Leu Ala

245 250 255

Ala Glu Gln Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

275 280 285

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Val Lys Leu Leu Arg

290 295 300

Ala Ala Val His Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys Ala

305 310 315 320

Gly Ala Asp Val Asn Ala Lys Asp Thr Asp Gly Glu Thr Pro Leu His

325 330 335

Tyr Ala Ala Gln Phe Gly His Leu Glu Ile Val Glu Val Leu Leu Lys

340 345 350

Ala Gly Ala Asp Val Asn Ala Lys Asp Ala Tyr Gly Ala Thr Pro Leu

355 360 365

His Trp Ala Ala Trp His Gly His Leu Glu Ile Val Glu Val Leu Leu

370 375 380

Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Val Ser Gly Ala Thr Pro

385 390 395 400

Ala Asp Leu Ala Ala Lys Val Gly His Gln Asp Ile Ala Glu Val Leu

405 410 415

Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

420 425 430

Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Gln

435 440 445

Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg Glu

450 455 460

Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly Trp

465 470 475 480

Thr Pro Leu His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe Glu

485 490 495

Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Asp Lys Gly

500 505 510

Val Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val

515 520 525

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ser Trp

530 535 540

Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Glu Asp Ile

545 550 555 560

Ala Glu Val Leu Gln Lys Ala Ala

565

<210> 63

<211> 5

<212> PRT

<213> artificial sequence

<220>

<223> common GS linker

<220>

<221> variant

<222> 1..5

<223> [ Gly-Gly-Gly-Gly-Ser ] n, wherein n is 1, 2, 3, 4, 5 or 6

<400> 63

Gly Gly Gly Gly Ser

1 5

<210> 64

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein#33

<400> 64

Asp Leu Gly Tyr Lys Leu Leu Gln Ala Ala Tyr Asp Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Ser Arg Gly Gln Thr Pro Leu His Tyr Ala Ala Ser Ile Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Asp His Gly Trp Thr Pro Leu His Leu Ala Ala Trp Ser Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Gln Glu Gly Thr Thr Pro Ala Asp Leu Ala Ala Val Gln Gly

100 105 110

His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120

<210> 65

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #34

<400> 65

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Leu Glu Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Gln Glu Gly Tyr Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Ser Ile Gly Arg Thr Pro Leu His Leu Ala Ala Tyr Lys Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Leu Leu Gly Glu Thr Pro Ala Asp Leu Ala Ala Glu Gln Gly

100 105 110

His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120

<210> 66

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #35

<400> 66

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Leu Glu Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Gln Glu Gly Tyr Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Ser Ile Gly Arg Thr Pro Leu His Leu Ala Ala Tyr Lys Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Leu Leu Gly Glu Thr Pro Ala Asp Leu Ala Ala Glu Gln Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120

<210> 67

<211> 125

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #36

<400> 67

Asp Leu Gly Val Lys Leu Leu Leu Ala Ala Ser Arg Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Ile Asp Glu Gly Tyr Thr Pro Leu His Ile Ala Ala Tyr Tyr Gly His

35 40 45

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

50 55 60

Lys Asp Arg Tyr Gly Lys Thr Pro Leu His Leu Ala Ala Ile Ser Gly

65 70 75 80

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

85 90 95

Ala Gln Asp Asp Lys Gly Asp Thr Pro Ala Asp Leu Ala Ala Asp Tyr

100 105 110

Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120 125

<210> 68

<211> 125

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #37

<400> 68

Asp Leu Gly Trp Lys Leu Leu Leu Ala Ala Ser Arg Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Ile Asp Glu Gly Tyr Thr Pro Leu His Ile Ala Ala Tyr Tyr Gly His

35 40 45

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

50 55 60

Lys Asp Arg Tyr Gly Lys Thr Pro Leu His Leu Ala Ala Ile Ser Gly

65 70 75 80

His Glu Asp Ile Ala Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

85 90 95

Ala Gln Asp Asp Lys Gly Asp Thr Pro Ala Asp Leu Ala Ala Asp Tyr

100 105 110

Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120 125

<210> 69

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #38

<400> 69

Asp Leu Gly Val Lys Leu Leu Arg Ala Ala Phe His Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Thr Asp Gly Glu Thr Pro Leu His Tyr Ala Ala Gln Phe Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Ala Tyr Gly Ala Thr Pro Leu His Trp Ala Ala Trp His Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Val Ser Gly Ala Thr Pro Ala Asp Leu Ala Ala Lys Val Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120

<210> 70

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #39

<400> 70

Asp Leu Gly Val Lys Leu Leu Arg Ala Ala Val His Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Thr Asp Gly Glu Thr Pro Leu His Tyr Ala Ala Gln Phe Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Ala Tyr Gly Ala Thr Pro Leu His Trp Ala Ala Trp His Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Val Ser Gly Ala Thr Pro Ala Asp Leu Ala Ala Lys Val Gly

100 105 110

His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120

<210> 71

<211> 421

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #40

<400> 71

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg Glu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg Glu Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Val Lys Leu Leu Leu Ala Ala Ser Arg

145 150 155 160

Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Ile Asp Glu Gly Tyr Thr Pro Leu His Ile Ala Ala

180 185 190

Tyr Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

195 200 205

Asp Val Asn Ala Lys Asp Arg Tyr Gly Lys Thr Pro Leu His Leu Ala

210 215 220

Ala Ile Ser Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly

225 230 235 240

Ala Asp Val Asn Ala Gln Asp Asp Lys Gly Asp Thr Pro Ala Asp Leu

245 250 255

Ala Ala Asp Tyr Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala

260 265 270

Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr

275 280 285

Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu

290 295 300

Glu Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys

305 310 315 320

Ala Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu

325 330 335

His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu

340 345 350

Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Asp Lys Gly Val Thr Pro

355 360 365

Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val Glu Val Leu

370 375 380

Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ser Trp Gly Thr Thr

385 390 395 400

Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Glu Asp Ile Ala Glu Val

405 410 415

Leu Gln Lys Ala Ala

420

<210> 72

<211> 420

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #41

<400> 72

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Leu Glu Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Gln Glu Gly Tyr Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Ser Ile Gly Arg Thr Pro Leu His Leu Ala Ala Tyr Lys Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Leu Leu Gly Glu Thr Pro Ala Asp Leu Ala Ala Glu Gln Gly

100 105 110

His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg

180 185 190

Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala

210 215 220

Arg Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala

245 250 255

Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

275 280 285

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu Glu

290 295 300

Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala

305 310 315 320

Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu His

325 330 335

Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu Lys

340 345 350

Ala Gly Ala Asp Val Asn Ala Lys Asp Asp Lys Gly Val Thr Pro Leu

355 360 365

His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val Glu Val Leu Leu

370 375 380

Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ser Trp Gly Thr Thr Pro

385 390 395 400

Ala Asp Leu Ala Ala Lys Tyr Gly His Glu Asp Ile Ala Glu Val Leu

405 410 415

Gln Lys Ala Ala

420

<210> 73

<211> 421

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #42

<400> 73

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg Glu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg Glu Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Val Lys Leu Leu Leu Ala Ala Ser Arg

145 150 155 160

Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Ile Asp Glu Gly Tyr Thr Pro Leu His Ile Ala Ala

180 185 190

Tyr Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

195 200 205

Asp Val Asn Ala Lys Asp Arg Tyr Gly Lys Thr Pro Leu His Leu Ala

210 215 220

Ala Ile Ser Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly

225 230 235 240

Ala Asp Val Asn Ala Gln Asp Asp Lys Gly Asp Thr Pro Ala Asp Leu

245 250 255

Ala Ala Asp Tyr Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala

260 265 270

Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr

275 280 285

Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu

290 295 300

Glu Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys

305 310 315 320

Ala Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu

325 330 335

His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu

340 345 350

Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Asp Lys Gly Val Thr Pro

355 360 365

Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val Glu Val Leu

370 375 380

Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ser Trp Gly Thr Thr

385 390 395 400

Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Glu Asp Ile Ala Glu Val

405 410 415

Leu Gln Lys Ala Ala

420

<210> 74

<211> 420

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #43

<400> 74

Asp Leu Gly Tyr Lys Leu Leu Gln Ala Ala Tyr Asp Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Ser Arg Gly Gln Thr Pro Leu His Tyr Ala Ala Ser Ile Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Asp His Gly Trp Thr Pro Leu His Leu Ala Ala Trp Ser Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Gln Glu Gly Thr Thr Pro Ala Asp Leu Ala Ala Val Gln Gly

100 105 110

His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg

180 185 190

Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala

210 215 220

Arg Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala

245 250 255

Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

275 280 285

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu Glu

290 295 300

Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala

305 310 315 320

Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu His

325 330 335

Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu Lys

340 345 350

Ala Gly Ala Asp Val Asn Ala Lys Asp Asp Lys Gly Val Thr Pro Leu

355 360 365

His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val Glu Val Leu Leu

370 375 380

Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ser Trp Gly Thr Thr Pro

385 390 395 400

Ala Asp Leu Ala Ala Lys Tyr Gly His Glu Asp Ile Ala Glu Val Leu

405 410 415

Gln Lys Ala Ala

420

<210> 75

<211> 568

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #44

<400> 75

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg Glu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg Glu Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg

180 185 190

Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala

210 215 220

Arg Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala

245 250 255

Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

275 280 285

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Val Lys Leu Leu Arg

290 295 300

Ala Ala Val His Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys Ala

305 310 315 320

Gly Ala Asp Val Asn Ala Lys Asp Thr Asp Gly Glu Thr Pro Leu His

325 330 335

Tyr Ala Ala Gln Phe Gly His Leu Glu Ile Val Glu Val Leu Leu Lys

340 345 350

Ala Gly Ala Asp Val Asn Ala Lys Asp Ala Tyr Gly Ala Thr Pro Leu

355 360 365

His Trp Ala Ala Trp His Gly His Leu Glu Ile Val Glu Val Leu Leu

370 375 380

Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Val Ser Gly Ala Thr Pro

385 390 395 400

Ala Asp Leu Ala Ala Lys Val Gly His Gln Asp Ile Ala Glu Val Leu

405 410 415

Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

420 425 430

Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Gln

435 440 445

Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg Glu

450 455 460

Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly Trp

465 470 475 480

Thr Pro Leu His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe Glu

485 490 495

Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Asp Lys Gly

500 505 510

Val Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val

515 520 525

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ser Trp

530 535 540

Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Glu Asp Ile

545 550 555 560

Ala Glu Val Leu Gln Lys Ala Ala

565

<210> 76

<211> 568

<212> PRT

<213> artificial sequence

<220>

<223> DARPin beta protein #45

<400> 76

Asp Leu Gly Tyr Lys Leu Leu Gln Ala Ala Tyr Asp Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Ser Arg Gly Gln Thr Pro Leu His Tyr Ala Ala Ser Ile Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Asp His Gly Trp Thr Pro Leu His Leu Ala Ala Trp Ser Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Gln Glu Gly Thr Thr Pro Ala Asp Leu Ala Ala Val Gln Gly

100 105 110

His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg

180 185 190

Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala

210 215 220

Arg Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala

245 250 255

Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

275 280 285

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu

290 295 300

Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala

305 310 315 320

Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His

325 330 335

Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys

340 345 350

Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu

355 360 365

His Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val Glu Val Leu Leu

370 375 380

Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro

385 390 395 400

Ala Asp Leu Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu

405 410 415

Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

420 425 430

Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Gln

435 440 445

Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg Glu

450 455 460

Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly Trp

465 470 475 480

Thr Pro Leu His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe Glu

485 490 495

Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Asp Lys Gly

500 505 510

Val Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val

515 520 525

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ser Trp

530 535 540

Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Glu Asp Ile

545 550 555 560

Ala Glu Val Leu Gln Lys Ala Ala

565

<210> 77

<211> 568

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #46

<400> 77

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg Glu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg Glu Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Leu Glu

145 150 155 160

Gly Gln Leu Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Gln Glu Gly Tyr Thr Pro Leu His Leu Ala Ala Ala

180 185 190

Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Ser Ile Gly Arg Thr Pro Leu His Leu Ala Ala

210 215 220

Tyr Lys Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Leu Leu Gly Glu Thr Pro Ala Asp Leu Ala

245 250 255

Ala Glu Gln Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

275 280 285

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu

290 295 300

Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala

305 310 315 320

Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His

325 330 335

Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys

340 345 350

Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu

355 360 365

His Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val Glu Val Leu Leu

370 375 380

Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro

385 390 395 400

Ala Asp Leu Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu

405 410 415

Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

420 425 430

Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Gln

435 440 445

Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg Glu

450 455 460

Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly Trp

465 470 475 480

Thr Pro Leu His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe Glu

485 490 495

Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Asp Lys Gly

500 505 510

Val Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val

515 520 525

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ser Trp

530 535 540

Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Glu Asp Ile

545 550 555 560

Ala Glu Val Leu Gln Lys Ala Ala

565

<210> 78

<211> 569

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #47

<400> 78

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Leu Glu

145 150 155 160

Gly Gln Leu Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Gln Glu Gly Tyr Thr Pro Leu His Leu Ala Ala Ala

180 185 190

Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Ser Ile Gly Arg Thr Pro Leu His Leu Ala Ala

210 215 220

Tyr Lys Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Leu Leu Gly Glu Thr Pro Ala Asp Leu Ala

245 250 255

Ala Glu Gln Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

275 280 285

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Val Lys Leu Leu Leu

290 295 300

Ala Ala Ser Arg Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys Ala

305 310 315 320

Gly Ala Asp Val Asn Ala Lys Asp Ile Asp Glu Gly Tyr Thr Pro Leu

325 330 335

His Ile Ala Ala Tyr Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu

340 345 350

Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Arg Tyr Gly Lys Thr Pro

355 360 365

Leu His Leu Ala Ala Ile Ser Gly His Leu Glu Ile Val Glu Val Leu

370 375 380

Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Asp Lys Gly Asp Thr

385 390 395 400

Pro Ala Asp Leu Ala Ala Asp Tyr Gly His Gln Asp Ile Ala Glu Val

405 410 415

Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr

420 425 430

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly

435 440 445

Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg

450 455 460

Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly

465 470 475 480

Trp Thr Pro Leu His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe

485 490 495

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Asp Lys

500 505 510

Gly Val Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile

515 520 525

Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ser

530 535 540

Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Glu Asp

545 550 555 560

Ile Ala Glu Val Leu Gln Lys Ala Ala

565

<210> 79

<211> 717

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #48

<400> 79

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg

180 185 190

Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala

210 215 220

Ala Asp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala

245 250 255

Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

275 280 285

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu

290 295 300

Ala Ala Leu Glu Gly Gln Leu Asp Glu Val Arg Glu Leu Leu Lys Ala

305 310 315 320

Gly Ala Asp Val Asn Ala Lys Asp Gln Glu Gly Tyr Thr Pro Leu His

325 330 335

Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys

340 345 350

Ala Gly Ala Asp Val Asn Ala Lys Asp Ser Ile Gly Arg Thr Pro Leu

355 360 365

His Leu Ala Ala Tyr Lys Gly His Leu Glu Ile Val Glu Val Leu Leu

370 375 380

Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Leu Leu Gly Glu Thr Pro

385 390 395 400

Ala Asp Leu Ala Ala Glu Gln Gly His Gln Asp Ile Ala Glu Val Leu

405 410 415

Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

420 425 430

Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Val

435 440 445

Lys Leu Leu Leu Ala Ala Ser Arg Gly Gln Leu Asp Glu Val Arg Ile

450 455 460

Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Ile Asp Glu Gly

465 470 475 480

Tyr Thr Pro Leu His Ile Ala Ala Tyr Tyr Gly His Leu Glu Ile Val

485 490 495

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Arg Tyr

500 505 510

Gly Lys Thr Pro Leu His Leu Ala Ala Ile Ser Gly His Leu Glu Ile

515 520 525

Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Asp

530 535 540

Lys Gly Asp Thr Pro Ala Asp Leu Ala Ala Asp Tyr Gly His Gln Asp

545 550 555 560

Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro

565 570 575

Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly

580 585 590

Ser Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Asp

595 600 605

Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

610 615 620

Asn Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala Gln Thr Gly His

625 630 635 640

Leu Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

645 650 655

Lys Asp Asp Lys Gly Val Thr Pro Leu His Leu Ala Ala Ala Leu Gly

660 665 670

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

675 680 685

Ala Gln Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr

690 695 700

Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

705 710 715

<210> 80

<211> 569

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #49

<400> 80

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Leu Glu Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Gln Glu Gly Tyr Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Ser Ile Gly Arg Thr Pro Leu His Leu Ala Ala Tyr Lys Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Leu Leu Gly Glu Thr Pro Ala Asp Leu Ala Ala Glu Gln Gly

100 105 110

His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Val Lys Leu Leu Leu Ala Ala Ser Arg

145 150 155 160

Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Ile Asp Glu Gly Tyr Thr Pro Leu His Ile Ala Ala

180 185 190

Tyr Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

195 200 205

Asp Val Asn Ala Lys Asp Arg Tyr Gly Lys Thr Pro Leu His Leu Ala

210 215 220

Ala Ile Ser Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly

225 230 235 240

Ala Asp Val Asn Ala Gln Asp Asp Lys Gly Asp Thr Pro Ala Asp Leu

245 250 255

Ala Ala Asp Tyr Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala

260 265 270

Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr

275 280 285

Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu

290 295 300

Glu Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys

305 310 315 320

Ala Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu

325 330 335

His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu

340 345 350

Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Asp Lys Gly Val Thr Pro

355 360 365

Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val Glu Val Leu

370 375 380

Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ser Trp Gly Thr Thr

385 390 395 400

Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Glu Asp Ile Ala Glu Val

405 410 415

Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr

420 425 430

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly

435 440 445

Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg

450 455 460

Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Tyr Phe Ser

465 470 475 480

His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu Lys Ile Val

485 490 495

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Phe Ala

500 505 510

Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His Leu Glu Ile

515 520 525

Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ile

530 535 540

Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly His Glu Asp

545 550 555 560

Ile Ala Glu Val Leu Gln Lys Ala Ala

565

<210> 81

<211> 569

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #50

<400> 81

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Tyr Lys Leu Leu Gln Ala Ala Tyr Asp

145 150 155 160

Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Ser Arg Gly Gln Thr Pro Leu His Tyr Ala Ala Ser

180 185 190

Ile Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Asp His Gly Trp Thr Pro Leu His Leu Ala Ala

210 215 220

Trp Ser Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Gln Glu Gly Thr Thr Pro Ala Asp Leu Ala

245 250 255

Ala Val Gln Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

275 280 285

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Val Lys Leu Leu Leu

290 295 300

Ala Ala Ser Arg Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys Ala

305 310 315 320

Gly Ala Asp Val Asn Ala Lys Asp Ile Asp Glu Gly Tyr Thr Pro Leu

325 330 335

His Ile Ala Ala Tyr Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu

340 345 350

Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Arg Tyr Gly Lys Thr Pro

355 360 365

Leu His Leu Ala Ala Ile Ser Gly His Leu Glu Ile Val Glu Val Leu

370 375 380

Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Asp Lys Gly Asp Thr

385 390 395 400

Pro Ala Asp Leu Ala Ala Asp Tyr Gly His Gln Asp Ile Ala Glu Val

405 410 415

Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr

420 425 430

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly

435 440 445

Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg

450 455 460

Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly

465 470 475 480

Trp Thr Pro Leu His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe

485 490 495

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Asp Lys

500 505 510

Gly Val Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile

515 520 525

Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ser

530 535 540

Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Glu Asp

545 550 555 560

Ile Ala Glu Val Leu Gln Lys Ala Ala

565

<210> 82

<211> 717

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #51

<400> 82

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg

180 185 190

Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala

210 215 220

Ala Asp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala

245 250 255

Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

275 280 285

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Tyr Lys Leu Leu Gln

290 295 300

Ala Ala Tyr Asp Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys Ala

305 310 315 320

Gly Ala Asp Val Asn Ala Lys Asp Ser Arg Gly Gln Thr Pro Leu His

325 330 335

Tyr Ala Ala Ser Ile Gly His Leu Glu Ile Val Glu Val Leu Leu Lys

340 345 350

Ala Gly Ala Asp Val Asn Ala Lys Asp Asp His Gly Trp Thr Pro Leu

355 360 365

His Leu Ala Ala Trp Ser Gly His Leu Glu Ile Val Glu Val Leu Leu

370 375 380

Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Gln Glu Gly Thr Thr Pro

385 390 395 400

Ala Asp Leu Ala Ala Val Gln Gly His Gln Asp Ile Ala Glu Val Leu

405 410 415

Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

420 425 430

Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Val

435 440 445

Lys Leu Leu Leu Ala Ala Ser Arg Gly Gln Leu Asp Glu Val Arg Ile

450 455 460

Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Ile Asp Glu Gly

465 470 475 480

Tyr Thr Pro Leu His Ile Ala Ala Tyr Tyr Gly His Leu Glu Ile Val

485 490 495

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Arg Tyr

500 505 510

Gly Lys Thr Pro Leu His Leu Ala Ala Ile Ser Gly His Leu Glu Ile

515 520 525

Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Asp

530 535 540

Lys Gly Asp Thr Pro Ala Asp Leu Ala Ala Asp Tyr Gly His Gln Asp

545 550 555 560

Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro

565 570 575

Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly

580 585 590

Ser Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Asp

595 600 605

Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

610 615 620

Asn Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala Gln Thr Gly His

625 630 635 640

Leu Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

645 650 655

Lys Asp Asp Lys Gly Val Thr Pro Leu His Leu Ala Ala Ala Leu Gly

660 665 670

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

675 680 685

Ala Gln Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr

690 695 700

Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

705 710 715

<210> 83

<211> 569

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #52

<400> 83

Asp Leu Gly Tyr Lys Leu Leu Gln Ala Ala Tyr Asp Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Ser Arg Gly Gln Thr Pro Leu His Tyr Ala Ala Ser Ile Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Asp His Gly Trp Thr Pro Leu His Leu Ala Ala Trp Ser Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Gln Glu Gly Thr Thr Pro Ala Asp Leu Ala Ala Val Gln Gly

100 105 110

His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Val Lys Leu Leu Leu Ala Ala Ser Arg

145 150 155 160

Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Ile Asp Glu Gly Tyr Thr Pro Leu His Ile Ala Ala

180 185 190

Tyr Tyr Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

195 200 205

Asp Val Asn Ala Lys Asp Arg Tyr Gly Lys Thr Pro Leu His Leu Ala

210 215 220

Ala Ile Ser Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly

225 230 235 240

Ala Asp Val Asn Ala Gln Asp Asp Lys Gly Asp Thr Pro Ala Asp Leu

245 250 255

Ala Ala Asp Tyr Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala

260 265 270

Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr

275 280 285

Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu

290 295 300

Glu Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys

305 310 315 320

Ala Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu

325 330 335

His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu

340 345 350

Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Asp Lys Gly Val Thr Pro

355 360 365

Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val Glu Val Leu

370 375 380

Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ser Trp Gly Thr Thr

385 390 395 400

Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Glu Asp Ile Ala Glu Val

405 410 415

Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr

420 425 430

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly

435 440 445

Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg

450 455 460

Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Tyr Phe Ser

465 470 475 480

His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu Lys Ile Val

485 490 495

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Phe Ala

500 505 510

Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His Leu Glu Ile

515 520 525

Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ile

530 535 540

Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly His Glu Asp

545 550 555 560

Ile Ala Glu Val Leu Gln Lys Ala Ala

565

<210> 84

<211> 716

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #53

<400> 84

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Tyr Lys Leu Leu Gln Ala Ala Tyr Asp

145 150 155 160

Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Ser Arg Gly Gln Thr Pro Leu His Tyr Ala Ala Ser

180 185 190

Ile Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Asp His Gly Trp Thr Pro Leu His Leu Ala Ala

210 215 220

Trp Ser Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Gln Glu Gly Thr Thr Pro Ala Asp Leu Ala

245 250 255

Ala Val Gln Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

275 280 285

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu

290 295 300

Ala Ala Leu Glu Gly Gln Leu Asp Glu Val Arg Glu Leu Leu Lys Ala

305 310 315 320

Gly Ala Asp Val Asn Ala Lys Asp Gln Glu Gly Tyr Thr Pro Leu His

325 330 335

Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys

340 345 350

Ala Gly Ala Asp Val Asn Ala Lys Asp Ser Ile Gly Arg Thr Pro Leu

355 360 365

His Leu Ala Ala Tyr Lys Gly His Leu Glu Ile Val Glu Val Leu Leu

370 375 380

Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Leu Leu Gly Glu Thr Pro

385 390 395 400

Ala Asp Leu Ala Ala Glu Gln Gly His Gln Asp Ile Ala Glu Val Leu

405 410 415

Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

420 425 430

Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Val

435 440 445

Lys Leu Leu Arg Ala Ala Val His Gly Gln Leu Asp Glu Val Arg Ile

450 455 460

Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Thr Asp Gly Glu

465 470 475 480

Thr Pro Leu His Tyr Ala Ala Gln Phe Gly His Leu Glu Ile Val Glu

485 490 495

Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Ala Tyr Gly

500 505 510

Ala Thr Pro Leu His Trp Ala Ala Trp His Gly His Leu Glu Ile Val

515 520 525

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Val Ser

530 535 540

Gly Ala Thr Pro Ala Asp Leu Ala Ala Lys Val Gly His Gln Asp Ile

545 550 555 560

Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr

565 570 575

Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser

580 585 590

Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Asp Asp

595 600 605

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asn

610 615 620

Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala Gln Thr Gly His Leu

625 630 635 640

Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

645 650 655

Asp Asp Lys Gly Val Thr Pro Leu His Leu Ala Ala Ala Leu Gly His

660 665 670

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

675 680 685

Gln Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly

690 695 700

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

705 710 715

<210> 85

<211> 864

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #54

<400> 85

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg

180 185 190

Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala

210 215 220

Ala Asp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala

245 250 255

Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

275 280 285

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Tyr Lys Leu Leu Gln

290 295 300

Ala Ala Tyr Asp Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys Ala

305 310 315 320

Gly Ala Asp Val Asn Ala Lys Asp Ser Arg Gly Gln Thr Pro Leu His

325 330 335

Tyr Ala Ala Ser Ile Gly His Leu Glu Ile Val Glu Val Leu Leu Lys

340 345 350

Ala Gly Ala Asp Val Asn Ala Lys Asp Asp His Gly Trp Thr Pro Leu

355 360 365

His Leu Ala Ala Trp Ser Gly His Leu Glu Ile Val Glu Val Leu Leu

370 375 380

Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Gln Glu Gly Thr Thr Pro

385 390 395 400

Ala Asp Leu Ala Ala Val Gln Gly His Gln Asp Ile Ala Glu Val Leu

405 410 415

Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

420 425 430

Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys

435 440 445

Lys Leu Leu Glu Ala Ala Leu Glu Gly Gln Leu Asp Glu Val Arg Glu

450 455 460

Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Gln Glu Gly Tyr

465 470 475 480

Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val Glu

485 490 495

Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Ser Ile Gly

500 505 510

Arg Thr Pro Leu His Leu Ala Ala Tyr Lys Gly His Leu Glu Ile Val

515 520 525

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Leu Leu

530 535 540

Gly Glu Thr Pro Ala Asp Leu Ala Ala Glu Gln Gly His Gln Asp Ile

545 550 555 560

Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr

565 570 575

Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser

580 585 590

Asp Leu Gly Val Lys Leu Leu Arg Ala Ala Val His Gly Gln Leu Asp

595 600 605

Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

610 615 620

Thr Asp Gly Glu Thr Pro Leu His Tyr Ala Ala Gln Phe Gly His Leu

625 630 635 640

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

645 650 655

Asp Ala Tyr Gly Ala Thr Pro Leu His Trp Ala Ala Trp His Gly His

660 665 670

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

675 680 685

Gln Asp Val Ser Gly Ala Thr Pro Ala Asp Leu Ala Ala Lys Val Gly

690 695 700

His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

705 710 715 720

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

725 730 735

Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala

740 745 750

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

755 760 765

Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala Gln

770 775 780

Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp

785 790 795 800

Val Asn Ala Lys Asp Asp Lys Gly Val Thr Pro Leu His Leu Ala Ala

805 810 815

Ala Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

820 825 830

Asp Val Asn Ala Gln Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala

835 840 845

Ala Lys Tyr Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

850 855 860

<210> 86

<211> 716

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #55

<400> 86

Asp Leu Gly Tyr Lys Leu Leu Gln Ala Ala Tyr Asp Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Ser Arg Gly Gln Thr Pro Leu His Tyr Ala Ala Ser Ile Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Asp His Gly Trp Thr Pro Leu His Leu Ala Ala Trp Ser Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Gln Glu Gly Thr Thr Pro Ala Asp Leu Ala Ala Val Gln Gly

100 105 110

His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Leu Glu

145 150 155 160

Gly Gln Leu Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Gln Glu Gly Tyr Thr Pro Leu His Leu Ala Ala Ala

180 185 190

Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Ser Ile Gly Arg Thr Pro Leu His Leu Ala Ala

210 215 220

Tyr Lys Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Leu Leu Gly Glu Thr Pro Ala Asp Leu Ala

245 250 255

Ala Glu Gln Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

275 280 285

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Val Lys Leu Leu Arg

290 295 300

Ala Ala Val His Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys Ala

305 310 315 320

Gly Ala Asp Val Asn Ala Lys Asp Thr Asp Gly Glu Thr Pro Leu His

325 330 335

Tyr Ala Ala Gln Phe Gly His Leu Glu Ile Val Glu Val Leu Leu Lys

340 345 350

Ala Gly Ala Asp Val Asn Ala Lys Asp Ala Tyr Gly Ala Thr Pro Leu

355 360 365

His Trp Ala Ala Trp His Gly His Leu Glu Ile Val Glu Val Leu Leu

370 375 380

Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Val Ser Gly Ala Thr Pro

385 390 395 400

Ala Asp Leu Ala Ala Lys Val Gly His Gln Asp Ile Ala Glu Val Leu

405 410 415

Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

420 425 430

Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Gln

435 440 445

Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln Asp Asp Glu Val Arg Glu

450 455 460

Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asn Ser Arg Gly Trp

465 470 475 480

Thr Pro Leu His Thr Ala Ala Gln Thr Gly His Leu Glu Ile Phe Glu

485 490 495

Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Asp Lys Gly

500 505 510

Val Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val

515 520 525

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ser Trp

530 535 540

Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys Tyr Gly His Glu Asp Ile

545 550 555 560

Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr

565 570 575

Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser

580 585 590

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

595 600 605

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

610 615 620

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

625 630 635 640

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

645 650 655

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

660 665 670

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

675 680 685

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

690 695 700

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

705 710 715

<210> 87

<211> 242

<212> PRT

<213> artificial sequence

<220>

<223> biotinylated extracellular domain of human CD33

<400> 87

Asp Pro Asn Phe Trp Leu Gln Val Gln Glu Ser Val Thr Val Gln Glu

1 5 10 15

Gly Leu Cys Val Leu Val Pro Cys Thr Phe Phe His Pro Ile Pro Tyr

20 25 30

Tyr Asp Lys Asn Ser Pro Val His Gly Tyr Trp Phe Arg Glu Gly Ala

35 40 45

Ile Ile Ser Gly Asp Ser Pro Val Ala Thr Asn Lys Leu Asp Gln Glu

50 55 60

Val Gln Glu Glu Thr Gln Gly Arg Phe Arg Leu Leu Gly Asp Pro Ser

65 70 75 80

Arg Asn Asn Cys Ser Leu Ser Ile Val Asp Ala Arg Arg Arg Asp Asn

85 90 95

Gly Ser Tyr Phe Phe Arg Met Glu Arg Gly Ser Thr Lys Tyr Ser Tyr

100 105 110

Lys Ser Pro Gln Leu Ser Val His Val Thr Asp Leu Thr His Arg Pro

115 120 125

Lys Ile Leu Ile Pro Gly Thr Leu Glu Pro Gly His Ser Lys Asn Leu

130 135 140

Thr Cys Ser Val Ser Trp Ala Cys Glu Gln Gly Thr Pro Pro Ile Phe

145 150 155 160

Ser Trp Leu Ser Ala Ala Pro Thr Ser Leu Gly Pro Arg Thr Thr His

165 170 175

Ser Ser Val Leu Ile Ile Thr Pro Arg Pro Gln Asp His Gly Thr Asn

180 185 190

Leu Thr Cys Gln Val Lys Phe Ala Gly Ala Gly Val Thr Thr Glu Arg

195 200 205

Thr Ile Gln Leu Asn Val Thr Tyr Val Pro Gln Asn Pro Thr Thr Gly

210 215 220

Ile Phe Pro Gly Asp Gly Ser Gly Lys Gln Glu Thr Arg Ala Gly Val

225 230 235 240

Val His

<210> 88

<211> 285

<212> PRT

<213> artificial sequence

<220>

<223> biotinylated extracellular domain of human CD123

<400> 88

Thr Lys Glu Asp Pro Asn Pro Pro Ile Thr Asn Leu Arg Met Lys Ala

1 5 10 15

Lys Ala Gln Gln Leu Thr Trp Asp Leu Asn Arg Asn Val Thr Asp Ile

20 25 30

Glu Cys Val Lys Asp Ala Asp Tyr Ser Met Pro Ala Val Asn Asn Ser

35 40 45

Tyr Cys Gln Phe Gly Ala Ile Ser Leu Cys Glu Val Thr Asn Tyr Thr

50 55 60

Val Arg Val Ala Asn Pro Pro Phe Ser Thr Trp Ile Leu Phe Pro Glu

65 70 75 80

Asn Ser Gly Lys Pro Trp Ala Gly Ala Glu Asn Leu Thr Cys Trp Ile

85 90 95

His Asp Val Asp Phe Leu Ser Cys Ser Trp Ala Val Gly Pro Gly Ala

100 105 110

Pro Ala Asp Val Gln Tyr Asp Leu Tyr Leu Asn Val Ala Asn Arg Arg

115 120 125

Gln Gln Tyr Glu Cys Leu His Tyr Lys Thr Asp Ala Gln Gly Thr Arg

130 135 140

Ile Gly Cys Arg Phe Asp Asp Ile Ser Arg Leu Ser Ser Gly Ser Gln

145 150 155 160

Ser Ser His Ile Leu Val Arg Gly Arg Ser Ala Ala Phe Gly Ile Pro

165 170 175

Cys Thr Asp Lys Phe Val Val Phe Ser Gln Ile Glu Ile Leu Thr Pro

180 185 190

Pro Asn Met Thr Ala Lys Cys Asn Lys Thr His Ser Phe Met His Trp

195 200 205

Lys Met Arg Ser His Phe Asn Arg Lys Phe Arg Tyr Glu Leu Gln Ile

210 215 220

Gln Lys Arg Met Gln Pro Val Ile Thr Glu Gln Val Arg Asp Arg Thr

225 230 235 240

Ser Phe Gln Leu Leu Asn Pro Gly Thr Tyr Thr Val Gln Ile Arg Ala

245 250 255

Arg Glu Arg Val Tyr Glu Phe Leu Ser Ala Trp Ser Thr Pro Gln Arg

260 265 270

Phe Glu Cys Asp Gln Glu Glu Gly Ala Asn Thr Arg Ala

275 280 285

<210> 89

<211> 142

<212> PRT

<213> artificial sequence

<220>

<223> biotinylated extracellular domain of human CD70

<400> 89

Ser Leu Gly Trp Asp Val Ala Glu Leu Gln Leu Asn His Thr Gly Pro

1 5 10 15

Gln Gln Asp Pro Arg Leu Tyr Trp Gln Gly Gly Pro Ala Leu Gly Arg

20 25 30

Ser Phe Leu His Gly Pro Glu Leu Asp Lys Gly Gln Leu Arg Ile His

35 40 45

Arg Asp Gly Ile Tyr Met Val His Ile Gln Val Thr Leu Ala Ile Cys

50 55 60

Ser Ser Thr Thr Ala Ser Arg His His Pro Thr Thr Leu Ala Val Gly

65 70 75 80

Ile Cys Ser Pro Ala Ser Arg Ser Ile Ser Leu Leu Arg Leu Ser Phe

85 90 95

His Gln Gly Cys Thr Ile Ala Ser Gln Arg Leu Thr Pro Leu Ala Arg

100 105 110

Gly Asp Thr Leu Cys Thr Asn Leu Thr Gly Thr Leu Leu Pro Ser Arg

115 120 125

Asn Thr Asp Glu Thr Phe Phe Gly Val Gln Trp Val Arg Pro

130 135 140

<210> 90

<211> 1308

<212> DNA

<213> artificial sequence

<220>

<223> nucleic acid encoding the domain of SEQ ID NO 58

<400> 90

atgagaggat cgcatcacca tcaccatcac ggttccgacc tgggtaaaaa gctgttggag 60

gccgcgttag agggtcaatt ggatgaagtt cgtgaactgt taaaagcggg cgccgatgta 120

aacgctaaag accaggaggg gtacactcct ttgcatctgg cagcagctct tggtcatctg 180

gaaattgtcg aggtgctgtt aaaggcagga gcagatgtaa atgcaaagga ctctattgga 240

cgtacaccac tgcacttggc tgcctacaaa ggtcacctgg aaattgtgga agtcttactg 300

aaagcgggcg ctgacgttaa cgcccaagac ctgctggggg aaacgccagc cgacctggcc 360

gccgagcagg gacatcagga tattgctgaa gttctgcaaa aggcagcagg ctcgccgact 420

ccgaccccga ccaccccaac tccaacaccg accaccccga cccctacccc aacaggatcc 480

gacctgggtg ttaaactttt gttagctgca tctcgtggtc aactggatga ggtgcgtatt 540

ttgctgaaag cgggtgcaga tgttaacgca aaggacatcg atgaaggata tacgccatta 600

cacattgcag catattatgg tcatttagag atcgtagagg ttcttttgaa ggcaggagcc 660

gatgttaacg ccaaggaccg ttatggaaag accccgttac atttagccgc aattagtggg 720

catcttgaaa ttgtcgaagt tttattaaag gctggggctg atgtaaatgc tcaggatgac 780

aagggcgaca ctcccgcaga tctggcggca gactatgggc accaggatat tgctgaagtt 840

ctgcagaagg ctgcggggag tccaaccccg acgccaacca cacccactcc tacgcctaca 900

actccaactc cgacgcctac cggatcagat ctgggtcaaa agttgttgga agctgcctgg 960

gcgggacagg atgatgaggt gcgcgaatta cttaaggcgg gagcagacgt gaatgcgaaa 1020

aactctcgtg gctggacacc actgcacacg gccgcgcaaa ctggtcacct tgaaattttc 1080

gaagtgcttc tgaaggcagg cgcagatgta aacgccaagg atgacaaagg ggtaacaccg 1140

cttcatctgg ctgctgcact gggacatctt gagattgtcg aagtactgct taaggcaggt 1200

gctgacgtaa acgctcagga ttcatggggg accacaccgg cggacctggc ggctaaatac 1260

ggacatgaag atattgctga agttcttcag aaggcagcat aatgatag 1308

<210> 91

<211> 1308

<212> DNA

<213> artificial sequence

<220>

<223> nucleic acid encoding the domain of SEQ ID NO 59

<400> 91

atgagaggat cgcatcacca tcaccatcac ggatccgacc tgggttataa acttcttcaa 60

gcggcctatg atgggcagct tgatgaggta cgcattttgt taaaggccgg cgcagatgtc 120

aacgcgaaag atagtcgcgg acagacgccc cttcattacg ccgcatcaat cggccatctg 180

gaaattgtag aggtactgct taaagccggt gctgatgtaa acgctaagga cgaccacgga 240

tggacgccac ttcatcttgc cgcgtggagt ggacacttgg agattgtcga agtcttgtta 300

aaagcgggcg ctgacgttaa cgcacaagac caggaaggta cgacgccagc agacttggct 360

gctgtccaag ggcaccagga cattgctgaa gttctgcaga aggcagcagg cagccctaca 420

cctacgccga ctacgcctac gccgactccg actaccccga ctccgacccc gaccggatca 480

gacctgggtt ggaaactgct gctggctgct tctcgtggtc aggacgacga agttcgtatc 540

ctgctggctg ctggcgccga cgttaatgct aaagacatcg acgaaggtta cactccgctg 600

cacatcgctg cttactacgg tcacctggaa atcgttgaag ttctgctgaa ggctggtgct 660

gacgttaatg ctaaagaccg ttacggtaaa actccgctgc acctggctgc tatctctggt 720

cacgaggata tcgctgaagt tctgctgaag gctggtgctg acgttaacgc tcaggacgac 780

aaaggtgaca ctccggctga tctggctgct gactacggtc acgaggatat cgctgaagtt 840

ctgcagaagg cagcaggttc cccgacccct acgccaacga ctccgacccc aactccaacg 900

acccctaccc cgaccccgac cggatcagac ctgggtcaaa agttgttgga agctgcctgg 960

gcgggacagg atgatgaggt gcgcgaatta cttaaggcgg gagcagacgt gaatgcgaaa 1020

aactctcgtg gctggacacc actgcacacg gccgcgcaaa ctggtcacct tgaaattttc 1080

gaagtgcttc tgaaggcagg cgcagatgta aacgccaagg atgacaaagg ggtaacaccg 1140

cttcatctgg ctgctgcact gggacatctt gagattgtcg aagtactgct taaggcaggt 1200

gctgacgtaa acgctcagga ttcatggggg accacaccgg cggacctggc ggctaaatac 1260

ggacatgaag atattgctga agttctgcag aaggcggcat aatgatag 1308

<210> 92

<211> 1308

<212> DNA

<213> artificial sequence

<220>

<223> nucleic acid encoding the domain of SEQ ID NO 60

<400> 92

atgagaggat cgcatcacca tcaccatcac ggatccgacc tgggttataa acttcttcaa 60

gcggcctatg atgggcagct tgatgaggta cgcattttgt taaaggccgg cgcagatgtc 120

aacgcgaaag atagtcgcgg acagacgccc cttcattacg ccgcatcaat cggccatctg 180

gaaattgtag aggtactgct taaagccggt gctgatgtaa acgctaagga cgaccacgga 240

tggacgccac ttcatcttgc cgcgtggagt ggacacttgg agattgtcga agtcttgtta 300

aaagcgggcg ctgacgttaa cgcacaagac caggaaggta cgacgccagc agacttggct 360

gctgtccaag ggcaccagga cattgctgaa gttctgcaga aggcagcagg cagccctaca 420

cctacgccga ctacgcctac gccgactccg actaccccga ctccgacccc gaccggatca 480

gacctgggtg ttaaactttt gttagctgca tctcgtggtc aactggatga ggtgcgtatt 540

ttgctgaaag cgggtgcaga tgttaacgca aaggacatcg atgaaggata tacgccatta 600

cacattgcag catattatgg tcatttagag atcgtagagg ttcttttgaa ggcaggagcc 660

gatgttaacg ccaaggaccg ttatggaaag accccgttac atttagccgc aattagtggg 720

catcttgaaa ttgtcgaagt tttattaaag gctggggctg atgtaaatgc tcaggatgac 780

aagggcgaca ctcccgcaga tctggcggca gactatgggc accaggatat tgctgaagtt 840

ctgcagaagg cagcaggttc cccgacccct acgccaacga ctccgacccc aactccaacg 900

acccctaccc cgaccccgac cggatcagac ctgggtcaaa agttgttgga agctgcctgg 960

gcgggacagg atgatgaggt gcgcgaatta cttaaggcgg gagcagacgt gaatgcgaaa 1020

aactctcgtg gctggacacc actgcacacg gccgcgcaaa ctggtcacct tgaaattttc 1080

gaagtgcttc tgaaggcagg cgcagatgta aacgccaagg atgacaaagg ggtaacaccg 1140

cttcatctgg ctgctgcact gggacatctt gagattgtcg aagtactgct taaggcaggt 1200

gctgacgtaa acgctcagga ttcatggggg accacaccgg cggacctggc ggctaaatac 1260

ggacatgaag atattgctga agttctgcag aaggcggcat aatgatag 1308

<210> 93

<211> 1749

<212> DNA

<213> artificial sequence

<220>

<223> nucleic acid encoding the domain of SEQ ID NO 61

<400> 93

atgagaggat cgcatcacca tcaccatcac ggatccgacc tgggttataa acttcttcaa 60

gcggcctatg atgggcagct tgatgaggta cgcattttgt taaaggccgg cgcagatgtc 120

aacgcgaaag atagtcgcgg acagacgccc cttcattacg ccgcatcaat cggccatctg 180

gaaattgtag aggtactgct taaagccggt gctgatgtaa acgctaagga cgaccacgga 240

tggacgccac ttcatcttgc cgcgtggagt ggacacttgg agattgtcga agtcttgtta 300

aaagcgggcg ctgacgttaa cgcacaagac caggaaggta cgacgccagc agacttggct 360

gctgtccaag ggcaccagga cattgctgaa gttctgcaga aggcagcagg ctcgccgact 420

ccgaccccga ccaccccaac tccaacaccg accaccccga cccctacccc aacaggatcc 480

gacctgggta agaagctgct ggaagctgct ctggaaggtc aggatgatga agttcgtgaa 540

ctgctgaaag caggcgccga tgttaatgca aaagatcaag agggctacac cccactgcat 600

ctggctgctg ctctgggtca cctggaaatt gttgaagttc tgctgaaagc cggtgcagat 660

gttaatgcaa aagattctat cggcagaacc ccgctgcatc tggctgctta caagggtcac 720

ctggaaattg ttgaagttct gctgaaagcc ggtgcagatg ttaacgcaca ggatctgctg 780

ggcgaaaccc ctgccgatct ggcagctgaa caaggtcatg aagatattgc agaagtgctg 840

cagaaggcag caggcagccc tacacctacg ccgactacgc ctacgccgac tccgactacc 900

ccgactccga ccccgaccgg atcagacctg ggtgttaaac tgctgcgtgc tgctttccat 960

ggtcaggacg acgaagttcg tatcctgctg gctgctggcg ctgacgttaa tgctaaagac 1020

actgacggtg aaactccgct gcactacgct gctcagttcg gtcacctgga aatcgttgaa 1080

gttctgctga aggctggtgc tgacgttaat gctaaagacg cttacggtgc tactccgctg 1140

cactgggctg cttggcatgg tcacctggaa atcgttgaag ttctgctgaa ggctggtgct 1200

gacgtcaacg ctcaggacgt ttctggtgct actccggctg atctggctgc taaagttggt 1260

cacgaggata tcgctgaagt tctgcagaag gcagcaggtt ccccgacccc tacgccaacg 1320

actccgaccc caactccaac gacccctacc ccgaccccga ccggatcaga cctgggtcaa 1380

aagttgttgg aagctgcctg ggcgggacag gatgatgagg tgcgcgaatt acttaaggcg 1440

ggagcagacg tgaatgcgaa aaactctcgt ggctggacac cactgcacac ggccgcgcaa 1500

actggtcacc ttgaaatttt cgaagtgctt ctgaaggcag gcgcagatgt aaacgccaag 1560

gatgacaaag gggtaacacc gcttcatctg gctgctgcac tgggacatct tgagattgtc 1620

gaagtactgc ttaaggcagg tgctgacgta aacgctcagg attcatgggg gaccacaccg 1680

gcggacctgg cggctaaata cggacatgaa gatattgctg aagttctgca gaaggcggca 1740

taatgatag 1749

<210> 94

<211> 1749

<212> DNA

<213> artificial sequence

<220>

<223> nucleic acid encoding the domain of SEQ ID NO 62

<400> 94

atgagaggat cgcatcacca tcaccatcac ggatccgacc tgggttataa acttcttcaa 60

gcggcctatg atgggcagct tgatgaggta cgcattttgt taaaggccgg cgcagatgtc 120

aacgcgaaag atagtcgcgg acagacgccc cttcattacg ccgcatcaat cggccatctg 180

gaaattgtag aggtactgct taaagccggt gctgatgtaa acgctaagga cgaccacgga 240

tggacgccac ttcatcttgc cgcgtggagt ggacacttgg agattgtcga agtcttgtta 300

aaagcgggcg ctgacgttaa cgcacaagac caggaaggta cgacgccagc agacttggct 360

gctgtccaag ggcaccagga cattgctgaa gttctgcaga aggcagcagg cagccctaca 420

cctacgccga ctacgcctac gccgactccg actaccccga ctccgacccc gaccggatca 480

gacctgggta aaaagctgtt ggaggccgcg ttagagggtc aattggatga agttcgtgaa 540

ctgttaaaag cgggcgccga tgtaaacgct aaagaccagg aggggtacac tcctttgcat 600

ctggcagcag ctcttggtca tctggaaatt gtcgaggtgc tgttaaaggc aggagcagat 660

gtaaatgcaa aggactctat tggacgtaca ccactgcact tggctgccta caaaggtcac 720

ctggaaattg tggaagtctt actgaaagcg ggcgctgacg ttaacgccca agacctgctg 780

ggggaaacgc cagccgacct ggccgccgag cagggacatc aggatattgc tgaagttctg 840

cagaaggcag caggctcgcc aacgccgacc cctacaacgc caaccccgac accaactaca 900

ccgaccccca caccaacggg atcagacctg ggtgttaagt tgcttcgtgc tgccgtccac 960

ggtcaattgg atgaagtacg catccttctg aaggctggtg cagacgtgaa cgcgaaagac 1020

actgacggcg aaacccccct tcattacgcg gcacaattcg gccacttgga gatcgttgag 1080

gtccttctga aagccggcgc agacgtgaat gcaaaggatg cttatggggc tacgccgtta 1140

cattgggctg cttggcacgg ccatcttgag attgttgagg tcctgttgaa agcgggggcg 1200

gatgtaaacg ctcaggacgt atccggcgcg acacctgctg acttagcagc taaagtcgga 1260

caccaggata ttgctgaagt tctgcagaag gcagcaggtt ccccgacccc tacgccaacg 1320

actccgaccc caactccaac gacccctacc ccgaccccga ccggatcaga cctgggtcaa 1380

aagttgttgg aagctgcctg ggcgggacag gatgatgagg tgcgcgaatt acttaaggcg 1440

ggagcagacg tgaatgcgaa aaactctcgt ggctggacac cactgcacac ggccgcgcaa 1500

actggtcacc ttgaaatttt cgaagtgctt ctgaaggcag gcgcagatgt aaacgccaag 1560

gatgacaaag gggtaacacc gcttcatctg gctgctgcac tgggacatct tgagattgtc 1620

gaagtactgc ttaaggcagg tgctgacgta aacgctcagg attcatgggg gaccacaccg 1680

gcggacctgg cggctaaata cggacatgaa gatattgctg aagttctgca gaaggcggca 1740

taatgatag 1749

<210> 95

<211> 865

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #56

<400> 95

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg

180 185 190

Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala

210 215 220

Ala Asp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala

245 250 255

Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

275 280 285

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Val Lys Leu Leu Leu

290 295 300

Ala Ala Glu Arg Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys Ala

305 310 315 320

Gly Ala Asp Val Asn Ala Lys Asp Ile Ala Glu Gly Tyr Thr Pro Leu

325 330 335

His Ile Ala Ala Tyr Gln Gly His Leu Glu Ile Val Glu Val Leu Leu

340 345 350

Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Arg Tyr Gly Lys Thr Pro

355 360 365

Leu His Leu Ala Ala Ile Gly Gly His Leu Glu Ile Val Glu Val Leu

370 375 380

Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Asn Lys Gly Ser Thr

385 390 395 400

Pro Ala Asp Leu Ala Ala Asp Tyr Gly His Gln Asp Ile Ala Glu Val

405 410 415

Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr

420 425 430

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly

435 440 445

Lys Lys Leu Leu Glu Ala Ala Leu Glu Gly Gln Leu Asp Glu Val Arg

450 455 460

Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Gln Glu Gly

465 470 475 480

Tyr Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val

485 490 495

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Gln Ile

500 505 510

Gly Arg Thr Pro Leu His Leu Ala Ala Tyr Lys Gly His Leu Glu Ile

515 520 525

Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ile

530 535 540

Ile Gly Gln Thr Pro Ala Asp Leu Ala Ala Gln Arg Gly His Gln Asp

545 550 555 560

Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro

565 570 575

Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly

580 585 590

Ser Asp Leu Gly Ile Lys Leu Leu Thr Ala Ala Tyr Asp Gly Gln Leu

595 600 605

Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

610 615 620

Asp Leu Arg Gly Gln Thr Pro Leu His Tyr Ala Ala Gly Leu Gly His

625 630 635 640

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

645 650 655

Lys Asp Leu His Gly Trp Thr Pro Leu His Leu Ala Ala Trp Ser Gly

660 665 670

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

675 680 685

Ala Gln Asp Val Glu Gly Val Thr Pro Ala Asp Leu Ala Ala Val Gln

690 695 700

Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro

705 710 715 720

Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

725 730 735

Thr Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp

740 745 750

Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp

755 760 765

Val Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala

770 775 780

Gln Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala

785 790 795 800

Asp Val Asn Ala Lys Asn Asp Lys Arg Val Thr Pro Leu His Leu Ala

805 810 815

Ala Ala Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly

820 825 830

Ala Asp Val Asn Ala Arg Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu

835 840 845

Ala Ala Lys Tyr Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala

850 855 860

Ala

865

<210> 96

<211> 847

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #57

<400> 96

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg

180 185 190

Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala

210 215 220

Ala Asp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala

245 250 255

Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

275 280 285

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Ile Lys Leu Leu Thr

290 295 300

Ala Ala Tyr Asp Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys Ala

305 310 315 320

Gly Ala Asp Val Asn Ala Lys Asp Leu Arg Gly Gln Thr Pro Leu His

325 330 335

Tyr Ala Ala Gly Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys

340 345 350

Ala Gly Ala Asp Val Asn Ala Lys Asp Leu His Gly Trp Thr Pro Leu

355 360 365

His Leu Ala Ala Trp Ser Gly His Leu Glu Ile Val Glu Val Leu Leu

370 375 380

Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Val Glu Gly Val Thr Pro

385 390 395 400

Ala Asp Leu Ala Ala Val Gln Gly His Gln Asp Ile Ala Glu Val Leu

405 410 415

Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

420 425 430

Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys

435 440 445

Lys Leu Leu Gln Ala Ala Leu Glu Gly Gln Leu Asp Glu Val Arg Glu

450 455 460

Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Ile Glu Gly Tyr

465 470 475 480

Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val Glu

485 490 495

Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Gln Ile Gly

500 505 510

Arg Thr Pro Leu His Leu Ala Ala Tyr Lys Gly His Leu Glu Ile Val

515 520 525

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ile Ile

530 535 540

Gly Gln Thr Pro Ala Asp Leu Ala Ala Gln Arg Gly His Gln Asp Ile

545 550 555 560

Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr

565 570 575

Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser

580 585 590

Asp Leu Gly Lys Lys Leu Leu Leu Ala Ala Glu Arg Gly Gln Leu Asp

595 600 605

Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

610 615 620

Arg Ala Glu Gly Tyr Thr Pro Leu His Ile Ala Ala Tyr Gln Gly His

625 630 635 640

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

645 650 655

Lys Asp Arg Tyr Gly Lys Thr Pro Leu His Leu Ala Ala Ile Ser Gly

660 665 670

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

675 680 685

Ala Gln Asp Asn Lys Gly Ser Thr Pro Ala Asp Leu Ala Ala Asp Tyr

690 695 700

Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro

705 710 715 720

Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly

725 730 735

Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn

740 745 750

Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala Gln Thr

755 760 765

Gly His Leu Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp Val

770 775 780

Asn Ala Lys Asn Asp Lys Arg Val Thr Pro Leu His Leu Ala Ala Ala

785 790 795 800

Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

805 810 815

Val Asn Ala Arg Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala

820 825 830

Lys Tyr Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

835 840 845

<210> 97

<211> 847

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #58

<400> 97

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg

180 185 190

Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala

210 215 220

Ala Asp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala

245 250 255

Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

275 280 285

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Ile Lys Leu Leu Thr

290 295 300

Ala Ala Tyr Asp Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys Ala

305 310 315 320

Gly Ala Asp Val Asn Ala Lys Asp Leu Arg Gly Gln Thr Pro Leu His

325 330 335

Tyr Ala Ala Gly Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys

340 345 350

Ala Gly Ala Asp Val Asn Ala Lys Asp Leu His Gly Trp Thr Pro Leu

355 360 365

His Leu Ala Ala Trp Ser Gly His Leu Glu Ile Val Glu Val Leu Leu

370 375 380

Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Val Glu Gly Val Thr Pro

385 390 395 400

Ala Asp Leu Ala Ala Val Gln Gly His Gln Asp Ile Ala Glu Val Leu

405 410 415

Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

420 425 430

Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Trp

435 440 445

Lys Leu Leu Asp Ala Ala Glu Ile Gly Gln Leu Asp Glu Val Arg Ile

450 455 460

Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Gln Gly Ile

465 470 475 480

Thr Pro Leu His Ile Ala Ala Ala His Gly His Leu Glu Ile Val Glu

485 490 495

Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Glu Ile Gly

500 505 510

Arg Thr Pro Leu His Leu Ala Ala Phe Lys Gly His Leu Glu Ile Val

515 520 525

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ile Ile

530 535 540

Gly Glu Thr Pro Ala Asp Leu Ala Ala Val Arg Gly His Gln Asp Ile

545 550 555 560

Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr

565 570 575

Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser

580 585 590

Asp Leu Gly Val Lys Leu Leu Leu Ala Ala Glu Arg Gly Gln Leu Asp

595 600 605

Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

610 615 620

Ile Ala Glu Gly Tyr Thr Pro Leu His Ile Ala Ala Tyr Gln Gly His

625 630 635 640

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

645 650 655

Lys Asp Arg Tyr Gly Lys Thr Pro Leu His Leu Ala Ala Ile Gly Gly

660 665 670

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

675 680 685

Ala Gln Asp Asn Lys Gly Ser Thr Pro Ala Asp Leu Ala Ala Asp Tyr

690 695 700

Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro

705 710 715 720

Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly

725 730 735

Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn

740 745 750

Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala Gln Thr

755 760 765

Gly His Leu Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp Val

770 775 780

Asn Ala Lys Asn Asp Lys Arg Val Thr Pro Leu His Leu Ala Ala Ala

785 790 795 800

Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

805 810 815

Val Asn Ala Arg Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala

820 825 830

Lys Tyr Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

835 840 845

<210> 98

<211> 847

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #59

<400> 98

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg

180 185 190

Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala

210 215 220

Ala Asp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala

245 250 255

Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

275 280 285

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Ile Lys Leu Leu Thr

290 295 300

Ala Ala Tyr Asp Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys Ala

305 310 315 320

Gly Ala Asp Val Asn Ala Lys Asp Leu Arg Gly Gln Thr Pro Leu His

325 330 335

Tyr Ala Ala Gly Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys

340 345 350

Ala Gly Ala Asp Val Asn Ala Lys Asp Leu His Gly Trp Thr Pro Leu

355 360 365

His Leu Ala Ala Trp Ser Gly His Leu Glu Ile Val Glu Val Leu Leu

370 375 380

Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Val Glu Gly Val Thr Pro

385 390 395 400

Ala Asp Leu Ala Ala Val Gln Gly His Gln Asp Ile Ala Glu Val Leu

405 410 415

Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

420 425 430

Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys

435 440 445

Lys Leu Leu Glu Ala Ala Leu Glu Gly Gln Leu Asp Glu Val Arg Glu

450 455 460

Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Gln Glu Gly Tyr

465 470 475 480

Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val Glu

485 490 495

Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Gln Ile Gly

500 505 510

Arg Thr Pro Leu His Leu Ala Ala Tyr Lys Gly His Leu Glu Ile Val

515 520 525

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ile Ile

530 535 540

Gly Gln Thr Pro Ala Asp Leu Ala Ala Gln Arg Gly His Gln Asp Ile

545 550 555 560

Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr

565 570 575

Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser

580 585 590

Asp Leu Gly Val Lys Leu Leu Leu Ala Ala Glu Arg Gly Gln Leu Asp

595 600 605

Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

610 615 620

Ile Ala Glu Gly Tyr Thr Pro Leu His Ile Ala Ala Tyr Gln Gly His

625 630 635 640

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

645 650 655

Lys Asp Arg Tyr Gly Lys Thr Pro Leu His Leu Ala Ala Ile Gly Gly

660 665 670

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

675 680 685

Ala Gln Asp Asn Lys Gly Ser Thr Pro Ala Asp Leu Ala Ala Asp Tyr

690 695 700

Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro

705 710 715 720

Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly

725 730 735

Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn

740 745 750

Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala Gln Thr

755 760 765

Gly His Leu Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp Val

770 775 780

Asn Ala Lys Asn Asp Lys Arg Val Thr Pro Leu His Leu Ala Ala Ala

785 790 795 800

Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

805 810 815

Val Asn Ala Arg Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala

820 825 830

Lys Tyr Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

835 840 845

<210> 99

<211> 880

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #60

<400> 99

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg

180 185 190

Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala

210 215 220

Ala Asp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala

245 250 255

Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

275 280 285

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Ile Lys Leu Leu Thr

290 295 300

Ala Ala Tyr Asp Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys Ala

305 310 315 320

Gly Ala Asp Val Asn Ala Lys Asp Leu Arg Gly Gln Thr Pro Leu His

325 330 335

Tyr Ala Ala Gly Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys

340 345 350

Ala Gly Ala Asp Val Asn Ala Lys Asp Leu His Gly Trp Thr Pro Leu

355 360 365

His Leu Ala Ala Trp Ser Gly His Leu Glu Ile Val Glu Val Leu Leu

370 375 380

Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Val Glu Gly Val Thr Pro

385 390 395 400

Ala Asp Leu Ala Ala Val Gln Gly His Gln Asp Ile Ala Glu Val Leu

405 410 415

Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

420 425 430

Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys

435 440 445

Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp Glu Val Arg Glu

450 455 460

Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Gln Tyr Gly Arg

465 470 475 480

Thr Pro Leu His Leu Ala Ala Ile Lys Gly His Leu Glu Ile Val Glu

485 490 495

Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Ser Leu Gly

500 505 510

Tyr Thr Pro Leu His Leu Ala Ala Val Glu Gly Pro Leu Glu Ile Val

515 520 525

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Ala Tyr

530 535 540

Gly Gln Thr Pro Leu His Ile Ala Ala Ala Trp Gly His Leu Glu Ile

545 550 555 560

Val Glu Val Leu Leu Lys Ala Val Ala Asp Val Asn Ala Gln Asp Lys

565 570 575

Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala Gly His Gln Asp

580 585 590

Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro

595 600 605

Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly

610 615 620

Ser Asp Leu Gly Val Lys Leu Leu Leu Ala Ala Glu Arg Gly Gln Leu

625 630 635 640

Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

645 650 655

Asp Ile Ala Glu Gly Tyr Thr Pro Leu His Ile Ala Ala Tyr Gln Gly

660 665 670

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

675 680 685

Ala Lys Asp Arg Tyr Gly Lys Thr Pro Leu His Leu Ala Ala Ile Gly

690 695 700

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val

705 710 715 720

Asn Ala Gln Asp Asn Lys Gly Ser Thr Pro Ala Asp Leu Ala Ala Asp

725 730 735

Tyr Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser

740 745 750

Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala

755 760 765

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

770 775 780

Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala Gln

785 790 795 800

Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp

805 810 815

Val Asn Ala Lys Asn Asp Lys Arg Val Thr Pro Leu His Leu Ala Ala

820 825 830

Ala Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

835 840 845

Asp Val Asn Ala Arg Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala

850 855 860

Ala Lys Tyr Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

865 870 875 880

<210> 100

<211> 880

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #61

<400> 100

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg

180 185 190

Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala

210 215 220

Ala Asp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala

245 250 255

Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

275 280 285

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Gln

290 295 300

Ala Ala Arg Ala Gly Gln Leu Asp Glu Val Arg Glu Leu Leu Lys Ala

305 310 315 320

Gly Ala Asp Val Asn Ala Lys Asp Gln Gln Gly Leu Thr Pro Leu His

325 330 335

Ile Ala Ala Asn Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys

340 345 350

Ala Gly Ala Asp Val Asn Ala Lys Asp Leu Phe Gly Leu Thr Pro Leu

355 360 365

His Leu Ala Ala Phe Glu Gly His Leu Glu Ile Val Glu Val Leu Leu

370 375 380

Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Gln His Gly Ala Thr Pro

385 390 395 400

Leu His Leu Ala Ala Trp Val Gly His Leu Glu Ile Val Glu Val Leu

405 410 415

Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr

420 425 430

Pro Ala Asp Leu Ala Ala Arg Ala Gly His Gln Asp Ile Ala Glu Val

435 440 445

Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr

450 455 460

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly

465 470 475 480

Lys Lys Leu Leu Gln Ala Ala Leu Glu Gly Gln Leu Asp Glu Val Arg

485 490 495

Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Ile Glu Gly

500 505 510

Tyr Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val

515 520 525

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Gln Ile

530 535 540

Gly Arg Thr Pro Leu His Leu Ala Ala Tyr Lys Gly His Leu Glu Ile

545 550 555 560

Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ile

565 570 575

Ile Gly Gln Thr Pro Ala Asp Leu Ala Ala Gln Arg Gly His Gln Asp

580 585 590

Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro

595 600 605

Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly

610 615 620

Ser Asp Leu Gly Lys Lys Leu Leu Leu Ala Ala Glu Arg Gly Gln Leu

625 630 635 640

Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

645 650 655

Asp Arg Ala Glu Gly Tyr Thr Pro Leu His Ile Ala Ala Tyr Gln Gly

660 665 670

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

675 680 685

Ala Lys Asp Arg Tyr Gly Lys Thr Pro Leu His Leu Ala Ala Ile Ser

690 695 700

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val

705 710 715 720

Asn Ala Gln Asp Asn Lys Gly Ser Thr Pro Ala Asp Leu Ala Ala Asp

725 730 735

Tyr Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser

740 745 750

Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala

755 760 765

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

770 775 780

Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala Gln

785 790 795 800

Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp

805 810 815

Val Asn Ala Lys Asn Asp Lys Arg Val Thr Pro Leu His Leu Ala Ala

820 825 830

Ala Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

835 840 845

Asp Val Asn Ala Arg Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala

850 855 860

Ala Lys Tyr Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

865 870 875 880

<210> 101

<211> 865

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #62

<400> 101

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg

180 185 190

Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala

210 215 220

Ala Asp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala

245 250 255

Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

275 280 285

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Ile Lys Leu Leu Thr

290 295 300

Ala Ala Tyr Asp Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys Ala

305 310 315 320

Gly Ala Asp Val Asn Ala Lys Asp Leu Arg Gly Gln Thr Pro Leu His

325 330 335

Tyr Ala Ala Gly Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys

340 345 350

Ala Gly Ala Asp Val Asn Ala Lys Asp Leu His Gly Trp Thr Pro Leu

355 360 365

His Leu Ala Ala Trp Ser Gly His Leu Glu Ile Val Glu Val Leu Leu

370 375 380

Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Val Glu Gly Val Thr Pro

385 390 395 400

Ala Asp Leu Ala Ala Val Gln Gly His Gln Asp Ile Ala Glu Val Leu

405 410 415

Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

420 425 430

Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Val

435 440 445

Lys Leu Leu Leu Ala Ala Glu Arg Gly Gln Leu Asp Glu Val Arg Ile

450 455 460

Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Ile Ala Glu Gly

465 470 475 480

Tyr Thr Pro Leu His Ile Ala Ala Tyr Gln Gly His Leu Glu Ile Val

485 490 495

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Arg Tyr

500 505 510

Gly Lys Thr Pro Leu His Leu Ala Ala Ile Gly Gly His Leu Glu Ile

515 520 525

Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Asn

530 535 540

Lys Gly Ser Thr Pro Ala Asp Leu Ala Ala Asp Tyr Gly His Gln Asp

545 550 555 560

Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro

565 570 575

Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly

580 585 590

Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Leu Glu Gly Gln Leu

595 600 605

Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

610 615 620

Asp Gln Glu Gly Tyr Thr Pro Leu His Leu Ala Ala Ala Leu Gly His

625 630 635 640

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

645 650 655

Lys Asp Gln Ile Gly Arg Thr Pro Leu His Leu Ala Ala Tyr Lys Gly

660 665 670

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

675 680 685

Ala Gln Asp Ile Ile Gly Gln Thr Pro Ala Asp Leu Ala Ala Gln Arg

690 695 700

Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro

705 710 715 720

Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

725 730 735

Thr Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp

740 745 750

Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp

755 760 765

Val Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala

770 775 780

Gln Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala

785 790 795 800

Asp Val Asn Ala Lys Asn Asp Lys Arg Val Thr Pro Leu His Leu Ala

805 810 815

Ala Ala Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly

820 825 830

Ala Asp Val Asn Ala Arg Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu

835 840 845

Ala Ala Lys Tyr Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala

850 855 860

Ala

865

<210> 102

<211> 157

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #63

<400> 102

Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Gln Tyr Gly Arg Thr Pro Leu His Leu Ala Ala Ile Lys Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Ser Leu Gly Tyr Thr Pro Leu His Leu Ala Ala Val Glu Gly Pro

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Lys Asp Ala Tyr Gly Gln Thr Pro Leu His Ile Ala Ala Ala Trp Gly

100 105 110

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Val Ala Asp Val Asn

115 120 125

Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala

130 135 140

Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

145 150 155

<210> 103

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #64

<400> 103

Asp Leu Gly Trp Lys Leu Leu Asp Ala Ala Glu Ile Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Lys Gln Gly Ile Thr Pro Leu His Ile Ala Ala Ala His Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Glu Ile Gly Arg Thr Pro Leu His Leu Ala Ala Phe Lys Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Ile Gly Glu Thr Pro Ala Asp Leu Ala Ala Val Arg Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120

<210> 104

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #65

<400> 104

Asp Leu Gly Trp Lys Leu Leu Asp Ala Ala Glu Ile Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Lys Gln Gly Ile Thr Pro Leu His Ile Ala Ala Ala His Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Glu Ile Gly Arg Thr Pro Leu His Leu Ala Ala Phe Lys Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Ile Gly Glu Thr Pro Ala Asp Leu Ala Ala Val Arg Gly

100 105 110

His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120

<210> 105

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #66

<400> 105

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Leu Glu Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Gln Glu Gly Tyr Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Gln Ile Gly Arg Thr Pro Leu His Leu Ala Ala Tyr Lys Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Ile Gly Gln Thr Pro Ala Asp Leu Ala Ala Gln Arg Gly

100 105 110

His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120

<210> 106

<211> 123

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #67

<400> 106

Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Leu Glu Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Ile Glu Gly Tyr Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Gln Ile Gly Arg Thr Pro Leu His Leu Ala Ala Tyr Lys Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Ile Gly Gln Thr Pro Ala Asp Leu Ala Ala Gln Arg Gly

100 105 110

His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala

115 120

<210> 107

<211> 157

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #68

<400> 107

Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Gln Ala Gly Leu Thr Pro Leu His Ile Ala Ala Ala Thr Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ser Gly Leu Thr Pro Leu His Leu Ala Ala Phe Glu Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Lys Asp Gln His Gly Gln Thr Pro Leu His Leu Ala Ala Trp Thr Gly

100 105 110

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

115 120 125

Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala

130 135 140

Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

145 150 155

<210> 108

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #69

<400> 108

Asp Leu Gly Tyr Lys Leu Leu Gln Ala Ala Tyr Asp Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Ala Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Ser Arg Gly Gln Thr Pro Leu His Tyr Ala Ala Ser Ile Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Asp His Gly Trp Thr Pro Leu His Leu Ala Ala Trp Ser Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Gln Glu Gly Thr Thr Pro Ala Asp Leu Ala Ala Val Gln Ser

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120

<210> 109

<211> 124

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #70

<400> 109

Asp Leu Gly Ile Lys Leu Leu Thr Ala Ala Tyr Asp Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Leu Arg Gly Gln Thr Pro Leu His Tyr Ala Ala Gly Leu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Leu His Gly Trp Thr Pro Leu His Leu Ala Ala Trp Ser Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Val Glu Gly Val Thr Pro Ala Asp Leu Ala Ala Val Gln Gly

100 105 110

His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120

<210> 110

<211> 157

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #71

<400> 110

Asp Leu Gly Lys Lys Leu Leu Gln Ala Ala Arg Ala Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Gln Gln Gly Leu Thr Pro Leu His Ile Ala Ala Asn Leu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Leu Phe Gly Leu Thr Pro Leu His Leu Ala Ala Phe Glu Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Lys Asp Gln His Gly Ala Thr Pro Leu His Leu Ala Ala Trp Val Gly

100 105 110

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

115 120 125

Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Arg Ala

130 135 140

Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

145 150 155

<210> 111

<211> 125

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #72

<400> 111

Asp Leu Gly Val Lys Leu Leu Leu Ala Ala Glu Arg Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Ile Ala Glu Gly Tyr Thr Pro Leu His Ile Ala Ala Tyr Gln Gly His

35 40 45

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

50 55 60

Lys Asp Arg Tyr Gly Lys Thr Pro Leu His Leu Ala Ala Ile Gly Gly

65 70 75 80

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

85 90 95

Ala Gln Asp Asn Lys Gly Ser Thr Pro Ala Asp Leu Ala Ala Asp Tyr

100 105 110

Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120 125

<210> 112

<211> 125

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #73

<400> 112

Asp Leu Gly Lys Lys Leu Leu Leu Ala Ala Glu Arg Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Arg Ala Glu Gly Tyr Thr Pro Leu His Ile Ala Ala Tyr Gln Gly His

35 40 45

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

50 55 60

Lys Asp Arg Tyr Gly Lys Thr Pro Leu His Leu Ala Ala Ile Ser Gly

65 70 75 80

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

85 90 95

Ala Gln Asp Asn Lys Gly Ser Thr Pro Ala Asp Leu Ala Ala Asp Tyr

100 105 110

Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

115 120 125

<210> 113

<211> 864

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #74

<400> 113

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg

180 185 190

Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala

210 215 220

Ala Asp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala

245 250 255

Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

275 280 285

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu

290 295 300

Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala

305 310 315 320

Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His

325 330 335

Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys

340 345 350

Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu

355 360 365

His Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val Glu Val Leu Leu

370 375 380

Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro

385 390 395 400

Ala Asp Leu Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu

405 410 415

Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

420 425 430

Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys

435 440 445

Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Glu

450 455 460

Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr

465 470 475 480

Thr Pro Leu His Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val Glu

485 490 495

Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly

500 505 510

Tyr Thr Pro Leu His Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val

515 520 525

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser

530 535 540

Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly His Glu Asp Ile

545 550 555 560

Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr

565 570 575

Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser

580 585 590

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

595 600 605

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

610 615 620

Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg Glu Gly His Leu

625 630 635 640

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

645 650 655

Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg Glu Gly His

660 665 670

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

675 680 685

Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly

690 695 700

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

705 710 715 720

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

725 730 735

Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala

740 745 750

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

755 760 765

Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala Gln

770 775 780

Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp

785 790 795 800

Val Asn Ala Lys Asn Asp Lys Arg Val Thr Pro Leu His Leu Ala Ala

805 810 815

Ala Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

820 825 830

Asp Val Asn Ala Arg Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala

835 840 845

Ala Lys Tyr Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

850 855 860

<210> 114

<211> 865

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #75

<400> 114

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg

180 185 190

Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala

210 215 220

Ala Asp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala

245 250 255

Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

275 280 285

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Val Lys Leu Leu Leu

290 295 300

Ala Ala Glu Arg Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys Ala

305 310 315 320

Gly Ala Asp Val Asn Ala Lys Asp Ile Ala Glu Gly Tyr Thr Pro Leu

325 330 335

His Ile Ala Ala Tyr Gln Gly His Leu Glu Ile Val Glu Val Leu Leu

340 345 350

Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Arg Tyr Gly Lys Thr Pro

355 360 365

Leu His Leu Ala Ala Ile Gly Gly His Leu Glu Ile Val Glu Val Leu

370 375 380

Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Asn Lys Gly Ser Thr

385 390 395 400

Pro Ala Asp Leu Ala Ala Asp Tyr Gly His Gln Asp Ile Ala Glu Val

405 410 415

Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr

420 425 430

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly

435 440 445

Lys Lys Leu Leu Glu Ala Ala Leu Glu Gly Gln Leu Asp Glu Val Arg

450 455 460

Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Gln Glu Gly

465 470 475 480

Tyr Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val

485 490 495

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Gln Ile

500 505 510

Gly Arg Thr Pro Leu His Leu Ala Ala Tyr Lys Gly His Leu Glu Ile

515 520 525

Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ile

530 535 540

Ile Gly Gln Thr Pro Ala Asp Leu Ala Ala Gln Arg Gly His Gln Asp

545 550 555 560

Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro

565 570 575

Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly

580 585 590

Ser Asp Leu Gly Ile Lys Leu Leu Thr Ala Ala Tyr Asp Gly Gln Leu

595 600 605

Asp Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

610 615 620

Asp Leu Arg Gly Gln Thr Pro Leu His Tyr Ala Ala Gly Leu Gly His

625 630 635 640

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

645 650 655

Lys Asp Leu His Gly Trp Thr Pro Leu His Leu Ala Ala Trp Ser Gly

660 665 670

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

675 680 685

Ala Gln Asp Val Glu Gly Val Thr Pro Ala Asp Leu Ala Ala Val Gln

690 695 700

Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro

705 710 715 720

Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

725 730 735

Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg

740 745 750

Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp

755 760 765

Val Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala

770 775 780

Arg Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

785 790 795 800

Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala

805 810 815

Ala Arg Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly

820 825 830

Ala Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu

835 840 845

Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala

850 855 860

Ala

865

<210> 115

<211> 846

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #76

<400> 115

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg

180 185 190

Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala

210 215 220

Ala Asp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala

245 250 255

Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

275 280 285

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu

290 295 300

Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala

305 310 315 320

Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His

325 330 335

Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys

340 345 350

Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu

355 360 365

His Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val Glu Val Leu Leu

370 375 380

Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro

385 390 395 400

Ala Asp Leu Ala Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu

405 410 415

Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

420 425 430

Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys

435 440 445

Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp Glu Val Arg Glu

450 455 460

Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly Tyr

465 470 475 480

Thr Pro Leu His Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val Glu

485 490 495

Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Lys Asp Gly

500 505 510

Tyr Thr Pro Leu His Leu Ala Ala Arg Glu Gly His Leu Glu Ile Val

515 520 525

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Lys Ser

530 535 540

Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly His Glu Asp Ile

545 550 555 560

Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr

565 570 575

Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser

580 585 590

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

595 600 605

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

610 615 620

Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg Glu Gly His Leu

625 630 635 640

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

645 650 655

Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg Glu Gly His

660 665 670

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

675 680 685

Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala Asp Ala Gly

690 695 700

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

705 710 715 720

Gly Ser Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala Gly Gln

725 730 735

Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

740 745 750

Lys Asn Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala Gln Thr Gly

755 760 765

His Leu Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

770 775 780

Ala Lys Asn Asp Lys Arg Val Thr Pro Leu His Leu Ala Ala Ala Leu

785 790 795 800

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val

805 810 815

Asn Ala Arg Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala Ala Lys

820 825 830

Tyr Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

835 840 845

<210> 116

<211> 847

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #77

<400> 116

Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly Gln Asp Asp

1 5 10 15

Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg Asn Gly His Leu

35 40 45

Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala Ala Asp Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala Ala Asp Ala Gly

100 105 110

His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asp Tyr Phe Ser His Thr Pro Leu His Leu Ala Ala Arg

180 185 190

Asn Gly His Leu Lys Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asp Phe Ala Gly Lys Thr Pro Leu His Leu Ala Ala

210 215 220

Ala Asp Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Gln Asp Ile Phe Gly Lys Thr Pro Ala Asp Ile Ala

245 250 255

Ala Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr

275 280 285

Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Ile Lys Leu Leu Thr

290 295 300

Ala Ala Tyr Asp Gly Gln Leu Asp Glu Val Arg Ile Leu Leu Lys Ala

305 310 315 320

Gly Ala Asp Val Asn Ala Lys Asp Leu Arg Gly Gln Thr Pro Leu His

325 330 335

Tyr Ala Ala Gly Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys

340 345 350

Ala Gly Ala Asp Val Asn Ala Lys Asp Leu His Gly Trp Thr Pro Leu

355 360 365

His Leu Ala Ala Trp Ser Gly His Leu Glu Ile Val Glu Val Leu Leu

370 375 380

Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Val Glu Gly Val Thr Pro

385 390 395 400

Ala Asp Leu Ala Ala Val Gln Gly His Gln Asp Ile Ala Glu Val Leu

405 410 415

Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro

420 425 430

Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser Asp Leu Gly Lys

435 440 445

Lys Leu Leu Gln Ala Ala Leu Glu Gly Gln Leu Asp Glu Val Arg Glu

450 455 460

Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Ile Glu Gly Tyr

465 470 475 480

Thr Pro Leu His Leu Ala Ala Ala Leu Gly His Leu Glu Ile Val Glu

485 490 495

Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp Gln Ile Gly

500 505 510

Arg Thr Pro Leu His Leu Ala Ala Tyr Lys Gly His Leu Glu Ile Val

515 520 525

Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Gln Asp Ile Ile

530 535 540

Gly Gln Thr Pro Ala Asp Leu Ala Ala Gln Arg Gly His Gln Asp Ile

545 550 555 560

Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr Pro Thr Pro Thr

565 570 575

Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Gly Ser

580 585 590

Asp Leu Gly Lys Lys Leu Leu Leu Ala Ala Glu Arg Gly Gln Leu Asp

595 600 605

Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

610 615 620

Arg Ala Glu Gly Tyr Thr Pro Leu His Ile Ala Ala Tyr Gln Gly His

625 630 635 640

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

645 650 655

Lys Asp Arg Tyr Gly Lys Thr Pro Leu His Leu Ala Ala Ile Ser Gly

660 665 670

His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn

675 680 685

Ala Gln Asp Asn Lys Gly Ser Thr Pro Ala Asp Leu Ala Ala Asp Tyr

690 695 700

Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro

705 710 715 720

Thr Gly Ser Asp Leu Gly Lys Lys Leu Leu Glu Ala Ala Arg Ala Gly

725 730 735

Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val Asn

740 745 750

Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg Glu

755 760 765

Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val

770 775 780

Asn Ala Lys Asp Lys Asp Gly Tyr Thr Pro Leu His Leu Ala Ala Arg

785 790 795 800

Glu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp

805 810 815

Val Asn Ala Gln Asp Lys Ser Gly Lys Thr Pro Ala Asp Leu Ala Ala

820 825 830

Asp Ala Gly His Glu Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

835 840 845

<210> 117

<211> 272

<212> PRT

<213> artificial sequence

<220>

<223> DARPin_protein #78

<400> 117

Asp Leu Gly Ile Lys Leu Leu Thr Ala Ala Tyr Asp Gly Gln Leu Asp

1 5 10 15

Glu Val Arg Ile Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys Asp

20 25 30

Leu Arg Gly Gln Thr Pro Leu His Tyr Ala Ala Gly Leu Gly His Leu

35 40 45

Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala Lys

50 55 60

Asp Leu His Gly Trp Thr Pro Leu His Leu Ala Ala Trp Ser Gly His

65 70 75 80

Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala Asp Val Asn Ala

85 90 95

Gln Asp Val Glu Gly Val Thr Pro Ala Asp Leu Ala Ala Val Gln Gly

100 105 110

His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala Gly Ser Pro Thr

115 120 125

Pro Thr Pro Thr Thr Pro Thr Pro Thr Pro Thr Thr Pro Thr Pro Thr

130 135 140

Pro Thr Gly Ser Asp Leu Gly Gln Lys Leu Leu Glu Ala Ala Trp Ala

145 150 155 160

Gly Gln Asp Asp Glu Val Arg Glu Leu Leu Lys Ala Gly Ala Asp Val

165 170 175

Asn Ala Lys Asn Ser Arg Gly Trp Thr Pro Leu His Thr Ala Ala Gln

180 185 190

Thr Gly His Leu Glu Ile Phe Glu Val Leu Leu Lys Ala Gly Ala Asp

195 200 205

Val Asn Ala Lys Asn Asp Lys Arg Val Thr Pro Leu His Leu Ala Ala

210 215 220

Ala Leu Gly His Leu Glu Ile Val Glu Val Leu Leu Lys Ala Gly Ala

225 230 235 240

Asp Val Asn Ala Arg Asp Ser Trp Gly Thr Thr Pro Ala Asp Leu Ala

245 250 255

Ala Lys Tyr Gly His Gln Asp Ile Ala Glu Val Leu Gln Lys Ala Ala

260 265 270

<210> 118

<211> 2631

<212> DNA

<213> artificial sequence

<220>

<223> nucleic acid encoding protein of SEQ ID NO. 95

<400> 118

atgagaggat cgcatcacca tcaccatcac ggatccgacc tgggtaaaaa gctgctggag 60

gcagcgcgtg ccggtcaaga cgacgaggtt cgcgaattgc ttaaagcggg tgcagacgtc 120

aacgccaaag attatttctc tcataccccg ttgcatttag ccgcgcgtaa tggccatctg 180

aagatcgtcg aggtcctctt gaaggcaggc gcggatgtca atgcgaagga ttttgcgggc 240

aaaacgccgc tgcacttagc ggcggcggac ggtcatttag aaatcgttga agtcctgtta 300

aaagcgggcg ccgatgtgaa tgcgcaggat attttcggta aaacgccggc ggacattgcg 360

gcagatgcgg gtcatgaaga tatcgcagaa gtcctgcaga aggcagcagg cagccctaca 420

cctacgccga ctacgcctac gccgactccg actaccccga ctccgacccc gaccggatca 480

gacctgggta aaaagctgct ggaggcagcg cgtgccggtc aagacgacga ggttcgcgaa 540

ttgcttaaag cgggtgcaga cgtcaacgcc aaagattatt tctctcatac cccgttgcat 600

ttagccgcgc gtaatggcca tctgaagatc gtcgaggtcc tcttgaaggc aggcgcggat 660

gtcaatgcga aggattttgc gggcaaaacg ccgctgcact tagcggcggc ggacggtcat 720

ttagaaatcg ttgaagtcct gttaaaagcg ggcgccgatg tgaatgcgca ggatattttc 780

ggtaaaacgc cggcggacat tgcggcagat gcgggtcatg aagatatcgc agaagtcctg 840

cagaaggcag caggctcgcc aacgccgacc cctacaacgc caaccccgac accaactaca 900

ccgaccccca caccaacggg atcagacctg ggtgttaagc tgttgcttgc agccgagcgt 960

gggcagcttg atgaagtacg cattcttctt aaagcggggg ctgatgtgaa cgcgaaagat 1020

attgctgagg gttacacacc gcttcacatt gccgcctacc agggtcatct ggagattgtt 1080

gaagtattac tgaaagcggg agcagatgtt aatgccaaag atcgctatgg aaaaactcct 1140

ttgcatttag ctgcaatcgg aggacacctg gaaatcgtcg aagtgttatt aaaagctgga 1200

gcggacgtaa acgcacaaga taacaagggc tcaactcccg cggaccttgc cgcagattac 1260

ggtcatcagg acattgctga agttctgcag aaggcagcag gttccccgac ccctacgcca 1320

acgactccga ccccaactcc aacgacccct accccgaccc cgaccggatc agacctgggt 1380

aaaaaattgc ttgaagccgc gttggaagga caattagacg aggtacgtga gctgttaaaa 1440

gcaggggccg atgtgaatgc taaagaccag gagggataca cccccttgca cctggctgcc 1500

gcgttgggcc acttagagat tgtagaggtt cttcttaagg cgggggcaga cgtgaatgca 1560

aaggaccaaa ttggacgtac tcctttgcat ctggcagcct ataaggggca cttggagatt 1620

gtcgaggtct tgttaaaggc gggtgccgat gtaaatgccc aggacatcat tgggcagact 1680

ccggcagatt tggccgccca acgtggccac caagatattg ctgaagttct gcagaaggca 1740

gcaggcagcc ccacgccaac tcctacaacc cccacaccta caccgacgac gccgacaccg 1800

actccaaccg gatcagacct gggtattaaa ctgttgacag ccgcttacga cgggcaatta 1860

gacgaagtgc gtattctgct taaagctgga gctgacgtga acgcgaaaga cttacgcggc 1920

caaacgcctt tacattacgc ggcgggactg ggccatcttg agattgttga ggtgcttctg 1980

aaggcaggcg cggatgtcaa tgcaaaagac ctgcacggat ggacacctct tcacttagct 2040

gcttggtctg ggcatttgga gattgtagag gttttattga aagcaggggc ggatgtgaat 2100

gcgcaagacg tagaaggagt caccccagct gacctggcag cggttcaagg gcatcaagac 2160

attgctgaag ttctgcagaa ggcagcaggt tcgccgaccc caacccctac cactccaacg 2220

ccgacgccta ccactccaac accaacacca acgggatcag acctgggtca aaagctgttg 2280

gaagccgcgt gggcgggtca ggacgatgaa gtccgtgagc tgcttaaagc aggagccgac 2340

gtgaacgcga agaactcacg cgggtggacg ccacttcaca cggccgcgca gacaggtcac 2400

cttgaaatct ttgaggttct tctgaaggca ggagcagacg ttaacgccaa aaacgacaag 2460

cgcgtgactc cgttgcacct tgccgcagct ctggggcatt tggagatcgt tgaggtactg 2520

ttgaaagcgg gagcagatgt taatgctcgc gacagttggg ggacgacacc agcagacctg 2580

gccgcaaaat acggacacca agacattgct gaagttctgc agaaggcggc a 2631

<210> 119

<211> 2577

<212> DNA

<213> artificial sequence

<220>

<223> nucleic acid encoding protein of SEQ ID NO. 96

<400> 119

atgagaggat cgcatcacca tcaccatcac ggatccgacc tgggtaagaa actgttggaa 60

gcagcacgtg cgggtcaaga cgatgaagtt cgtgagctgt taaaggctgg cgccgacgtg 120

aacgcgaagg actactttag ccacaccccg ctgcacttgg cagcgcgcaa cggtcacctg 180

aaaattgtcg aggtcctgtt gaaggctggt gcggatgtga acgcaaaaga ttttgcgggt 240

aagacgccgc tgcatctggc ggcggctgat ggtcacttag agatcgtaga ggttctgttg 300

aaagcgggcg ccgatgtgaa tgcccaggac atcttcggca agaccccggc agacattgcc 360

gcggatgctg gtcacgaaga tatcgcagag gtcctgcaaa aagcggcagg cagcccgacc 420

ccgacgccga ccactccgac cccgacccca accaccccga ctccgactcc gaccggatct 480

gacctgggta aaaaactgct ggaagcagca cgtgccggcc aggatgatga agttcgtgaa 540

ctgctgaaag caggcgccga tgttaatgca aaggattatt ttagccacac accgctgcat 600

ctggcagccc gtaatggtca cctaaagatt gttgaagttc tgctgaaggc tggtgcagac 660

gttaacgcca aagatttcgc gggcaaaacc cctctgcatt tagccgcagc ggacggtcac 720

ctggagatcg tagaggtgct gcttaaggcg ggtgcggatg ttaatgcaca ggatattttc 780

ggtaaaaccc ctgccgatat tgcagctgat gccggtcatg aagatatcgc agaagtgctg 840

cagaaggcag caggatcacc aacaccaacc ccgaccaccc caactccaac accgaccacc 900

ccgaccccta ccccaacagg atccgacctg ggtattaaac tgttgacagc cgcttacgac 960

gggcaattag acgaagtgcg tattctgctt aaagctggag ctgacgtgaa cgcgaaagac 1020

ttacgcggcc aaacgccttt acattacgcg gcgggactgg gccatcttga gattgttgag 1080

gtgcttctga aggcaggcgc ggatgtcaat gcaaaagacc tgcacggatg gacacctctt 1140

cacttagctg cttggtctgg gcatttggag attgtagagg ttttattgaa agcaggggcg 1200

gatgtgaatg cgcaagacgt agaaggagtc accccagctg acctggcagc ggttcaaggg 1260

catcaagaca ttgctgaagt tctgcagaag gcagcaggct cgccgactcc gaccccgacc 1320

accccaactc caacaccgac caccccgacc cctaccccaa caggatctga cctgggtaaa 1380

aagttgttac aggcagcatt ggagggccaa cttgacgagg tgcgcgagtt actgaaagct 1440

ggtgcagatg tcaacgcgaa ggacattgaa ggatatactc cgctgcacct tgccgcggct 1500

ttggggcatc ttgagattgt ggaggtgctt cttaaggcgg gagctgatgt caatgctaaa 1560

gaccaaatcg ggcgcacacc gttacacttg gctgcgtaca aaggtcactt agaaatcgtg 1620

gaagtgcttc tgaaggctgg cgctgatgtc aacgcccaag acattatcgg ccagacaccg 1680

gcggacctgg cagcgcaacg tgggcatcag gatattgctg aagttctgca gaaggcagca 1740

ggctcgccga ctccgacccc gaccacccca actccaacac cgaccacccc gacccctacc 1800

ccaacaggat ctgacctggg taaaaagttg ttattagctg cggagcgcgg gcagttagac 1860

gaagtgcgta ttctgctgaa ggccggggcc gacgttaacg caaaggatcg tgcagagggt 1920

tacacccccc tgcacatcgc cgcttatcaa ggtcacttgg agattgttga ggtcttactg 1980

aaagcggggg ccgacgtgaa tgccaaagat cgctatggaa aaacaccgtt acacttagca 2040

gctatttcgg ggcatctgga gatcgtggaa gtcctgttaa aggctggtgc cgatgttaat 2100

gcacaagata ataaaggcag cactccagcc gatctggccg ctgattatgg gcaccaggac 2160

attgctgaag ttctgcagaa ggcagcaggc tcgccaaccg gatcagatct gggtcaaaag 2220

ctgttggaag ccgcgtgggc gggtcaggac gatgaagtcc gtgagctgct taaagcagga 2280

gccgacgtga acgcgaagaa ctcacgcggg tggacgccac ttcacacggc cgcgcagaca 2340

ggtcaccttg aaatctttga ggttcttctg aaggcaggag cagacgttaa cgccaaaaac 2400

gacaagcgcg tgactccgtt gcaccttgcc gcagctctgg ggcatttgga gatcgttgag 2460

gtactgttga aagcgggagc agatgttaat gctcgcgaca gttgggggac gacaccagca 2520

gacctggccg caaaatacgg acaccaagac attgctgaag ttctgcaaaa ggcagca 2577

<210> 120

<211> 2586

<212> DNA

<213> artificial sequence

<220>

<223> nucleic acid encoding protein of SEQ ID NO. 97

<400> 120

atgagaggat cgcatcacca tcaccatcac ggatccgacc tgggtaagaa actgttggaa 60

gcagcacgtg cgggtcaaga cgatgaagtt cgtgagctgt taaaggctgg cgccgacgtg 120

aacgcgaagg actactttag ccacaccccg ctgcacttgg cagcgcgcaa cggtcacctg 180

aaaattgtcg aggtcctgtt gaaggctggt gcggatgtga acgcaaaaga ttttgcgggt 240

aagacgccgc tgcatctggc ggcggctgat ggtcacttag agatcgtaga ggttctgttg 300

aaagcgggcg ccgatgtgaa tgcccaggac atcttcggca agaccccggc agacattgcc 360

gcggatgctg gtcacgaaga tatcgcagag gtcctgcaaa aagcggcagg cagcccgacc 420

ccgacgccga ccactccgac cccgacccca accaccccga ctccgactcc gaccggatct 480

gacctgggta aaaaactgct ggaagcagca cgtgccggcc aggatgatga agttcgtgaa 540

ctgctgaaag caggcgccga tgttaatgca aaggattatt ttagccacac accgctgcat 600

ctggcagccc gtaatggtca cctaaagatt gttgaagttc tgctgaaggc tggtgcagac 660

gttaacgcca aagatttcgc gggcaaaacc cctctgcatt tagccgcagc ggacggtcac 720

ctggagatcg tagaggtgct gcttaaggcg ggtgcggatg ttaatgcaca ggatattttc 780

ggtaaaaccc ctgccgatat tgcagctgat gccggtcatg aagatatcgc agaagtgctg 840

cagaaggcag caggatcacc aacaccaacc ccgaccaccc caactccaac accgaccacc 900

ccgaccccta ccccaacagg atccgacctg ggtattaaac tgttgacagc cgcttacgac 960

gggcaattag acgaagtgcg tattctgctt aaagctggag ctgacgtgaa cgcgaaagac 1020

ttacgcggcc aaacgccttt acattacgcg gcgggactgg gccatcttga gattgttgag 1080

gtgcttctga aggcaggcgc ggatgtcaat gcaaaagacc tgcacggatg gacacctctt 1140

cacttagctg cttggtctgg gcatttggag attgtagagg ttttattgaa agcaggggcg 1200

gatgtgaatg cgcaagacgt agaaggagtc accccagctg acctggcagc ggttcaaggg 1260

catcaagaca ttgctgaagt tctgcagaag gcagcaggct cgccgactcc gaccccgacc 1320

accccaactc caacaccgac caccccgacc cctaccccaa caggatctga cctgggttgg 1380

aaactgcttg atgccgccga gattggtcag cttgacgaag tccgtattct tttgaaggca 1440

ggggccgacg ttaatgccaa agacaaacag ggtatcacgc cgttacatat tgccgcagcg 1500

catggtcact tagagatcgt agaagtactt ctgaaagcag gtgctgacgt taatgcaaag 1560

gatgagatcg gccgcacccc gcttcatctt gctgccttta agggccattt ggaaatcgta 1620

gaggtgctgt taaaggctgg cgctgatgtc aatgcacaag acatcatcgg ggagacgcct 1680

gccgacctgg cggcggtacg cgggcatcag gatattgctg aagttctgca gaaggcagca 1740

ggctcgccga ctccgacccc gaccacccca actccaacac cgaccacccc gacccctacc 1800

ccaacaggat ctgacctggg tgttaagctg ttgcttgcag ccgagcgtgg gcagcttgat 1860

gaagtacgca ttcttcttaa agcgggggct gatgtgaacg cgaaagatat tgctgagggt 1920

tacacaccgc ttcacattgc cgcctaccag ggtcatctgg agattgttga agtattactg 1980

aaagcgggag cagatgttaa tgccaaagat cgctatggaa aaactccttt gcatttagct 2040

gcaatcggag gacacctgga aatcgtcgaa gtgttattaa aagctggagc ggacgtaaac 2100

gcacaagata acaagggctc aactcccgcg gaccttgccg cagattacgg tcatcaggac 2160

attgctgaag ttctgcagaa ggcagcaggc tcgccaaccg gatcagatct gggtcaaaag 2220

ctgttggaag ccgcgtgggc gggtcaggac gatgaagtcc gtgagctgct taaagcagga 2280

gccgacgtga acgcgaagaa ctcacgcggg tggacgccac ttcacacggc cgcgcagaca 2340

ggtcaccttg aaatctttga ggttcttctg aaggcaggag cagacgttaa cgccaaaaac 2400

gacaagcgcg tgactccgtt gcaccttgcc gcagctctgg ggcatttgga gatcgttgag 2460

gtactgttga aagcgggagc agatgttaat gctcgcgaca gttgggggac gacaccagca 2520

gacctggccg caaaatacgg acaccaagac attgctgaag ttctgcaaaa ggcagcataa 2580

tgatag 2586

<210> 121

<211> 2586

<212> DNA

<213> artificial sequence

<220>

<223> nucleic acid encoding the protein of SEQ ID NO. 98

<400> 121

atgagaggat cgcatcacca tcaccatcac ggatccgacc tgggtaagaa actgttggaa 60

gcagcacgtg cgggtcaaga cgatgaagtt cgtgagctgt taaaggctgg cgccgacgtg 120

aacgcgaagg actactttag ccacaccccg ctgcacttgg cagcgcgcaa cggtcacctg 180

aaaattgtcg aggtcctgtt gaaggctggt gcggatgtga acgcaaaaga ttttgcgggt 240

aagacgccgc tgcatctggc ggcggctgat ggtcacttag agatcgtaga ggttctgttg 300

aaagcgggcg ccgatgtgaa tgcccaggac atcttcggca agaccccggc agacattgcc 360

gcggatgctg gtcacgaaga tatcgcagag gtcctgcaaa aagcggcagg cagcccgacc 420

ccgacgccga ccactccgac cccgacccca accaccccga ctccgactcc gaccggatct 480

gacctgggta aaaaactgct ggaagcagca cgtgccggcc aggatgatga agttcgtgaa 540

ctgctgaaag caggcgccga tgttaatgca aaggattatt ttagccacac accgctgcat 600

ctggcagccc gtaatggtca cctaaagatt gttgaagttc tgctgaaggc tggtgcagac 660

gttaacgcca aagatttcgc gggcaaaacc cctctgcatt tagccgcagc ggacggtcac 720

ctggagatcg tagaggtgct gcttaaggcg ggtgcggatg ttaatgcaca ggatattttc 780

ggtaaaaccc ctgccgatat tgcagctgat gccggtcatg aagatatcgc agaagtgctg 840

cagaaggcag caggatcacc aacaccaacc ccgaccaccc caactccaac accgaccacc 900

ccgaccccta ccccaacagg atccgacctg ggtattaaac tgttgacagc cgcttacgac 960

gggcaattag acgaagtgcg tattctgctt aaagctggag ctgacgtgaa cgcgaaagac 1020

ttacgcggcc aaacgccttt acattacgcg gcgggactgg gccatcttga gattgttgag 1080

gtgcttctga aggcaggcgc ggatgtcaat gcaaaagacc tgcacggatg gacacctctt 1140

cacttagctg cttggtctgg gcatttggag attgtagagg ttttattgaa agcaggggcg 1200

gatgtgaatg cgcaagacgt agaaggagtc accccagctg acctggcagc ggttcaaggg 1260

catcaagaca ttgctgaagt tctgcagaag gcagcaggct cgccgactcc gaccccgacc 1320

accccaactc caacaccgac caccccgacc cctaccccaa caggatctga cctgggtaaa 1380

aaattgcttg aagccgcgtt ggaaggacaa ttagacgagg tacgtgagct gttaaaagca 1440

ggggccgatg tgaatgctaa agaccaggag ggatacaccc ccttgcacct ggctgccgcg 1500

ttgggccact tagagattgt agaggttctt cttaaggcgg gggcagacgt gaatgcaaag 1560

gaccaaattg gacgtactcc tttgcatctg gcagcctata aggggcactt ggagattgtc 1620

gaggtcttgt taaaggcggg tgccgatgta aatgcccagg acatcattgg gcagactccg 1680

gcagatttgg ccgcccaacg tggccaccaa gatattgctg aagttctgca gaaggcagca 1740

ggctcgccga ctccgacccc gaccacccca actccaacac cgaccacccc gacccctacc 1800

ccaacaggat ctgacctggg tgttaagctg ttgcttgcag ccgagcgtgg gcagcttgat 1860

gaagtacgca ttcttcttaa agcgggggct gatgtgaacg cgaaagatat tgctgagggt 1920

tacacaccgc ttcacattgc cgcctaccag ggtcatctgg agattgttga agtattactg 1980

aaagcgggag cagatgttaa tgccaaagat cgctatggaa aaactccttt gcatttagct 2040

gcaatcggag gacacctgga aatcgtcgaa gtgttattaa aagctggagc ggacgtaaac 2100

gcacaagata acaagggctc aactcccgcg gaccttgccg cagattacgg tcatcaggac 2160

attgctgaag ttctgcagaa ggcagcaggc tcgccaaccg gatcagatct gggtcaaaag 2220

ctgttggaag ccgcgtgggc gggtcaggac gatgaagtcc gtgagctgct taaagcagga 2280

gccgacgtga acgcgaagaa ctcacgcggg tggacgccac ttcacacggc cgcgcagaca 2340

ggtcaccttg aaatctttga ggttcttctg aaggcaggag cagacgttaa cgccaaaaac 2400

gacaagcgcg tgactccgtt gcaccttgcc gcagctctgg ggcatttgga gatcgttgag 2460

gtactgttga aagcgggagc agatgttaat gctcgcgaca gttgggggac gacaccagca 2520

gacctggccg caaaatacgg acaccaagac attgctgaag ttctgcaaaa ggcagcataa 2580

tgatag 2586

<210> 122

<211> 2676

<212> DNA

<213> artificial sequence

<220>

<223> nucleic acid encoding protein of SEQ ID NO. 99

<400> 122

atgagaggat cgcatcacca tcaccatcac ggatccgacc tgggtaagaa actgttggaa 60

gcagcacgtg cgggtcaaga cgatgaagtt cgtgagctgt taaaggctgg cgccgacgtg 120

aacgcgaagg actactttag ccacaccccg ctgcacttgg cagcgcgcaa cggtcacctg 180

aaaattgtcg aggtcctgtt gaaggctggt gcggatgtga acgcaaaaga ttttgcgggt 240

aagacgccgc tgcatctggc ggcggctgat ggtcacttag agatcgtaga ggttctgttg 300

aaagcgggcg ccgatgtgaa tgcccaggac atcttcggca agaccccggc agacattgcc 360

gcggatgctg gtcacgaaga tatcgcagag gtcctgcaaa aagcggcagg cagcccgacc 420

ccgacgccga ccactccgac cccgacccca accaccccga ctccgactcc gaccggatct 480

gacctgggta aaaaactgct ggaagcagca cgtgccggcc aggatgatga agttcgtgaa 540

ctgctgaaag caggcgccga tgttaatgca aaggattatt ttagccacac accgctgcat 600

ctggcagccc gtaatggtca cctaaagatt gttgaagttc tgctgaaggc tggtgcagac 660

gttaacgcca aagatttcgc gggcaaaacc cctctgcatt tagccgcagc ggacggtcac 720

ctggagatcg tagaggtgct gcttaaggcg ggtgcggatg ttaatgcaca ggatattttc 780

ggtaaaaccc ctgccgatat tgcagctgat gccggtcatg aagatatcgc agaagtgctg 840

cagaaggcag caggatcacc aacaccaacc ccgaccaccc caactccaac accgaccacc 900

ccgaccccta ccccaacagg atccgacctg ggtattaaac tgttgacagc cgcttacgac 960

gggcaattag acgaagtgcg tattctgctt aaagctggag ctgacgtgaa cgcgaaagac 1020

ttacgcggcc aaacgccttt acattacgcg gcgggactgg gccatcttga gattgttgag 1080

gtgcttctga aggcaggcgc ggatgtcaat gcaaaagacc tgcacggatg gacacctctt 1140

cacttagctg cttggtctgg gcatttggag attgtagagg ttttattgaa agcaggggcg 1200

gatgtgaatg cgcaagacgt agaaggagtc accccagctg acctggcagc ggttcaaggg 1260

catcaagaca ttgctgaagt tctgcagaag gcagcaggct cgccgactcc gaccccgacc 1320

accccaactc caacaccgac caccccgacc cctaccccaa caggatctga cctgggtaaa 1380

aaactgctgc aagcagcacg tgcaggtcag ctggatgaag ttcgtgaact gctgaaagca 1440

ggcgccgatg ttaatgcaaa agatcaatac ggcagaaccc cgctgcatct ggctgctatc 1500

aagggtcacc tggaaattgt tgaagttctg ctgaaagccg gtgcagatgt taatgcaaaa 1560

gattctctgg gctacacccc gctgcatctg gctgctgtgg agggtcccct ggaaattgtt 1620

gaagttctgc tgaaagccgg tgcagatgtt aatgcaaaag atgcttacgg ccaaaccccg 1680

ctgcatatcg ctgctgcttg gggtcacctg gaaattgttg aagttctgct gaaagccgtt 1740

gcagatgtta acgcacagga taaaagcggt aaaacccctg ccgatctggc agctcgcgcc 1800

ggtcatcaag atattgctga agtgctgcag aaggcagcag gctcgccgac tccgaccccg 1860

accaccccaa ctccaacacc gaccaccccg acccctaccc caacaggatc tgacctgggt 1920

gttaagctgt tgcttgcagc cgagcgtggg cagcttgatg aagtacgcat tcttcttaaa 1980

gcgggggctg atgtgaacgc gaaagatatt gctgagggtt acacaccgct tcacattgcc 2040

gcctaccagg gtcatctgga gattgttgaa gtattactga aagcgggagc agatgttaat 2100

gccaaagatc gctatggaaa aactcctttg catttagctg caatcggagg acacctggaa 2160

atcgtcgaag tgttattaaa agctggagcg gacgtaaacg cacaagataa caagggctca 2220

actcccgcgg accttgccgc agattacggt catcaggaca ttgctgaagt tctgcagaag 2280

gcagcaggct cgccaaccgg atcagatctg ggtcaaaagc tgttggaagc cgcgtgggcg 2340

ggtcaggacg atgaagtccg tgagctgctt aaagcaggag ccgacgtgaa cgcgaagaac 2400

tcacgcgggt ggacgccact tcacacggcc gcgcagacag gtcaccttga aatctttgag 2460

gttcttctga aggcaggagc agacgttaac gccaaaaacg acaagcgcgt gactccgttg 2520

caccttgccg cagctctggg gcatttggag atcgttgagg tactgttgaa agcgggagca 2580

gatgttaatg ctcgcgacag ttgggggacg acaccagcag acctggccgc aaaatacgga 2640

caccaagaca ttgctgaagt tctgcaaaag gcagca 2676

<210> 123

<211> 2676

<212> DNA

<213> artificial sequence

<220>

<223> nucleic acid encoding protein of SEQ ID NO. 100

<400> 123

atgagaggat cgcatcacca tcaccatcac ggatccgacc tgggtaagaa actgttggaa 60

gcagcacgtg cgggtcaaga cgatgaagtt cgtgagctgt taaaggctgg cgccgacgtg 120

aacgcgaagg actactttag ccacaccccg ctgcacttgg cagcgcgcaa cggtcacctg 180

aaaattgtcg aggtcctgtt gaaggctggt gcggatgtga acgcaaaaga ttttgcgggt 240

aagacgccgc tgcatctggc ggcggctgat ggtcacttag agatcgtaga ggttctgttg 300

aaagcgggcg ccgatgtgaa tgcccaggac atcttcggca agaccccggc agacattgcc 360

gcggatgctg gtcacgaaga tatcgcagag gtcctgcaaa aagcggcagg cagcccgacc 420

ccgacgccga ccactccgac cccgacccca accaccccga ctccgactcc gaccggatct 480

gacctgggta aaaaactgct ggaagcagca cgtgccggcc aggatgatga agttcgtgaa 540

ctgctgaaag caggcgccga tgttaatgca aaggattatt ttagccacac accgctgcat 600

ctggcagccc gtaatggtca cctaaagatt gttgaagttc tgctgaaggc tggtgcagac 660

gttaacgcca aagatttcgc gggcaaaacc cctctgcatt tagccgcagc ggacggtcac 720

ctggagatcg tagaggtgct gcttaaggcg ggtgcggatg ttaatgcaca ggatattttc 780

ggtaaaaccc ctgccgatat tgcagctgat gccggtcatg aagatatcgc agaagtgctg 840

cagaaggcag caggatcacc aacaccaacc ccgaccaccc caactccaac accgaccacc 900

ccgaccccta ccccaacagg atccgacctg ggtaaaaaat tgcttcaggc cgctcgcgcc 960

gggcaacttg acgaagtacg tgaattactg aaagcaggcg cagacgtgaa cgctaaggat 1020

cagcagggct taactccgtt acacatcgcc gctaatctgg gtcatctgga gattgttgaa 1080

gtattattaa aggccggagc ggatgtaaat gccaaggatc tgtttggact tacgccgctt 1140

catctggccg cttttgaggg tcacttagaa attgtggagg tcttacttaa ggcgggggct 1200

gacgtaaatg cgaaagatca gcatggtgcc acgcctcttc atttggctgc ttgggtcgga 1260

catttggaaa ttgtggaagt gttattgaaa gctggagcag atgtgaacgc gcaggacaag 1320

tcaggtaaaa ccccggcgga tctggcggca cgcgcaggac atcaagatat tgctgaagtt 1380

ctgcagaagg cagcaggctc gccgactccg accccgacca ccccaactcc aacaccgacc 1440

accccgaccc ctaccccaac aggatctgac ctgggtaaaa agttgttaca ggcagcattg 1500

gagggccaac ttgacgaggt gcgcgagtta ctgaaagctg gtgcagatgt caacgcgaag 1560

gacattgaag gatatactcc gctgcacctt gccgcggctt tggggcatct tgagattgtg 1620

gaggtgcttc ttaaggcggg agctgatgtc aatgctaaag accaaatcgg gcgcacaccg 1680

ttacacttgg ctgcgtacaa aggtcactta gaaatcgtgg aagtgcttct gaaggctggc 1740

gctgatgtca acgcccaaga cattatcggc cagacaccgg cggacctggc agcgcaacgt 1800

gggcatcagg atattgctga agttctgcag aaggcagcag gctcgccgac tccgaccccg 1860

accaccccaa ctccaacacc gaccaccccg acccctaccc caacaggatc tgacctgggt 1920

aaaaagttgt tattagctgc ggagcgcggg cagttagacg aagtgcgtat tctgctgaag 1980

gccggggccg acgttaacgc aaaggatcgt gcagagggtt acacccccct gcacatcgcc 2040

gcttatcaag gtcacttgga gattgttgag gtcttactga aagcgggggc cgacgtgaat 2100

gccaaagatc gctatggaaa aacaccgtta cacttagcag ctatttcggg gcatctggag 2160

atcgtggaag tcctgttaaa ggctggtgcc gatgttaatg cacaagataa taaaggcagc 2220

actccagccg atctggccgc tgattatggg caccaggaca ttgctgaagt tctgcagaag 2280

gcagcaggct cgccaaccgg atcagatctg ggtcaaaagc tgttggaagc cgcgtgggcg 2340

ggtcaggacg atgaagtccg tgagctgctt aaagcaggag ccgacgtgaa cgcgaagaac 2400

tcacgcgggt ggacgccact tcacacggcc gcgcagacag gtcaccttga aatctttgag 2460

gttcttctga aggcaggagc agacgttaac gccaaaaacg acaagcgcgt gactccgttg 2520

caccttgccg cagctctggg gcatttggag atcgttgagg tactgttgaa agcgggagca 2580

gatgttaatg ctcgcgacag ttgggggacg acaccagcag acctggccgc aaaatacgga 2640

caccaagaca ttgctgaagt tctgcaaaag gcagca 2676

<210> 124

<211> 2631

<212> DNA

<213> artificial sequence

<220>

<223> nucleic acid encoding protein of SEQ ID NO. 101

<400> 124

atgagaggat cgcatcacca tcaccatcac ggatccgacc tgggtaaaaa gctgctggag 60

gcagcgcgtg ccggtcaaga cgacgaggtt cgcgaattgc ttaaagcggg tgcagacgtc 120

aacgccaaag attatttctc tcataccccg ttgcatttag ccgcgcgtaa tggccatctg 180

aagatcgtcg aggtcctctt gaaggcaggc gcggatgtca atgcgaagga ttttgcgggc 240

aaaacgccgc tgcacttagc ggcggcggac ggtcatttag aaatcgttga agtcctgtta 300

aaagcgggcg ccgatgtgaa tgcgcaggat attttcggta aaacgccggc ggacattgcg 360

gcagatgcgg gtcatgaaga tatcgcagaa gtcctgcaga aggcagcagg cagccctaca 420

cctacgccga ctacgcctac gccgactccg actaccccga ctccgacccc gaccggatca 480

gacctgggta aaaagctgct ggaggcagcg cgtgccggtc aagacgacga ggttcgcgaa 540

ttgcttaaag cgggtgcaga cgtcaacgcc aaagattatt tctctcatac cccgttgcat 600

ttagccgcgc gtaatggcca tctgaagatc gtcgaggtcc tcttgaaggc aggcgcggat 660

gtcaatgcga aggattttgc gggcaaaacg ccgctgcact tagcggcggc ggacggtcat 720

ttagaaatcg ttgaagtcct gttaaaagcg ggcgccgatg tgaatgcgca ggatattttc 780

ggtaaaacgc cggcggacat tgcggcagat gcgggtcatg aagatatcgc agaagtcctg 840

cagaaggcag caggctcgcc aacgccgacc cctacaacgc caaccccgac accaactaca 900

ccgaccccca caccaacggg atcagacctg ggtattaaac tgttgacagc cgcttacgac 960

gggcaattag acgaagtgcg tattctgctt aaagctggag ctgacgtgaa cgcgaaagac 1020

ttacgcggcc aaacgccttt acattacgcg gcgggactgg gccatcttga gattgttgag 1080

gtgcttctga aggcaggcgc ggatgtcaat gcaaaagacc tgcacggatg gacacctctt 1140

cacttagctg cttggtctgg gcatttggag attgtagagg ttttattgaa agcaggggcg 1200

gatgtgaatg cgcaagacgt agaaggagtc accccagctg acctggcagc ggttcaaggg 1260

catcaagaca ttgctgaagt tctgcagaag gcagcaggtt ccccgacccc tacgccaacg 1320

actccgaccc caactccaac gacccctacc ccgaccccga ccggatcaga cctgggtgtt 1380

aagctgttgc ttgcagccga gcgtgggcag cttgatgaag tacgcattct tcttaaagcg 1440

ggggctgatg tgaacgcgaa agatattgct gagggttaca caccgcttca cattgccgcc 1500

taccagggtc atctggagat tgttgaagta ttactgaaag cgggagcaga tgttaatgcc 1560

aaagatcgct atggaaaaac tcctttgcat ttagctgcaa tcggaggaca cctggaaatc 1620

gtcgaagtgt tattaaaagc tggagcggac gtaaacgcac aagataacaa gggctcaact 1680

cccgcggacc ttgccgcaga ttacggtcat caggacattg ctgaagttct gcagaaggca 1740

gcaggcagcc ccacgccaac tcctacaacc cccacaccta caccgacgac gccgacaccg 1800

actccaaccg gatcagacct gggtaaaaaa ttgcttgaag ccgcgttgga aggacaatta 1860

gacgaggtac gtgagctgtt aaaagcaggg gccgatgtga atgctaaaga ccaggaggga 1920

tacaccccct tgcacctggc tgccgcgttg ggccacttag agattgtaga ggttcttctt 1980

aaggcggggg cagacgtgaa tgcaaaggac caaattggac gtactccttt gcatctggca 2040

gcctataagg ggcacttgga gattgtcgag gtcttgttaa aggcgggtgc cgatgtaaat 2100

gcccaggaca tcattgggca gactccggca gatttggccg cccaacgtgg ccaccaagat 2160

attgctgaag ttctgcagaa ggcagcaggt tcgccgaccc caacccctac cactccaacg 2220

ccgacgccta ccactccaac accaacacca acgggatcag acctgggtca aaagctgttg 2280

gaagccgcgt gggcgggtca ggacgatgaa gtccgtgagc tgcttaaagc aggagccgac 2340

gtgaacgcga agaactcacg cgggtggacg ccacttcaca cggccgcgca gacaggtcac 2400

cttgaaatct ttgaggttct tctgaaggca ggagcagacg ttaacgccaa aaacgacaag 2460

cgcgtgactc cgttgcacct tgccgcagct ctggggcatt tggagatcgt tgaggtactg 2520

ttgaaagcgg gagcagatgt taatgctcgc gacagttggg ggacgacacc agcagacctg 2580

gccgcaaaat acggacacca agacattgct gaagttctgc agaaggcggc a 2631

Claims

1. A recombinant protein comprising (1) a first binding agent that specifically binds to a protein expressed on the surface of an immune cell and (2) at least two binding agents that specifically bind to tumor-associated antigens, wherein the two binding agents specifically bind to different tumor-associated antigens.

2. The recombinant protein according to claim 1, wherein said tumor-associated antigen specifically bound by said two binding agents is co-expressed in tumor cells.

3. A recombinant protein comprising (1) a first binding agent that specifically binds to a protein expressed on the surface of an immune cell and (2) at least three binding agents that specifically bind to tumor-associated antigens, wherein the three binding agents specifically bind to different tumor-associated antigens.

4. The recombinant protein according to claim 3, wherein said tumor-associated antigen specifically bound by said three binding agents is co-expressed in tumor cells.

5. The recombinant protein according to any one of claims 2 and 4, wherein said tumor cell is a tumor cell from a liquid tumor.

6. The recombinant protein according to any one of claims 2, 4, and 5, wherein said tumor cell is from leukemia.

7. The recombinant protein according to claim 6, wherein said leukemia is Acute Myelogenous Leukemia (AML).

8. The recombinant protein according to any preceding claim, wherein said recombinant protein is capable of having a lower dissociation constant (K _D ) Binding to a surface displaying the tumor-associated antigen.

9. The recombinant protein according to claim 8, wherein said surface displaying said tumor-associated antigen is a surface of a tumor cell.

10. The recombinant protein according to any one of claims 8 and 9, wherein said lower dissociation constant (K _D ) Corresponding solution of the recombinant protein comprising only one of the binding agents specifically binding to a tumor-associated antigenAt least about 1/2, at least about 1/4, at least about 1/10, at least about 1/20, at least about 1/40, or at least about 1/100 of the dissociation constant.

11. The recombinant protein according to any preceding claim, wherein said immune cell is a T cell.

12. The recombinant protein according to claim 11, wherein said T cell is a cd8+ cytotoxic T cell.

13. The recombinant protein according to any preceding claim, wherein said protein expressed on an immune cell surface is a protein that is part of a T cell receptor complex.

14. The recombinant protein according to any preceding claim, wherein said protein expressed on the surface of an immune cell is CD3.

15. The recombinant protein according to any preceding claim, wherein said first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

16. The recombinant protein according to any preceding claim, wherein said first binding agent is a designed ankyrin repeat domain having binding specificity for said protein expressed on an immune cell surface.

17. The recombinant protein according to any preceding claim, wherein said first binding agent is a designed ankyrin repeat domain having binding specificity for CD3.

18. The recombinant protein according to claim 17, wherein said ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID NOs 1 to 5.

19. The recombinant protein according to any one of claims 17 and 18, wherein said ankyrin repeat domain having binding specificity for CD3 comprises any one of the amino acid sequences of SEQ ID NOs 1 to 5.

20. The recombinant protein according to any preceding claim, wherein said binding agent that specifically binds a tumor-associated antigen is selected from the group consisting of (i) a second binding agent that specifically binds a first tumor-associated antigen (TAA 1), (ii) a third binding agent that specifically binds a second tumor-associated antigen (TAA 2), and (iii) a fourth binding agent that specifically binds a third tumor-associated antigen (TAA 3).

21. The recombinant protein according to claim 20, wherein said second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

22. The recombinant protein according to any one of claims 20 and 21, wherein said second binding agent is a designed ankyrin repeat domain having binding specificity for said TAA 1.

23. The recombinant protein according to any one of claims 20 to 22, wherein said TAA1 is CD33.

24. The recombinant protein according to any one of claims 20 to 23, wherein said second binding agent is a designed ankyrin repeat domain having binding specificity for CD33.

25. The recombinant protein according to claim 24, wherein said ankyrin repeat domain having binding specificity for CD33 includes an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NOs 15, 67 to 70 and 111 to 112.

26. The recombinant protein according to any one of claims 24 and 25, wherein said ankyrin repeat domain having binding specificity for CD33 comprises the amino acid sequence of any one of SEQ ID NOs 15, 67 to 70 and 111 to 112.

27. The recombinant protein according to any one of claims 20 to 26, wherein said third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

28. The recombinant protein according to any one of claims 20 to 27, wherein said third binding agent is a designed ankyrin repeat domain having binding specificity for said TAA 2.

29. The recombinant protein according to any one of claims 20 to 28, wherein said TAA2 is CD123.

30. The recombinant protein according to any one of claims 20 to 29, wherein said third binding agent is a designed ankyrin repeat domain having binding specificity for CD123.

31. The recombinant protein according to claim 30, wherein said ankyrin repeat domain having binding specificity for CD123 includes an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NOs 6, 65 to 66 and 102 to 106.

32. The recombinant protein according to any one of claims 30 and 31, wherein said ankyrin repeat domain having binding specificity for CD123 comprises the amino acid sequence of any one of SEQ ID NOs 6, 65 to 66 and 102 to 106.

33. The recombinant protein according to any one of claims 20 to 32, wherein said fourth binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

34. The recombinant protein according to any one of claims 20 to 33, wherein said fourth binding agent is a designed ankyrin repeat domain having binding specificity for said TAA 3.

35. The recombinant protein according to any one of claims 20 to 34, wherein said TAA3 is CD70.

36. The recombinant protein according to any one of claims 20 to 35, wherein said fourth binding agent is a designed ankyrin repeat domain having binding specificity for CD70.

37. The recombinant protein according to claim 36, wherein said ankyrin repeat domain having binding specificity for CD70 comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NOs 64 and 107 to 110.

38. The recombinant protein according to any one of claims 36 and 37, wherein said ankyrin repeat domain having binding specificity for CD70 comprises the amino acid sequence of any one of SEQ ID NOs 64 and 107 to 110.

39. The recombinant protein according to any one of claims 36 and 37, wherein said ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID NOs 1 to 5, preferably SEQ ID NOs 2 or 3, wherein said ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID NOs 15, 67 to 70 and 111 to 112; wherein the ankyrin repeat domain with binding specificity for CD123 comprises an amino acid sequence having at least 85% identity to any one of the amino acid sequences of SEQ ID nos. 6, 65 to 66 and 102 to 106, and/or wherein the ankyrin repeat domain with binding specificity for CD70 comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID nos. 64 and 107 to 110.

40. The recombinant protein according to any one of claims 20 to 39, wherein said recombinant protein comprises said first binding agent, said second binding agent, and said third binding agent, and wherein said first binding agent, said second binding agent, and said third binding agent are arranged from N-terminus to C-terminus according to the formula: (third binder) - (second binder) - (first binder).

41. The recombinant protein according to any one of claims 20 to 39, wherein said recombinant protein comprises said first binding agent, said second binding agent, and said third binding agent, and wherein said first binding agent, said second binding agent, and said third binding agent are arranged from said N-terminus to said C-terminus according to the formula: (second binding agent) - (third binding agent) - (first binding agent).

42. The recombinant protein according to any one of claims 20 to 39, wherein said recombinant protein comprises said first binding agent, said second binding agent, and said third binding agent, and wherein said first binding agent, said second binding agent, and said third binding agent are arranged from N-terminus to said C-terminus according to the formula: (first binding agent) - (second binding agent) - (third binding agent).

43. The recombinant protein according to any one of claims 20 to 39, wherein said recombinant protein comprises said first binding agent, said second binding agent, and said third binding agent, and wherein said first binding agent, said second binding agent, and said third binding agent are arranged from said N-terminus to said C-terminus according to the formula: (first binding agent) - (third binding agent) - (second binding agent).

44. The recombinant protein according to any one of claims 20 to 39, wherein said recombinant protein comprises said first binding agent, said second binding agent, and said fourth binding agent, and wherein said first binding agent, said second binding agent, and said fourth binding agent are arranged from said N-terminus to said C-terminus according to the formula: (fourth binder) - (second binder) - (first binder).

45. The recombinant protein according to any one of claims 20 to 39, wherein said recombinant protein comprises said first binding agent, said second binding agent, and said fourth binding agent, and wherein said first binding agent, said second binding agent, and said fourth binding agent are arranged from said N-terminus to said C-terminus according to the formula: (second binding agent) - (fourth binding agent) - (first binding agent).

46. The recombinant protein according to any one of claims 20 to 39, wherein said recombinant protein comprises said first binding agent, said second binding agent, and said fourth binding agent, and wherein said first binding agent, said second binding agent, and said fourth binding agent are arranged from said N-terminus to said C-terminus according to the formula: (first binding agent) - (second binding agent) - (fourth binding agent).

47. The recombinant protein according to any one of claims 20 to 39, wherein said recombinant protein comprises said first binding agent, said second binding agent, and said fourth binding agent, and wherein said first binding agent, said second binding agent, and said fourth binding agent are arranged from said N-terminus to said C-terminus according to the formula: (first binding agent) - (fourth binding agent) - (second binding agent).

48. The recombinant protein according to any one of claims 20 to 39, wherein said recombinant protein comprises said first binding agent, said third binding agent, and said fourth binding agent, and wherein said first binding agent, said third binding agent, and said fourth binding agent are arranged from said N-terminus to said C-terminus according to the formula: (fourth binder) - (third binder) - (first binder).

49. The recombinant protein according to any one of claims 20 to 39, wherein said recombinant protein comprises said first binding agent, said third binding agent, and said fourth binding agent, and wherein said first binding agent, said third binding agent, and said fourth binding agent are arranged from said N-terminus to said C-terminus according to the formula: (third binder) - (fourth binder) - (first binder).

50. The recombinant protein according to any one of claims 20 to 39, wherein said recombinant protein comprises said first binding agent, said third binding agent, and said fourth binding agent, and wherein said first binding agent, said third binding agent, and said fourth binding agent are arranged from said N-terminus to said C-terminus according to the formula: (first binding agent) - (third binding agent) - (fourth binding agent).

51. The recombinant protein according to any one of claims 20 to 39, wherein said recombinant protein comprises said first binding agent, said third binding agent, and said fourth binding agent, and wherein said first binding agent, said third binding agent, and said fourth binding agent are arranged from said N-terminus to said C-terminus according to the formula: (first binding agent) - (fourth binding agent) - (third binding agent).

52. The recombinant protein according to any one of claims 20 to 39, wherein said recombinant protein comprises said first binding agent, said second binding agent, said third binding agent, and said fourth binding agent, and wherein said first binding agent, said second binding agent, said third binding agent, and said fourth binding agent are arranged from said N-terminus to said C-terminus according to the formula: (fourth binder) - (third binder) - (second binder) - (first binder).

53. The recombinant protein according to any one of claims 20-52, wherein said first binding agent, said second binding agent, said third binding agent, and/or said fourth binding agent is covalently linked to a peptide linker.

54. The recombinant protein according to claim 53, wherein said peptide linker is a proline-threonine rich peptide linker.

55. The recombinant protein according to any one of claims 53 and 54, wherein an amino acid sequence of said peptide linker has a length of 1 to 50 amino acids.

56. The recombinant protein according to any one of claims 18 to 55, wherein a at the penultimate position of any one of SEQ ID NOs 1 to 4, 6, 15, 64 to 70 and 102 to 112 is optionally substituted with L, and/or a at the last position of any one of SEQ ID NOs 1 to 4, 6 and 15 is optionally substituted with N; or wherein L at the penultimate position of SEQ ID NO. 5 is optionally substituted with A and/or N at the last position of SEQ ID NO. 5 is optionally substituted with A.

57. The recombinant protein according to any one of claims 18-56, wherein any one of said ankyrin repeat domains further comprises G, S or GS at said N-terminus.

58. The recombinant protein according to any preceding claim, wherein said recombinant protein comprises a polypeptide having an amino acid sequence having at least 80% identity to any one of the amino acid sequences of SEQ ID NOs 7 to 10 and 58 to 62, preferably wherein said recombinant protein comprises a polypeptide having the amino acid sequence of any one of SEQ ID NOs 7 to 10 and 58 to 62.

59. The recombinant protein according to any preceding claim, wherein said recombinant protein is at less than 10 in PBS ^-6 Dissociation constant of M (K _D ) Binds human CD3.

60. The recombinant protein according to any one of claims 20-59,wherein the recombinant protein comprises the second binding agent, and wherein the recombinant protein is present at less than 10 in PBS ^-7 Dissociation constant of M (K _D ) Binds human CD33.

61. The recombinant protein according to any one of claims 20 to 60, wherein said recombinant protein comprises said third binding agent, and wherein said recombinant protein is at less than 10 in PBS ^-7 Dissociation constant of M (K _D ) Binds human CD123.

62. The recombinant protein according to any one of claims 20 to 61, wherein said recombinant protein comprises said fourth binding agent, and wherein said recombinant protein is at less than 10 in PBS ^-7 Dissociation constant of M (K _D ) Binds human CD70.

63. The recombinant protein according to any preceding claim, wherein said recombinant protein has an EC in the range of 1nM to 400nM ₅₀ Binds human CD3.

64. The recombinant protein according to any one of claims 20-63, wherein said recombinant protein includes said second binding agent and said third binding agent, and wherein said recombinant protein is at 10 in PBS ^-6 M to 10 ^-12 Dissociation constant between M (K _D ) Binds to said TAA1 and wherein said recombinant protein is present at 10 in PBS ^-6 M to 10 ^-12 Dissociation constant between M (K _D ) Binding to said TAA2.

65. The recombinant protein according to any one of claims 20 to 64, wherein said recombinant protein comprises said second binding agent and said fourth binding agent, and wherein said recombinant protein is at 10 in PBS ^-6 M to 10 ^-12 Dissociation constant between M (K _D ) Binds to said TAA1 and wherein said recombinant protein is present at 10 in PBS ^-6 M to 10 ^-12 Dissociation constant between M (K _D ) Binding to said TAA3.

66. The recombinant protein according to any one of claims 20 to 65, wherein said recombinant protein comprises said third binding agent and said fourth binding agent, and wherein said recombinant protein is at 10 in PBS ^-6 M to 10 ^-12 Dissociation constant between M (K _D ) Binds to said TAA2 and wherein said recombinant protein is present at 10 in PBS ^-6 M to 10 ^-12 Dissociation constant between M (K _D ) Binding to said TAA3.

67. The recombinant protein according to any one of claims 20-63, wherein said recombinant protein comprises said second binding agent, said third binding agent, and said fourth binding agent, and wherein said recombinant protein is at 10 in PBS ^-6 M to 10 ^- ¹² Dissociation constant between M (K _D ) Binds to said TAA1 and wherein said recombinant protein is present at 10 in PBS ^-6 M to 10 ^-12 Dissociation constant between M (K _D ) Binds to said TAA2 and wherein said recombinant protein is present at 10 in PBS ^-6 M to 10 ^-12 Dissociation constant between M (K _D ) Binding to said TAA3.

68. The recombinant protein according to any preceding claim, wherein said recombinant protein further comprises a half-life extending moiety.

69. The recombinant protein according to claim 68, wherein the half-life extending moiety is a binding agent that specifically binds human serum albumin.

70. The recombinant protein according to claim 69, wherein said binding agent that specifically binds human serum albumin is a designed ankyrin repeat domain having binding specificity for human serum albumin.

71. The recombinant protein according to claim 70, wherein said ankyrin repeat domain having binding specificity for human serum albumin comprises an amino acid sequence having at least 85% identity to the amino acid sequence of any one of SEQ ID NOs 34 to 36.

72. The recombinant protein according to any one of claims 70 and 71, wherein said ankyrin repeat domain having binding specificity for human serum albumin comprises the amino acid sequence of any one of SEQ ID NOs 34 to 36.

73. The recombinant protein according to any one of claims 69-72, wherein the recombinant protein is at less than 10 in PBS ^-7 Dissociation constant of M (K _D ) Binding human serum albumin.

74. The recombinant protein according to any one of claims 1 to 39, 53 to 57, and 59 to 73, wherein said recombinant protein comprises a polypeptide having an amino acid sequence having at least 80% identity to any one of the amino acid sequences of SEQ ID NOs 11 to 14, 78 to 86, and 95 to 101, preferably wherein said recombinant protein comprises a polypeptide having the amino acid sequence of any one of the SEQ ID NOs 11 to 14, 78 to 86, and 95 to 101.

75. The recombinant protein according to any one of claims 1-39, 53-57, and 59-73, wherein said recombinant protein comprises a polypeptide having an amino acid sequence having at least 80% identity to any one of the amino acid sequences of SEQ ID NOs 95-101, preferably wherein said recombinant protein comprises a polypeptide having the amino acid sequence of any one of SEQ ID NOs 95-101.

76. The recombinant protein according to any one of claims 1 to 39, 53 to 57, and 59 to 73, wherein said recombinant protein comprises a polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID No. 95, preferably wherein said recombinant protein comprises a polypeptide having the amino acid sequence of SEQ ID No. 95.

77. The recombinant protein according to any one of claims 1 to 39, 53 to 57, and 59 to 73, wherein said recombinant protein comprises a polypeptide having an amino acid sequence having at least 80% identity to the amino acid sequence of SEQ ID No. 96, preferably wherein said recombinant protein comprises a polypeptide having the amino acid sequence of SEQ ID No. 96.

78. The recombinant protein according to any one of claims 68-73, wherein said half-life extending moiety is located on an N-terminal side of said first binding agent and said binding agent that specifically binds a tumor-associated antigen.

79. The recombinant protein according to any preceding claim, wherein said recombinant protein is capable of binding simultaneously said protein expressed on the surface of an immune cell and said tumor-associated antigen, said protein expressed on the surface of an immune cell being specifically bound by said first binding agent, said tumor-associated antigen being specifically bound by said at least two binding agents that specifically bind to tumor-associated antigen or said at least three binding agents that specifically bind to tumor-associated antigen.

80. A nucleic acid encoding the recombinant protein of any preceding claim.

81. A nucleic acid encoding an ankyrin repeat domain according to any one of claims 16 to 78.

82. A pharmaceutical composition comprising the recombinant protein according to any one of claims 1 to 79 or the nucleic acid according to any one of claims 80 and 81, and a pharmaceutically acceptable carrier and/or diluent.

83. A method of activating immune cells in a tumor tissue of a mammal, including a human, the method comprising the step of administering to the mammal the recombinant protein of any one of claims 1 to 79.

84. The method of claim 83, wherein the immune cell is a T cell.

85. A method of treating a medical condition, the method comprising the step of administering to a patient in need thereof a therapeutically effective amount of the recombinant protein of any one of claims 1-79, the nucleic acid of any one of claims 80 and 81, or the pharmaceutical composition of claim 82.

86. The method of claim 85, wherein the medical condition is cancer.

87. The method of claim 85, wherein the medical condition is a cancer characterized by a liquid tumor.

88. The method of claim 85, wherein the medical condition is leukemia.

89. The method of claim 85, wherein the medical condition is acute myelogenous leukemia.

90. The recombinant protein according to any one of claims 1 to 79, the nucleic acid according to any one of claims 80 and 81, or the pharmaceutical composition according to claim 82, for use in therapy.

91. The recombinant protein according to any one of claims 1 to 79, the nucleic acid according to any one of claims 80 and 81, or the pharmaceutical composition according to claim 82, for use in the treatment of cancer, optionally in the treatment of cancer characterized by a liquid tumor.

92. The recombinant protein, nucleic acid, or pharmaceutical composition for use according to claim 91, wherein the cancer is leukemia, optionally wherein the cancer is acute myelogenous leukemia.