Novel DARPin-based multispecific T cell engagers

Abstract

The present invention relates to recombinant multispecific proteins comprising binding agents with binding specificities for different targets, such as, for example, CD3, CD33, CD123, and CD70. Additionally, the present invention 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 mammals, including humans, in methods for treating or diagnosing diseases, such as cancer, for example acute myeloid leukemia (AML).

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 63 / 158,539, filed March 9, 2021, U.S. Patent No. 63 / 172,973, filed April 9, 2021, and U.S. Patent Application No. 63 / 265,184, filed December 9, 2021. The disclosures of these patent applications are incorporated herein by reference in their entirety for all purposes.

[0002] The present invention relates to recombinant multispecific proteins comprising binding agents with binding specificities for different targets, such as, for example, CD3, CD33, CD70, and CD 123. In addition, the present invention relates to nucleic acids encoding such multispecific proteins, pharmaceutical compositions, pharmaceutical compositions comprising such proteins or nucleic acids, and the use of such binding proteins, nucleic acids, or pharmaceutical compositions in mammals, including humans, in methods for treating or diagnosing diseases such as cancer, e.g., acute myeloid leukemia (AML). [Background technology]

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

[0004] Monoclonal antibody-based therapies primarily involve anti-CD33 or anti-CD123 antibodies, either as monotherapy or conjugated with cytotoxic agents (Winer & Stone, Ther Adv Hematol; 2019; 10). However, these drugs have demonstrated either significant adverse effects or low efficacy. For example, treatment with gemtuzumab ozogamicin, a humanized anti-CD33 monoclonal antibody conjugated to the antibiotic calicheamicin, resulted in significant hematologic and hepatotoxicity, while treatment with talcotuzumab, a humanized anti-CD123 monoclonal antibody, failed to provide significant therapeutic benefit. Another human monoclonal antibody targeting CD70 is currently in clinical trials (cusatuzumab), and although initial findings seem promising, experts still express concerns about its potential safety profile (see, for example, www.clinicaltrialsarena.com / comment / argenxs-cusatuzumab-in-previously-untreated-aml-draws-varied-expert-forecasts).

[0005] CAR-T cell therapy is an approach that has had a profound impact on the management of lymphoid malignancies. There has been considerable interest in applying this technology to AML, but in practice, this has proven challenging. Regarding monoclonal antibodies, CD33 and CD123 are considered the most promising targets for CAR-T cell therapy in AML. However, preclinical models for these targets have demonstrated a wide range of side effects (on-target and off-tumor toxicity) against non-AML cells, with cytokine release syndrome (CRS) being another recognized side effect.

[0006] T cell-directed killing of tumor cells using bispecific antibodies is another recent therapeutic tool being utilized in the treatment of various cancer types, including AML. These T cell engager (TCE) bispecific antibodies contain two distinct variable regions: one that binds to the T cell receptor complex subunit CD3 and the other that binds to tumor cell surface antigens. TCE binding to these two targets provides a functional connection between the cells, resulting in 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 that can trigger T cell cytotoxicity against AML cells. Similarly, flotetuzumab is a dual affinity retargeting antibody (DART®) that uses two independent polypeptides to connect CD3 on T cells and CD123 on AML cells, fusing the heavy chain variable domain of one antibody to the light chain variable domain of the other antibody.

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

[0008] As mentioned above, acute myeloid leukemia (AML) is a type of cancer that exemplifies the challenges of cancer therapy and the shortcomings of currently available cancer therapies in many ways. AML remains a significant medical need due to its high mortality rate, and treatment of relapsed or refractory AML remains difficult to treat. While numerous antibody-drug conjugate (ADC) and targeted T-cell engager therapies are currently in clinical development for AML, these therapies often suffer from dose-limiting toxicities. The greatest challenges appear to be limited target specificity and overstimulation of the immune system, resulting in, for example, myelotoxicity and cytokine release syndrome (CRS), respectively. Furthermore, resistance to targeted cancer therapies can evolve due to downregulation of individual targets in tumor cells. Therefore, novel approaches or drug molecules are needed to address these challenges and shortcomings of currently available cancer therapies, as exemplified by AML.

[0009] Thus, there remains a need for new molecules, such as immune cell engagers, e.g., TCE proteins, that have beneficial properties that address one or more of the shortcomings of the therapeutic proteins described above. Such novel molecules may be useful in therapeutic approaches for the treatment of neoplastic diseases, e.g., acute myeloid leukemia. Summary of the Invention

[0010] 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 a tumor-associated antigen, wherein the two binding agents specifically bind to different tumor-associated antigens. In addition, the present invention provides nucleic acids encoding such binding proteins and pharmaceutical compositions comprising such binding proteins or nucleic acids. The present invention also provides the use of such binding proteins, nucleic acids, or pharmaceutical compositions for the local activation of immune cells, such as T cells, in the tumor environment and in methods for treating and diagnosing diseases, such as acute myeloid leukemia, in mammals, including humans.

[0011] The recombinant proteins of the present invention target at least two different tumor-associated antigens (TAAs). When at least two TAAs are simultaneously present in tumor cells, the recombinant proteins of the present invention may exhibit substantially increased tumor specificity due to avidity gain. Due to such avidity gain, the multispecific proteins of the present invention may have a larger effective window compared to their respective single-target specificity counterparts. Furthermore, the proteins of the present invention may induce significantly less cytokine release compared to current therapeutic molecules, demonstrating an improved therapeutic window. By targeting at least two different TAAs, the proteins of the present invention may also be more resistant to the development of tumor resistance due to target downregulation.

[0012] 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 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 present invention provides such a recombinant protein that can simultaneously bind to the protein expressed on the surface of an immune cell and to the two different tumor-associated antigens.

[0013] In one aspect, the invention provides such a recombinant protein, wherein the tumor-associated antigens specifically bound by the two binding agents are co-expressed in tumor cells.

[0014] 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 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 present invention provides such a recombinant protein that can simultaneously bind to the protein expressed on the surface of an immune cell and to the three different tumor-associated antigens.

[0015] In one aspect, the invention provides such a recombinant protein, wherein the tumor-associated antigens specifically bound by the three binding agents are co-expressed in tumor cells.

[0016] In one aspect, the present invention provides such recombinant proteins, wherein the tumor cells are tumor cells derived from a liquid tumor, preferably the liquid tumor is a leukemia, more preferably the leukemia is acute myeloid leukemia (AML).

[0017] In one aspect, the present invention provides a method for producing a surface that displays a tumor-associated antigen with a lower dissociation constant (K) when compared to a recombinant protein that comprises only one of the binding agents that specifically binds to the tumor-associated antigen. D The present invention provides such recombinant proteins that can be bound by the nucleotide sequence nucleotides (nucleotides 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 2

[0018] In one aspect, the invention provides such a recombinant protein, wherein said surface presenting said tumor-associated antigen is the surface of said tumor cell.

[0019] In one aspect, the invention provides such a recombinant protein, wherein the immune cell is a T cell.

[0020] 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 the T cell receptor complex, preferably wherein said part of the T cell receptor complex is CD3.

[0021] In one aspect, the present invention provides such a recombinant protein, wherein said first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or an engineered repeat domain.

[0022] In one aspect, the present invention provides such a recombinant protein, wherein said first binding agent is an engineered ankyrin repeat domain that has binding specificity for said protein expressed on the surface of an immune cell.

[0023] In one aspect, the present invention provides such a recombinant protein, wherein said first binding agent comprises an engineered ankyrin repeat domain that has binding specificity for CD3.

[0024] In one aspect, the present invention provides such a recombinant protein, wherein the ankyrin repeat domain with binding specificity for CD3 comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 1 to 5, preferably SEQ ID NO: 2. The present invention further provides such a recombinant protein, wherein the ankyrin repeat domain with binding specificity for CD3 comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 3.

[0025] In one aspect, the present 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 (TAA1), (ii) a third binding agent that specifically binds to a second tumor-associated antigen (TAA2), and (iii) a fourth binding agent that specifically binds to a third tumor-associated antigen (TAA3).

[0026] In one aspect, the present invention provides such a recombinant protein, wherein said second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or an engineered repeat domain.

[0027] In one aspect, the present invention provides such a recombinant protein, wherein said second binding agent is a designed ankyrin repeat domain that has binding specificity for said TAA1, preferably wherein the TAA1 is CD33.

[0028] In one aspect, the invention provides such recombinant proteins, wherein the ankyrin repeat domain with binding specificity for CD33 comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 15 and 67 to 70. The invention further provides such recombinant proteins, wherein the ankyrin repeat domain with binding specificity for CD33 comprises an amino acid sequence that is at least 85% identical 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.

[0029] In one aspect, the present invention provides such a recombinant protein, wherein said third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or an engineered repeat domain.

[0030] In one aspect, the present invention provides such a recombinant protein, wherein said third binding agent is a designed ankyrin repeat domain that has binding specificity for said TAA2, preferably wherein the TAA2 is CD123.

[0031] In one aspect, the invention provides such recombinant proteins, wherein the ankyrin repeat domain with binding specificity for CD123 comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 6 and 65 to 66. The invention further provides such recombinant proteins, wherein the ankyrin repeat domain with binding specificity for CD123 comprises an amino acid sequence that is at least 85% identical 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.

[0032] In one aspect, the present invention provides such a recombinant protein, wherein said fourth binding agent is a designed ankyrin repeat domain that has binding specificity for said TAA3, preferably wherein the TAA3 is CD70.

[0033] In one aspect, the invention provides such recombinant proteins, wherein the ankyrin repeat domain with binding specificity for CD70 comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 64. The invention further provides such recombinant proteins, wherein the ankyrin repeat domain with binding specificity for CD70 comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 64 and 107 to 110, preferably SEQ ID NO: 109.

[0034] In one aspect, the present invention provides such recombinant proteins, wherein the ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 1 to 5, preferably SEQ ID NO: 2; the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 15 and 67 to 70; and the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 6, 65 to 66. The present invention further provides such recombinant proteins, wherein the ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 1 to 5, preferably SEQ ID NO: 3; the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence that is at least 85% identical 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 the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence that is at least 85% identical 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.

[0035] In one aspect, the invention provides such a recombinant protein, wherein the ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 1 to 5, preferably SEQ ID NO: 2; the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 15 and 67 to 70; and the ankyrin repeat domain having binding specificity for CD70 comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 64. The present invention further provides such recombinant proteins, wherein the ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 1 to 5, preferably SEQ ID NO: 3; the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence that is at least 85% identical 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 the ankyrin repeat domain having binding specificity for CD70 comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 64 and 107 to 110, preferably SEQ ID NO: 109.

[0036] In one aspect, the invention provides such a recombinant protein, wherein the ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 1 to 5, preferably SEQ ID NO: 2; the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 6 and 65 to 66; and the ankyrin repeat domain having binding specificity for CD70 comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 64. The present invention further provides such recombinant proteins, wherein the ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 1 to 5, preferably SEQ ID NO: 3; the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence that is at least 85% identical 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 the ankyrin repeat domain having binding specificity for CD70 comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 64 and 107 to 110, preferably SEQ ID NO: 109.

[0037] In one aspect, the invention provides such recombinant proteins, wherein the ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 1 to 5, preferably SEQ ID NO: 2; the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 15 and 67 to 70; the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 6 and 65 to 66; and the ankyrin repeat domain having binding specificity for CD70 comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of SEQ ID NO: 64. The present invention further provides recombinant proteins in which the ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 1 to 5, preferably SEQ ID NO: 3; the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence that is at least 85% identical 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; the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence that is at least 85% identical 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 the ankyrin repeat domain having binding specificity for CD70 comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 64 and 107 to 110, preferably SEQ ID NO: 109.

[0038] In one aspect, the present invention provides such a recombinant protein, wherein the first, second, and / or third binding agents are covalently attached to a peptide linker.

[0039] In one aspect, the present invention provides a recombinant protein, wherein the protein comprises a polypeptide having an amino acid sequence at least 80% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 7 to 10 and 58 to 62, preferably, the protein comprises a polypeptide having an amino acid sequence at least 80% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 7 to 10 and 58 to 62. The present invention further provides a recombinant protein, wherein the protein comprises a polypeptide having an amino acid sequence at least 80% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 11 to 14, 78 to 86, and 95 to 101, preferably, the protein comprises a polypeptide having any one of the amino acid sequences set forth in SEQ ID NOs: 11 to 14, 78 to 86, and 95 to 101. The present invention further provides a recombinant protein, wherein the protein comprises a polypeptide having an amino acid sequence at least 80% identical to any one of the amino acid sequences set forth in SEQ ID NOs: 95 to 101, preferably, SEQ ID NO: 95 or SEQ ID NO: 96 ...

[0040] In one aspect, the present invention relates to a method for producing a protein comprising the steps of: -6 A dissociation constant (K D ) binds to human CD3 in PBS.

[0041] In one aspect, the present invention relates to a method for producing a protein comprising the steps of: -7 A dissociation constant (K D ) binds to human CD33 in PBS.

[0042] In one aspect, the present invention relates to a method for producing a protein comprising the steps of: -7 A dissociation constant (K D ) that binds to human CD123 in PBS.

[0043] In one aspect, the present invention relates to a method for producing a protein comprising the steps of: -7 A dissociation constant (K D ) binds to human CD70 in PBS.

[0044] In one aspect, the present invention relates to a method for treating atopic dermatitis, comprising administering to a subject the method of the present invention a method for treating atopic dermatitis, the method ... 50 The present invention provides such recombinant proteins that bind to human CD3 at the

[0045] In one aspect, the present invention provides such a recombinant protein, wherein the protein further comprises a half-life extending moiety, preferably, the half-life extending moiety is a binding agent that specifically binds to human serum albumin.

[0046] In one aspect, the present invention provides such a recombinant protein, wherein said half-life extending moiety is an engineered ankyrin repeat domain with binding specificity for human serum albumin.

[0047] In one aspect, the present invention provides such a recombinant protein, wherein the ankyrin repeat domain having binding specificity for human serum albumin comprises an amino acid sequence that is at least 85% identical to the amino acid sequence of any one of SEQ ID NOs: 34 to 36.

[0048] In one aspect, the present invention relates to a recombinant protein comprising: -7 A dissociation constant (K D The present invention provides such a recombinant protein that binds to human serum albumin in PBS at 1000 kJ / ml.

[0049] In one aspect, the present invention further provides nucleic acids encoding such recombinant proteins.

[0050] In one aspect, the present invention further provides a pharmaceutical composition comprising a recombinant protein, or a nucleic acid encoding such a recombinant protein, and a pharmaceutically acceptable carrier and / or diluent.

[0051] In one aspect, the present invention further provides a method of treating a medical condition comprising administering to a patient in need thereof a therapeutically effective amount of such a recombinant protein, or a pharmaceutical composition comprising said recombinant protein.

[0052] In one aspect, the present invention provides a method of treating a medical condition, wherein the medical condition is cancer, preferably a liquid tumor, more preferably leukemia, even more preferably acute myeloid leukemia.

[0053] 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.

[0054] 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 for use in the treatment of liquid tumors.

[0055] 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 said cancer is acute myeloid leukemia. [Brief explanation of the drawings]

[0056] [Figure 1A] Figure 1 shows the group mean serum concentration-time profiles of DARPin® Protein #16, DARPin® Protein #17, DARPin® Protein #18, and DARPin® Protein #19 in female BALB / c mice after a single intravenous bolus dose of 1 mg / kg (mean ± max / min, N=3 per group). [Figure 1B]Pharmacokinetic analysis of exemplary specifically designed ankyrin repeat proteins in female BALB / c mice. Figure shows the group mean serum concentration-time profiles of DARPin® Protein #51, DARPin® Protein #50, DARPin® Protein #54, DARPin® Protein #54, and DARPin® Protein #55 in female BALB / c mice after a single intravenous bolus dose of 1 mg / kg (mean ± max / min, N=3 per group). [Figure 2A] Surface plasmon resonance (SPR) analysis of ankyrin repeat protein binding to human CD3, exemplified by DARPin® protein #53 (Figure 2A), DARPin® protein #54 (Figure 2B), and DARPin® protein #9 (Figure 2C). Various concentrations of purified ankyrin repeat proteins were applied to a GLC chip with immobilized human CD3 for on- and off-rate measurements. Analysis of the resulting SPR traces was used to determine ankyrin repeat protein-CD3 interactions. RU, resonance units; s, time (seconds). [Figure 2B] Surface plasmon resonance (SPR) analysis of ankyrin repeat protein binding to human CD3, exemplified by DARPin® protein #53 (Figure 2A), DARPin® protein #54 (Figure 2B), and DARPin® protein #9 (Figure 2C). Various concentrations of purified ankyrin repeat proteins were applied to a GLC chip with immobilized human CD3 for on- and off-rate measurements. Analysis of the resulting SPR traces was used to determine ankyrin repeat protein-CD3 interactions. RU, resonance units; s, time (seconds). [Figure 2C]Surface plasmon resonance (SPR) analysis of ankyrin repeat protein binding to human CD3, exemplified by DARPin® protein #53 (Figure 2A), DARPin® protein #54 (Figure 2B), and DARPin® protein #9 (Figure 2C). Various concentrations of purified ankyrin repeat proteins were applied to a GLC chip with immobilized human CD3 for on- and off-rate measurements. Analysis of the resulting SPR traces was used to determine ankyrin repeat protein-CD3 interactions. RU, resonance units; s, time (seconds). [Figure 2D] Shown are SPR traces obtained for DARPin® protein #56 and DARPin® protein #57. [Figure 3A]Short-term T cell activation measured by the activation marker CD25. 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 coculture in the presence of serial dilutions of the indicated molecules. Activated T cells were gated as viable CD8+ / CD25+ cells. Selected recombinant proteins (without half-life extension) that bind to MOLM-13 cells (Figure 3A) include known benchmark T cell engagers AMG330, which has binding specificity for CD33, and flotetuzumab, which has binding specificity for CD123, as well as DARPin® Protein #27, which has two tumor antigen-specific binding domains, CD123 and CD33, compared to proteins with only one tumor antigen-specific binding domain (DARPin® Protein 40 and DARPin® Protein 41). (Figure 3B) DARPin® Protein #27, which has two tumor antigen-specific binding domains, CD123 and CD33, compared to proteins with only one tumor antigen-specific binding domain (DARPin® Protein 40 and DARPin® Protein 41). 3C shows two tumor antigen-specific binding domains with binding specificity for CD70 and CD33, DARPin® Protein #28 and DARPin® Protein #29, compared to proteins with binding specificity for CD70 and CD33 (DARPin® Protein 42 and DARPin® Protein 43), (Figure 3C) and three tumor antigen-specific binding domains with binding specificity for CD70, CD123, and CD33, DARPin® Protein #30, compared to proteins with only one tumor antigen-specific binding domain (DARPin® Protein 44, DARPin® Protein 45, and DARPin® Protein 46). [Figure 3B]Short-term T cell activation measured by the activation marker CD25. 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 coculture in the presence of serial dilutions of the indicated molecules. Activated T cells were gated as viable CD8+ / CD25+ cells. Selected recombinant proteins (without half-life extension) that bind to MOLM-13 cells (Figure 3A) include known benchmark T cell engagers AMG330, which has binding specificity for CD33, and flotetuzumab, which has binding specificity for CD123, as well as DARPin® Protein #27, which has two tumor antigen-specific binding domains, CD123 and CD33, compared to proteins with only one tumor antigen-specific binding domain (DARPin® Protein 40 and DARPin® Protein 41). (Figure 3B) DARPin® Protein #27, which has two tumor antigen-specific binding domains, CD123 and CD33, compared to proteins with only one tumor antigen-specific binding domain (DARPin® Protein 40 and DARPin® Protein 41). 3C shows two tumor antigen-specific binding domains with binding specificity for CD70 and CD33, DARPin® Protein #28 and DARPin® Protein #29, compared to proteins with binding specificity for CD70 and CD33 (DARPin® Protein 42 and DARPin® Protein 43), (Figure 3C) and three tumor antigen-specific binding domains with binding specificity for CD70, CD123, and CD33, DARPin® Protein #30, compared to proteins with only one tumor antigen-specific binding domain (DARPin® Protein 44, DARPin® Protein 45, and DARPin® Protein 46). [Figure 3C]Short-term T cell activation measured by the activation marker CD25. 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 coculture in the presence of serial dilutions of the indicated molecules. Activated T cells were gated as viable CD8+ / CD25+ cells. Selected recombinant proteins (without half-life extension) that bind to MOLM-13 cells (Figure 3A) include known benchmark T cell engagers AMG330, which has binding specificity for CD33, and flotetuzumab, which has binding specificity for CD123, as well as DARPin® Protein #27, which has two tumor antigen-specific binding domains, CD123 and CD33, compared to proteins with only one tumor antigen-specific binding domain (DARPin® Protein 40 and DARPin® Protein 41). (Figure 3B) DARPin® Protein #27, which has two tumor antigen-specific binding domains, CD123 and CD33, compared to proteins with only one tumor antigen-specific binding domain (DARPin® Protein 40 and DARPin® Protein 41). 3C shows two tumor antigen-specific binding domains with binding specificity for CD70 and CD33, DARPin® Protein #28 and DARPin® Protein #29, compared to proteins with binding specificity for CD70 and CD33 (DARPin® Protein 42 and DARPin® Protein 43), (Figure 3C) and three tumor antigen-specific binding domains with binding specificity for CD70, CD123, and CD33, DARPin® Protein #30, compared to proteins with only one tumor antigen-specific binding domain (DARPin® Protein 44, DARPin® Protein 45, and DARPin® Protein 46). [Figure 4A]Short-term T cell activation measured by the activation marker CD25. 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 coculture in the presence of serial dilutions of the indicated molecules. Activated T cells were gated as viable CD8+ / CD25+ cells. DARPin® Protein #27 compared to known benchmark T cell engagers AMG330, which has binding specificity for CD33, and flotetuzumab, which has binding specificity for CD123, as well as selected recombinant proteins binding to MOLM-13 cells. (FIG. 4A) DARPin® Protein #27 compared to a similar protein with two tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus (DARPin® Protein #47), a similar protein with two half-life prolonging domains located at the N-terminus (DARPin® Protein #48), and a similar protein with one half-life prolonging domain located at the C-terminus (DARPin® Protein #49). (FIG. 4B) DARPin® Protein #27 compared to a similar protein with two tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus (DARPin® Protein #49). 4C shows DARPin® Protein #29 compared to a similar protein with three tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus (DARPin® Protein #50), a similar protein with two half-life prolonging domains located at the N-terminus (DARPin® Protein #51), and a similar protein with one half-life prolonging domain located at the C-terminus (DARPin® Protein #52); (FIG. 4C) shows DARPin® Protein #31 compared to a similar protein with three tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus (DARPin® Protein #53), a similar protein with two half-life prolonging domains located at the N-terminus (DARPin® Protein #54), and a similar protein with one half-life prolonging domain located at the C-terminus (DARPin® Protein #55). [Figure 4B]Short-term T cell activation measured by the activation marker CD25. 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 coculture in the presence of serial dilutions of the indicated molecules. Activated T cells were gated as viable CD8+ / CD25+ cells. DARPin® Protein #27 compared to known benchmark T cell engagers AMG330, which has binding specificity for CD33, and flotetuzumab, which has binding specificity for CD123, as well as selected recombinant proteins binding to MOLM-13 cells. (FIG. 4A) DARPin® Protein #27 compared to a similar protein with two tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus (DARPin® Protein #47), a similar protein with two half-life prolonging domains located at the N-terminus (DARPin® Protein #48), and a similar protein with one half-life prolonging domain located at the C-terminus (DARPin® Protein #49). (FIG. 4B) DARPin® Protein #27 compared to a similar protein with two tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus (DARPin® Protein #49). 4C shows DARPin® Protein #29 compared to a similar protein with three tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus (DARPin® Protein #50), a similar protein with two half-life prolonging domains located at the N-terminus (DARPin® Protein #51), and a similar protein with one half-life prolonging domain located at the C-terminus (DARPin® Protein #52); (FIG. 4C) shows DARPin® Protein #31 compared to a similar protein with three tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus (DARPin® Protein #53), a similar protein with two half-life prolonging domains located at the N-terminus (DARPin® Protein #54), and a similar protein with one half-life prolonging domain located at the C-terminus (DARPin® Protein #55). [Figure 4C]Short-term T cell activation measured by the activation marker CD25. 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 coculture in the presence of serial dilutions of the indicated molecules. Activated T cells were gated as viable CD8+ / CD25+ cells. DARPin® Protein #27 compared to known benchmark T cell engagers AMG330, which has binding specificity for CD33, and flotetuzumab, which has binding specificity for CD123, as well as selected recombinant proteins binding to MOLM-13 cells. (FIG. 4A) DARPin® Protein #27 compared to a similar protein with two tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus (DARPin® Protein #47), a similar protein with two half-life prolonging domains located at the N-terminus (DARPin® Protein #48), and a similar protein with one half-life prolonging domain located at the C-terminus (DARPin® Protein #49). (FIG. 4B) DARPin® Protein #27 compared to a similar protein with two tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus (DARPin® Protein #49). 4C shows DARPin® Protein #29 compared to a similar protein with three tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus (DARPin® Protein #50), a similar protein with two half-life prolonging domains located at the N-terminus (DARPin® Protein #51), and a similar protein with one half-life prolonging domain located at the C-terminus (DARPin® Protein #52); (FIG. 4C) shows DARPin® Protein #31 compared to a similar protein with three tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus (DARPin® Protein #53), a similar protein with two half-life prolonging domains located at the N-terminus (DARPin® Protein #54), and a similar protein with one half-life prolonging domain located at the C-terminus (DARPin® Protein #55). [Figure 5A]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 48 hours of coculture in the presence of serial dilutions of the indicated molecules. Benchmark control molecules (known benchmark T cell engagers, AMG330 with binding specificity for CD33 and flotetuzumab with binding specificity for CD123) and multispecific recombinant proteins with two or three tumor-specific binding domains, DARPin® Protein #27 (Figure 5A), compared with proteins with only one tumor antigen-specific binding domain (DARPin® Protein 40 and DARPin® Protein 41), (Figure 5B) compared with proteins with only one tumor antigen-specific binding domain (DARPin® Protein 42). (FIG. 5C) shows two tumor antigen-specific binding domains with binding specificity for CD70 and CD33, DARPin® Protein #28 and DARPin® Protein #29, compared to proteins with binding specificity for CD70 and CD33 (DARPin® Protein 42 and DARPin® Protein 43), (FIG. 5C) and three tumor antigen-specific binding domains with binding specificity for CD70, CD123, and CD33, DARPin® Protein #30, compared to proteins with only one tumor antigen-specific binding domain (DARPin® Protein 44, DARPin® Protein 45, and DARPin® Protein 46). [Figure 5B]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 48 hours of coculture in the presence of serial dilutions of the indicated molecules. Benchmark control molecules (known benchmark T cell engagers, AMG330 with binding specificity for CD33 and flotetuzumab with binding specificity for CD123) and multispecific recombinant proteins with two or three tumor-specific binding domains, DARPin® Protein #27 (Figure 5A), compared with proteins with only one tumor antigen-specific binding domain (DARPin® Protein 40 and DARPin® Protein 41), (Figure 5B) compared with proteins with only one tumor antigen-specific binding domain (DARPin® Protein 42). (FIG. 5C) shows two tumor antigen-specific binding domains with binding specificity for CD70 and CD33, DARPin® Protein #28 and DARPin® Protein #29, compared to proteins with binding specificity for CD70 and CD33 (DARPin® Protein 42 and DARPin® Protein 43), (FIG. 5C) and three tumor antigen-specific binding domains with binding specificity for CD70, CD123, and CD33, DARPin® Protein #30, compared to proteins with only one tumor antigen-specific binding domain (DARPin® Protein 44, DARPin® Protein 45, and DARPin® Protein 46). [Figure 5C]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 48 hours of coculture in the presence of serial dilutions of the indicated molecules. Benchmark control molecules (known benchmark T cell engagers, AMG330 with binding specificity for CD33 and flotetuzumab with binding specificity for CD123) and multispecific recombinant proteins with two or three tumor-specific binding domains, DARPin® Protein #27 (Figure 5A), compared with proteins with only one tumor antigen-specific binding domain (DARPin® Protein 40 and DARPin® Protein 41), (Figure 5B) compared with proteins with only one tumor antigen-specific binding domain (DARPin® Protein 42). (FIG. 5C) shows two tumor antigen-specific binding domains with binding specificity for CD70 and CD33, DARPin® Protein #28 and DARPin® Protein #29, compared to proteins with binding specificity for CD70 and CD33 (DARPin® Protein 42 and DARPin® Protein 43), (FIG. 5C) and three tumor antigen-specific binding domains with binding specificity for CD70, CD123, and CD33, DARPin® Protein #30, compared to proteins with only one tumor antigen-specific binding domain (DARPin® Protein 44, DARPin® Protein 45, and DARPin® Protein 46). [Figure 6A]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 48 hours of co-culture in the presence of serial dilutions of the indicated molecules. DARPin® Protein #27, (FIG. 6B) a similar protein with two tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus, compared with benchmark control molecules (known benchmark T cell engagers, AMG330 with binding specificity for CD33 and flotetuzumab with binding specificity for CD123), and multispecific recombinant proteins with two or three tumor-specific binding domains: (FIG. 6A) a similar protein with two tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus (DARPin® Protein #47), (FIG. 6B) a similar protein with two tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus (DARPin® Protein #48), and (FIG. 6C) a similar protein with one half-life prolonging domain located at the C-terminus (DARPin® Protein #49). 6C shows DARPin® Protein #29 compared to a similar protein with three tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus (DARPin® Protein #50), a similar protein with two half-life prolonging domains located at the N-terminus (DARPin® Protein #51), and a similar protein with one half-life prolonging domain located at the C-terminus (DARPin® Protein #52); (FIG. 6C) shows DARPin® Protein #31 compared to a similar protein with three tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus (DARPin® Protein #63), a similar protein with two half-life prolonging domains located at the N-terminus (DARPin® Protein #54), and a similar protein with one half-life prolonging domain located at the C-terminus (DARPin® Protein #55). [Figure 6B]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 48 hours of co-culture in the presence of serial dilutions of the indicated molecules. DARPin® Protein #27, (FIG. 6B) a similar protein with two tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus, compared with benchmark control molecules (known benchmark T cell engagers, AMG330 with binding specificity for CD33 and flotetuzumab with binding specificity for CD123), and multispecific recombinant proteins with two or three tumor-specific binding domains: (FIG. 6A) a similar protein with two tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus (DARPin® Protein #47), (FIG. 6B) a similar protein with two tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus (DARPin® Protein #48), and (FIG. 6C) a similar protein with one half-life prolonging domain located at the C-terminus (DARPin® Protein #49). 6C shows DARPin® Protein #29 compared to a similar protein with three tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus (DARPin® Protein #50), a similar protein with two half-life prolonging domains located at the N-terminus (DARPin® Protein #51), and a similar protein with one half-life prolonging domain located at the C-terminus (DARPin® Protein #52); (FIG. 6C) shows DARPin® Protein #31 compared to a similar protein with three tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus (DARPin® Protein #63), a similar protein with two half-life prolonging domains located at the N-terminus (DARPin® Protein #54), and a similar protein with one half-life prolonging domain located at the C-terminus (DARPin® Protein #55). [Figure 6C]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 48 hours of co-culture in the presence of serial dilutions of the indicated molecules. DARPin® Protein #27, (FIG. 6B) a similar protein with two tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus, compared with benchmark control molecules (known benchmark T cell engagers, AMG330 with binding specificity for CD33 and flotetuzumab with binding specificity for CD123), and multispecific recombinant proteins with two or three tumor-specific binding domains: (FIG. 6A) a similar protein with two tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus (DARPin® Protein #47), (FIG. 6B) a similar protein with two tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus (DARPin® Protein #48), and (FIG. 6C) a similar protein with one half-life prolonging domain located at the C-terminus (DARPin® Protein #49). 6C shows DARPin® Protein #29 compared to a similar protein with three tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus (DARPin® Protein #50), a similar protein with two half-life prolonging domains located at the N-terminus (DARPin® Protein #51), and a similar protein with one half-life prolonging domain located at the C-terminus (DARPin® Protein #52); (FIG. 6C) shows DARPin® Protein #31 compared to a similar protein with three tumor antigen-specific binding domains and one half-life prolonging domain located at the N-terminus (DARPin® Protein #63), a similar protein with two half-life prolonging domains located at the N-terminus (DARPin® Protein #54), and a similar protein with one half-life prolonging domain located at the C-terminus (DARPin® Protein #55). [Figure 7]Target cell killing. Benchmark control molecules (known benchmark T cell engagers AMG330, which has binding specificity for CD33, and flotetuzumab, which has binding specificity for CD123) and DARPin® Protein #31 are shown. The data show that in tumor cell killing assays, DARPin® Protein #5 exerts similar potency and efficacy when compared to the clinical benchmarks. [Figure 8] Target cell killing. Curve 7 represents Molm13 parental cells expressing all three targets (CD70, CD123, and CD33). DARPin® Protein #31 shows full efficacy. Curves 4-6 represent single KO cells, meaning only two targets are still expressed. Here, DARPin® Protein #5 is still active, suggesting its potential to combat tumor heterogeneity. Curves 1-3 represent double KO cells, meaning only one target is still expressed to mimic the healthy tissue compartment. Here, DARPin® Protein #31 has significantly less activity, meaning improved selectivity for healthy tissue. [Figure 9A] IFNγ levels (average of three donors). The mean IFNγ concentration values ​​for all three donors (D1-3) in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. The calculated LLOQ is indicated by the dotted line. Each point is the average of three donors, and the error bars are SD. Flotetuzumab is shown in Figure 9A, DARPin® Protein #27 in Figure 9B, DARPin® Protein #29 in Figure 9C, and DARPin® Protein #31 in Figure 9D. Data and values ​​are above the ULOQ. [Figure 9B]IFNγ levels (average of three donors). The mean IFNγ concentration values ​​for all three donors (D1-3) in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. The calculated LLOQ is indicated by the dotted line. Each point is the average of three donors, and the error bars are SD. Flotetuzumab is shown in Figure 9A, DARPin® Protein #27 in Figure 9B, DARPin® Protein #29 in Figure 9C, and DARPin® Protein #31 in Figure 9D. Data and values ​​are above the ULOQ. [Figure 9C] IFNγ levels (average of three donors). The mean IFNγ concentration values ​​for all three donors (D1-3) in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. The calculated LLOQ is indicated by the dotted line. Each point is the average of three donors, and the error bars are SD. Flotetuzumab is shown in Figure 9A, DARPin® Protein #27 in Figure 9B, DARPin® Protein #29 in Figure 9C, and DARPin® Protein #31 in Figure 9D. Data and values ​​are above the ULOQ. [Figure 9D] IFNγ levels (average of three donors). The mean IFNγ concentration values ​​for all three donors (D1-3) in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. The calculated LLOQ is indicated by the dotted line. Each point is the average of three donors, and the error bars are SD. Flotetuzumab is shown in Figure 9A, DARPin® Protein #27 in Figure 9B, DARPin® Protein #29 in Figure 9C, and DARPin® Protein #31 in Figure 9D. Data and values ​​are above the ULOQ. [Figure 10A]IL-2 levels (average of three donors). The average IL-2 concentration values ​​for all three donors (D1-3) in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0, 2, 4, 8, and 24 hours. The calculated LLOQ is indicated by the dotted line. Each point is the average of three donors, and the error bars are SD. Flotetuzumab is shown in Figure 10A, DARPin® Protein #27 in Figure 10B, DARPin® Protein #29 in Figure 10C, and DARPin® Protein #31 in Figure 10D. [Figure 10B] IL-2 levels (average of three donors). The average IL-2 concentration values ​​for all three donors (D1-3) in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0, 2, 4, 8, and 24 hours. The calculated LLOQ is indicated by the dotted line. Each point is the average of three donors, and the error bars are SD. Flotetuzumab is shown in Figure 10A, DARPin® Protein #27 in Figure 10B, DARPin® Protein #29 in Figure 10C, and DARPin® Protein #31 in Figure 10D. [Figure 10C] IL-2 levels (average of three donors). The average IL-2 concentration values ​​for all three donors (D1-3) in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0, 2, 4, 8, and 24 hours. The calculated LLOQ is indicated by the dotted line. Each point is the average of three donors, and the error bars are SD. Flotetuzumab is shown in Figure 10A, DARPin® Protein #27 in Figure 10B, DARPin® Protein #29 in Figure 10C, and DARPin® Protein #31 in Figure 10D. [Figure 10D]IL-2 levels (average of three donors). The average IL-2 concentration values ​​for all three donors (D1-3) in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0, 2, 4, 8, and 24 hours. The calculated LLOQ is indicated by the dotted line. Each point is the average of three donors, and the error bars are SD. Flotetuzumab is shown in Figure 10A, DARPin® Protein #27 in Figure 10B, DARPin® Protein #29 in Figure 10C, and DARPin® Protein #31 in Figure 10D. [Figure 11A] IL-6 levels (average of three donors). The average IL-6 concentration values ​​for all three donors (D1-3) in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. The calculated LLOQ is indicated by the dotted line. Each point is the average of three donors, and the error bars are SD. Flotetuzumab is shown in Figure 11A, DARPin® Protein #27 in Figure 11B, DARPin® Protein #29 in Figure 11C, and DARPin® Protein #31 in Figure 11D. Data and values ​​are above the ULOQ. [Figure 11B] IL-6 levels (average of three donors). The average IL-6 concentration values ​​for all three donors (D1-3) in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. The calculated LLOQ is indicated by the dotted line. Each point is the average of three donors, and the error bars are SD. Flotetuzumab is shown in Figure 11A, DARPin® Protein #27 in Figure 11B, DARPin® Protein #29 in Figure 11C, and DARPin® Protein #31 in Figure 11D. Data and values ​​are above the ULOQ. [Figure 11C]IL-6 levels (average of three donors). The average IL-6 concentration values ​​for all three donors (D1-3) in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. The calculated LLOQ is indicated by the dotted line. Each point is the average of three donors, and the error bars are SD. Flotetuzumab is shown in Figure 11A, DARPin® Protein #27 in Figure 11B, DARPin® Protein #29 in Figure 11C, and DARPin® Protein #31 in Figure 11D. Data and values ​​are above the ULOQ. [Figure 11D] IL-6 levels (average of three donors). The average IL-6 concentration values ​​for all three donors (D1-3) in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. The calculated LLOQ is indicated by the dotted line. Each point is the average of three donors, and the error bars are SD. Flotetuzumab is shown in Figure 11A, DARPin® Protein #27 in Figure 11B, DARPin® Protein #29 in Figure 11C, and DARPin® Protein #31 in Figure 11D. Data and values ​​are above the ULOQ. [Figure 12A] IL-8 levels (average of three donors). The mean IL-8 concentration values ​​for all three donors (D1-3) in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. The calculated LLOQ is indicated by the dotted line. Each point is the average of three donors, and the error bars are SD. Flotetuzumab is shown in Figure 11A, DARPin® Protein #27 in Figure 11B, DARPin® Protein #29 in Figure 11C, and DARPin® Protein #31 in Figure 11D. Data and values ​​are above the ULOQ. [Figure 12B]IL-8 levels (average of three donors). The mean IL-8 concentration values ​​for all three donors (D1-3) in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. The calculated LLOQ is indicated by the dotted line. Each point is the average of three donors, and the error bars are SD. Flotetuzumab is shown in Figure 11A, DARPin® Protein #27 in Figure 11B, DARPin® Protein #29 in Figure 11C, and DARPin® Protein #31 in Figure 11D. Data and values ​​are above the ULOQ. [Figure 12C] IL-8 levels (average of three donors). The mean IL-8 concentration values ​​for all three donors (D1-3) in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. The calculated LLOQ is indicated by the dotted line. Each point is the average of three donors, and the error bars are SD. Flotetuzumab is shown in Figure 11A, DARPin® Protein #27 in Figure 11B, DARPin® Protein #29 in Figure 11C, and DARPin® Protein #31 in Figure 11D. Data and values ​​are above the ULOQ. [Figure 12D] IL-8 levels (average of three donors). The mean IL-8 concentration values ​​for all three donors (D1-3) in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. The calculated LLOQ is indicated by the dotted line. Each point is the average of three donors, and the error bars are SD. Flotetuzumab is shown in Figure 11A, DARPin® Protein #27 in Figure 11B, DARPin® Protein #29 in Figure 11C, and DARPin® Protein #31 in Figure 11D. Data and values ​​are above the ULOQ. [Figure 13A]TNFα levels (average of three donors). The mean TNFα concentration values ​​for all three donors (D1-3) in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. The calculated LLOQ is indicated by the dotted line. Each point is the average of three donors, and the error bars are SD. Flotetuzumab is shown in Figure 13A, DARPin® Protein #27 in Figure 13B, DARPin® Protein #29 in Figure 13C, and DARPin® Protein #31 in Figure 13D. Data and values ​​are above the ULOQ. [Figure 13B] TNFα levels (average of three donors). The mean TNFα concentration values ​​for all three donors (D1-3) in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. The calculated LLOQ is indicated by the dotted line. Each point is the average of three donors, and the error bars are SD. Flotetuzumab is shown in Figure 13A, DARPin® Protein #27 in Figure 13B, DARPin® Protein #29 in Figure 13C, and DARPin® Protein #31 in Figure 13D. Data and values ​​are above the ULOQ. [Figure 13C] TNFα levels (average of three donors). The mean TNFα concentration values ​​for all three donors (D1-3) in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. The calculated LLOQ is indicated by the dotted line. Each point is the average of three donors, and the error bars are SD. Flotetuzumab is shown in Figure 13A, DARPin® Protein #27 in Figure 13B, DARPin® Protein #29 in Figure 13C, and DARPin® Protein #31 in Figure 13D. Data and values ​​are above the ULOQ. [Figure 13D]TNFα levels (average of three donors). The mean TNFα concentration values ​​for all three donors (D1-3) in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. The calculated LLOQ is indicated by the dotted line. Each point is the average of three donors, and the error bars are SD. Flotetuzumab is shown in Figure 13A, DARPin® Protein #27 in Figure 13B, DARPin® Protein #29 in Figure 13C, and DARPin® Protein #31 in Figure 13D. Data and values ​​are above the ULOQ. [Figure 14A] Platelet counts. Blood was drawn from the loops at time 0 (zero sample), 2, 4, 8, and 24 hours, and PLT counts were automatically counted using a Sysmex Hematology Analyzer. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab is shown in Figure 14A, DARPin® protein #27 in Figure 14B, DARPin® protein #29 in Figure 14C, and DARPin® protein #31 in Figure 14D. [Figure 14B] Platelet counts. Blood was drawn from the loops at time 0 (zero sample), 2, 4, 8, and 24 hours, and PLT counts were automatically counted using a Sysmex Hematology Analyzer. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab is shown in Figure 14A, DARPin® protein #27 in Figure 14B, DARPin® protein #29 in Figure 14C, and DARPin® protein #31 in Figure 14D. [Figure 14C] Platelet counts. Blood was drawn from the loops at time 0 (zero sample), 2, 4, 8, and 24 hours, and PLT counts were automatically counted using a Sysmex Hematology Analyzer. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab is shown in Figure 14A, DARPin® protein #27 in Figure 14B, DARPin® protein #29 in Figure 14C, and DARPin® protein #31 in Figure 14D. [Figure 14D]Platelet counts. Blood was drawn from the loops at time 0 (zero sample), 2, 4, 8, and 24 hours, and PLT counts were automatically counted using a Sysmex Hematology Analyzer. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab is shown in Figure 14A, DARPin® protein #27 in Figure 14B, DARPin® protein #29 in Figure 14C, and DARPin® protein #31 in Figure 14D. [Figure 15A] On-rate / off-rate constants for the CD33 target measured for selected recombinant proteins DARPin® Protein 27, DARPin® Protein 40 and DARPin® Protein 41. [Figure 15B] On-rate / off-rate constants for the CD33 target measured for selected recombinant proteins DARPin® Protein 27, DARPin® Protein 40 and DARPin® Protein 41. [Figure 16A] On-rate / off-rate constants measured for selected recombinant proteins DARPin® Protein 27, DARPin® Protein 40 and DARPin® Protein 41 for the CD123 target. [Figure 16B] On-rate / off-rate constants measured for selected recombinant proteins DARPin® Protein 27, DARPin® Protein 40 and DARPin® Protein 41 for the CD123 target. [Figure 17] KD values ​​for the interaction of recombinant proteins with human CD33 [Figure 18] KD values ​​for the interaction of recombinant proteins with human CD123 [Figure 19]SDS-PAGE gel analysis of the purification of four selected ankyrin repeat proteins with binding specificity for human CD3, DARPin® Protein #1, DARPin® Protein #2, DARPin® Protein #3, and DARPin® Protein #4. M indicates protein size marker (reducing SDS-PAGE, NuPAGE 4-12%, Bis Tris (Invitrogen) gel; 5 μg / lane; MES buffer; Instant Blue stain). The molecular weight (kDa) of the marker proteins is indicated. Lane 1: protein size marker; Lane 2: purified DARPin® Protein #1; Lane 3: purified DARPin® Protein #2; Lane 4: purified DARPin® Protein #3; Lane 5: purified DARPin® Protein #4. [Figure 20A]Figures 20A and 20B. CD3 binding of exemplary ankyrin repeat proteins to T cells. Binding to CD3 on Pan-T cells was assessed by Mirrorball. Benchmark control molecules (known benchmark T cell engagers, AMG330, which has binding specificity for CD33, and flotetuzumab, which has binding specificity for CD123) are shown, as well as selected ankyrin repeat proteins DARPin® Protein 7, DARPin® Protein 8, DARPin® Protein 9, and DARPin® Protein 10 without half-life extension (HLE) (Figure 20A) or DARPin® Protein 11, DARPin® Protein 12, DARPin® Protein 13, and DARPin® Protein 14 with half-life extension (HLE) (Figure 20B). Half-life-extending proteins (B) show similar binding compared to the corresponding non-HLE molecules shown in (A). Pan-T cells from five different donors were tested, one representative donor is shown here. (*Negative control: designed ankyrin repeat proteins with binding specificity for only CD33 and CD123, respectively, with or without half-life extension. Figure 20C shows tumor cell binding of selected multispecific binding proteins DARPin® Protein #56, DARPin® Protein #57, DARPin® Protein #58, DARPin® Protein #59, DARPin® Protein #60, DARPin® Protein #61, and DARPin® Protein #62 to Molm-13 N1 cells. [Figure 20B]Figures 20A and 20B. CD3 binding of exemplary ankyrin repeat proteins to T cells. Binding to CD3 on Pan-T cells was assessed by Mirrorball. Benchmark control molecules (known benchmark T cell engagers, AMG330, which has binding specificity for CD33, and flotetuzumab, which has binding specificity for CD123) are shown, as well as selected ankyrin repeat proteins DARPin® Protein 7, DARPin® Protein 8, DARPin® Protein 9, and DARPin® Protein 10 without half-life extension (HLE) (Figure 20A) or DARPin® Protein 11, DARPin® Protein 12, DARPin® Protein 13, and DARPin® Protein 14 with half-life extension (HLE) (Figure 20B). Half-life-extending proteins (B) show similar binding compared to the corresponding non-HLE molecules shown in (A). Pan-T cells from five different donors were tested, one representative donor is shown here. (*Negative control: designed ankyrin repeat proteins with binding specificity for only CD33 and CD123, respectively, with or without half-life extension. Figure 20C shows tumor cell binding of selected multispecific binding proteins DARPin® Protein #56, DARPin® Protein #57, DARPin® Protein #58, DARPin® Protein #59, DARPin® Protein #60, DARPin® Protein #61, and DARPin® Protein #62 to Molm-13 N1 cells. [Figure 20C]Figures 20A and 20B. CD3 binding of exemplary ankyrin repeat proteins to T cells. Binding to CD3 on Pan-T cells was assessed by Mirrorball. Benchmark control molecules (known benchmark T cell engagers, AMG330, which has binding specificity for CD33, and flotetuzumab, which has binding specificity for CD123) are shown, as well as selected ankyrin repeat proteins DARPin® Protein 7, DARPin® Protein 8, DARPin® Protein 9, and DARPin® Protein 10 without half-life extension (HLE) (Figure 20A) or DARPin® Protein 11, DARPin® Protein 12, DARPin® Protein 13, and DARPin® Protein 14 with half-life extension (HLE) (Figure 20B). Half-life-extending proteins (B) show similar binding compared to the corresponding non-HLE molecules shown in (A). Pan-T cells from five different donors were tested, one representative donor is shown here. (*Negative control: designed ankyrin repeat proteins with binding specificity for only CD33 and CD123, respectively, with or without half-life extension. Figure 20C shows tumor cell binding of selected multispecific binding proteins DARPin® Protein #56, DARPin® Protein #57, DARPin® Protein #58, DARPin® Protein #59, DARPin® Protein #60, DARPin® Protein #61, and DARPin® Protein #62 to Molm-13 N1 cells. [Figure 21]Short-term T cell activation measured by the activation marker CD25. 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 coculture in the presence of serial dilutions of the indicated molecules. Activated T cells were gated as viable CD8+ / CD25+ cells. Shown are known benchmark T cell engagers, AMG330, which has binding specificity for CD33, and flotetuzumab, which has binding specificity for CD123, as well as selected recombinant proteins that bind to MOLM-13 cells with either two tumor-specific binding domains (DARPin® Protein 8) or only one tumor-specific binding domain (DARPin® Protein 23 and DARPin® Protein 24). T cell activation induced by DARPin® Protein 8 is comparable to the benchmark molecules, while DARPin® Protein 23 and DARPin® Protein 24 showed lower potency. Representative data using pan T cells from one donor are shown. [Figure 22A]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 48 hours of coculture in the presence of serial dilutions of the indicated molecules. Benchmark control molecules (known benchmark T cell engagers, AMG330, which has binding specificity for CD33, and flotetuzumab, which has binding specificity for CD123) are shown, as well as selected ankyrin repeat proteins DARPin® Protein 7, DARPin® Protein 8, DARPin® Protein 9, and DARPin® Protein 10 without half-life extension (HLE) (Figure 22A), or DARPin® Protein 11, DARPin® Protein 12, DARPin® Protein 13, and DARPin® Protein 14 with half-life extension (HLE) (Figure 22B). (Figure 22A) For proteins without half-life extension, tumor cell killing induced by DARPin® Protein 8 and DARPin® Protein 9 is comparable to the benchmark molecules, whereas DARPin® Protein 10 and DARPin® Protein 7 show lower potency in cytotoxicity. (Figure 22B) The half-life extension proteins show a 4- to 70-fold decrease in potency compared to the corresponding non-HLE molecules shown in (A). Pan-T cells from five different donors were tested, and one representative donor is shown here. (*Negative control: designed ankyrin repeat proteins with binding specificity for CD33 and CD123, respectively, with or without half-life extension). [Figure 22B]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 48 hours of coculture in the presence of serial dilutions of the indicated molecules. Benchmark control molecules (known benchmark T cell engagers, AMG330, which has binding specificity for CD33, and flotetuzumab, which has binding specificity for CD123) are shown, as well as selected ankyrin repeat proteins DARPin® Protein 7, DARPin® Protein 8, DARPin® Protein 9, and DARPin® Protein 10 without half-life extension (HLE) (Figure 22A), or DARPin® Protein 11, DARPin® Protein 12, DARPin® Protein 13, and DARPin® Protein 14 with half-life extension (HLE) (Figure 22B). (Figure 22A) For proteins without half-life extension, tumor cell killing induced by DARPin® Protein 8 and DARPin® Protein 9 is comparable to the benchmark molecules, whereas DARPin® Protein 10 and DARPin® Protein 7 show lower potency in cytotoxicity. (Figure 22B) The half-life extension proteins show a 4- to 70-fold decrease in potency compared to the corresponding non-HLE molecules shown in (A). Pan-T cells from five different donors were tested, and one representative donor is shown here. (*Negative control: designed ankyrin repeat proteins with binding specificity for CD33 and CD123, respectively, with or without half-life extension). [Figure 23A]Short-term T cell activation measured by the activation marker CD25. 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 coculture in the presence of serial dilutions of the indicated molecules. Activated T cells were gated as viable CD8+ / CD25+ cells. A benchmark control molecule (AMG330, a known benchmark T cell engager with binding specificity for CD33) is shown, as well as selected ankyrin repeat proteins DARPin® Protein 7, DARPin® Protein 8, DARPin® Protein 9, and DARPin® Protein 10 without half-life extension (HLE) (Figure 23A) or DARPin® Protein 11, DARPin® Protein 12, DARPin® Protein 13, and DARPin® Protein 14 with half-life extension (HLE) (Figure 23B). (Figure 23A) T cell activation induced by DARPin® Protein 8 and DARPin® Protein 9 without half-life extension is comparable to the benchmark, whereas DARPin® Protein 10 and DARPin® Protein 7 show lower potency. (Figure 23B) The half-life extended proteins show a 4- to 100-fold decrease in potency compared to the corresponding non-HLE molecules shown in (A). Pan-T cells from seven different donors were tested, and one representative donor is shown here. (*Negative control: designed ankyrin repeat proteins with binding specificity for only CD33 and CD123, respectively, with or without half-life extension). [Figure 23B]Short-term T cell activation measured by the activation marker CD25. 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 coculture in the presence of serial dilutions of the indicated molecules. Activated T cells were gated as viable CD8+ / CD25+ cells. A benchmark control molecule (AMG330, a known benchmark T cell engager with binding specificity for CD33) is shown, as well as selected ankyrin repeat proteins DARPin® Protein 7, DARPin® Protein 8, DARPin® Protein 9, and DARPin® Protein 10 without half-life extension (HLE) (Figure 23A) or DARPin® Protein 11, DARPin® Protein 12, DARPin® Protein 13, and DARPin® Protein 14 with half-life extension (HLE) (Figure 23B). (Figure 23A) T cell activation induced by DARPin® Protein 8 and DARPin® Protein 9 without half-life extension is comparable to the benchmark, whereas DARPin® Protein 10 and DARPin® Protein 7 show lower potency. (Figure 23B) The half-life extended proteins show a 4- to 100-fold decrease in potency compared to the corresponding non-HLE molecules shown in (A). Pan-T cells from seven different donors were tested, and one representative donor is shown here. (*Negative control: designed ankyrin repeat proteins with binding specificity for only CD33 and CD123, respectively, with or without half-life extension). [Figure 24A]Short-term T cell activation measured by IFNγ secretion. Pan-T and Molm-13 cells were incubated at an E:T ratio of 1:1. After 24 h of coculture in the presence of serial dilutions of the indicated molecules, IFNγ secretion in the culture supernatants was analyzed by ELISA. A benchmark control molecule (AMG330, a known benchmark T cell engager with binding specificity for CD33) is shown, as well as selected ankyrin repeat proteins DARPin® Protein 7, DARPin® Protein 8, DARPin® Protein 9, and DARPin® Protein 10 without half-life extension (HLE) (Figure 24A) or DARPin® Protein 11, DARPin® Protein 12, DARPin® Protein 13, and DARPin® Protein 14 with half-life extension (HLE) (Figure 24B). (Figure 24A) T cell activation induced by DARPin® Protein 8 and DARPin® Protein 9 without half-life extension is comparable to the benchmark molecules, whereas DARPin® Protein 10 and DARPin® Protein 7 show lower potency. (Figure 24B) The half-life extended proteins show a 3- to 20-fold decrease in potency compared to the corresponding non-HLE molecules shown in (A). Pan-T cells from four different donors were tested, and one representative donor is shown here. (*Negative control: designed ankyrin repeat proteins with binding specificity for only CD33 and CD123, respectively, with or without half-life extension). [Figure 24B]Short-term T cell activation measured by IFNγ secretion. Pan-T and Molm-13 cells were incubated at an E:T ratio of 1:1. After 24 h of coculture in the presence of serial dilutions of the indicated molecules, IFNγ secretion in the culture supernatants was analyzed by ELISA. A benchmark control molecule (AMG330, a known benchmark T cell engager with binding specificity for CD33) is shown, as well as selected ankyrin repeat proteins DARPin® Protein 7, DARPin® Protein 8, DARPin® Protein 9, and DARPin® Protein 10 without half-life extension (HLE) (Figure 24A) or DARPin® Protein 11, DARPin® Protein 12, DARPin® Protein 13, and DARPin® Protein 14 with half-life extension (HLE) (Figure 24B). (Figure 24A) T cell activation induced by DARPin® Protein 8 and DARPin® Protein 9 without half-life extension is comparable to the benchmark molecules, whereas DARPin® Protein 10 and DARPin® Protein 7 show lower potency. (Figure 24B) The half-life extended proteins show a 3- to 20-fold decrease in potency compared to the corresponding non-HLE molecules shown in (A). Pan-T cells from four different donors were tested, and one representative donor is shown here. (*Negative control: designed ankyrin repeat proteins with binding specificity for only CD33 and CD123, respectively, with or without half-life extension). [Figure 25]Long-term tumor cell killing. Pan-T cells and Molm-13 cells were incubated at a 5:1 E:T ratio, and tumor cell killing was assessed by IncuCyte over 6 days of co-culture in the presence of serial dilutions of the indicated molecules. Tumor cell killing is calculated as the ratio between the area under the curve of Annexin V staining and cell proliferation. Two benchmark control molecules (known benchmark T cell engagers, AMG330, which has binding specificity for CD33, and flotetuzumab, which has binding specificity for CD123) are shown, as well as selected ankyrin repeat proteins DARPin® Protein 7, DARPin® Protein 8, DARPin® Protein 9, and DARPin® Protein 10 without half-life extension (HLE), or DARPin® Protein 11, DARPin® Protein 12, DARPin® Protein 13, and DARPin® Protein 14 with half-life extension (HLE). The data show potent and specific tumor cell killing comparable to the benchmark molecule for most tested proteins, regardless of half-life extension. Only the lower affinity DARPin® Protein 7 shows a significant decrease in killing efficacy. Concentrations, from left to right, are 2 nM diluted 1 / 10 for the benchmark and test proteins and 20 nM diluted 1 / 10 for the half-life extension molecule. Pan-T cells from five different donors were used, with one representative donor shown here. [Figure 26A]Long-term T cell activation. 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 5 days of coculture in the presence of serial dilutions of the indicated molecules. Activated T cells were gated as viable CD8+ / CD25+ cells. Benchmark control molecules (known benchmark T cell engagers, AMG330, which has binding specificity for CD33, and flotetuzumab, which has binding specificity for CD123) are shown, as well as selected ankyrin repeat proteins DARPin® Protein 7, DARPin® Protein 8, DARPin® Protein 9, and DARPin® Protein 10 without half-life extension (HLE), or DARPin® Protein 11, DARPin® Protein 12, DARPin® Protein 13, and DARPin® Protein 14 with half-life extension (HLE). (Figure 26A) T cell activation induced by DARPin® Protein 8 and DARPin® Protein 9 without half-life extension is comparable to the benchmark molecules, whereas DARPin® Protein 7 and DARPin® Protein 10 show a more than 100-fold decrease in potency. (Figure 26B) The half-life extended proteins show a more than 10-fold decrease in potency compared to the corresponding non-HLE molecules shown in (A). Pan-T cells from two different donors were used, with one representative donor shown here. [Figure 26B]Long-term T cell activation. 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 5 days of coculture in the presence of serial dilutions of the indicated molecules. Activated T cells were gated as viable CD8+ / CD25+ cells. Benchmark control molecules (known benchmark T cell engagers, AMG330, which has binding specificity for CD33, and flotetuzumab, which has binding specificity for CD123) are shown, as well as selected ankyrin repeat proteins DARPin® Protein 7, DARPin® Protein 8, DARPin® Protein 9, and DARPin® Protein 10 without half-life extension (HLE), or DARPin® Protein 11, DARPin® Protein 12, DARPin® Protein 13, and DARPin® Protein 14 with half-life extension (HLE). (Figure 26A) T cell activation induced by DARPin® Protein 8 and DARPin® Protein 9 without half-life extension is comparable to the benchmark molecules, whereas DARPin® Protein 7 and DARPin® Protein 10 show a more than 100-fold decrease in potency. (Figure 26B) The half-life extended proteins show a more than 10-fold decrease in potency compared to the corresponding non-HLE molecules shown in (A). Pan-T cells from two different donors were used, with one representative donor shown here. [Figure 27A]Long-term T cell proliferation. Pan T cells and Molm-13 cells were incubated at a 1:1 E:T ratio, and T cell proliferation was assessed by FACS after 5 days of coculture in the presence of serial dilutions of the indicated molecules. Proliferating T cells were gated as CellTrace Violet-positive cells showing at least one cell division. Two benchmark control molecules (known benchmark T cell engagers, AMG330, which has binding specificity for CD33, and flotetuzumab, which has binding specificity for CD123) are shown, as well as selected ankyrin repeat proteins DARPin® Protein 7, DARPin® Protein 8, DARPin® Protein 9, and DARPin® Protein 10 without half-life extension (HLE), or DARPin® Protein 11, DARPin® Protein 12, DARPin® Protein 13, and DARPin® Protein 14 with half-life extension (HLE). (Figure 27A) T cell proliferation induced by DARPin® Protein 8 and DARPin® Protein 9 without half-life extension is comparable to the benchmark molecules, whereas DARPin® Protein 7 and DARPin® Protein 10 show a more than 100-fold decrease in potency. (Figure 27B) The half-life extended DARPins show a more than 30-fold decrease in potency compared to the corresponding non-HLE molecules shown in (A). Pan T cells from two different donors were used, one representative donor is shown here. [Figure 27B]Long-term T cell proliferation. Pan T cells and Molm-13 cells were incubated at a 1:1 E:T ratio, and T cell proliferation was assessed by FACS after 5 days of coculture in the presence of serial dilutions of the indicated molecules. Proliferating T cells were gated as CellTrace Violet-positive cells showing at least one cell division. Two benchmark control molecules (known benchmark T cell engagers, AMG330, which has binding specificity for CD33, and flotetuzumab, which has binding specificity for CD123) are shown, as well as selected ankyrin repeat proteins DARPin® Protein 7, DARPin® Protein 8, DARPin® Protein 9, and DARPin® Protein 10 without half-life extension (HLE), or DARPin® Protein 11, DARPin® Protein 12, DARPin® Protein 13, and DARPin® Protein 14 with half-life extension (HLE). (Figure 27A) T cell proliferation induced by DARPin® Protein 8 and DARPin® Protein 9 without half-life extension is comparable to the benchmark molecules, whereas DARPin® Protein 7 and DARPin® Protein 10 show a more than 100-fold decrease in potency. (Figure 27B) The half-life extended DARPins show a more than 30-fold decrease in potency compared to the corresponding non-HLE molecules shown in (A). Pan T cells from two different donors were used, one representative donor is shown here. [Figure 28A] Levels of IFNγ. Plasma samples were collected from the whole blood loop system at 0 (zero), 4 hours, 8 hours, 24 hours, and 48 hours. Vehicle is the negative control. The average of two donors is shown over time for AMG330 analog (Figure 28A), flotetuzumab analog (Figure 28B), DARPin® Protein #7 (Figure 28C), DARPin® Protein #8 (Figure 28D), DARPin® Protein #9 (Figure 28E), and DARPin® Protein 10 (Figure 28F). Zero values ​​are not shown. The dotted line represents the lower limit of quantification (LLOQ). All values ​​are below the upper limit of quantification (below ULOQ). [Figure 28B] Levels of IFNγ. Plasma samples were collected from the whole blood loop system at 0 (zero), 4 hours, 8 hours, 24 hours, and 48 hours. Vehicle is the negative control. The average of two donors is shown over time for AMG330 analog (Figure 28A), flotetuzumab analog (Figure 28B), DARPin® Protein #7 (Figure 28C), DARPin® Protein #8 (Figure 28D), DARPin® Protein #9 (Figure 28E), and DARPin® Protein 10 (Figure 28F). Zero values ​​are not shown. The dotted line represents the lower limit of quantification (LLOQ). All values ​​are below the upper limit of quantification (below ULOQ). [Figure 28C] Levels of IFNγ. Plasma samples were collected from the whole blood loop system at 0 (zero), 4 hours, 8 hours, 24 hours, and 48 hours. Vehicle is the negative control. The average of two donors is shown over time for AMG330 analog (Figure 28A), flotetuzumab analog (Figure 28B), DARPin® Protein #7 (Figure 28C), DARPin® Protein #8 (Figure 28D), DARPin® Protein #9 (Figure 28E), and DARPin® Protein 10 (Figure 28F). Zero values ​​are not shown. The dotted line represents the lower limit of quantification (LLOQ). All values ​​are below the upper limit of quantification (below ULOQ). [Figure 28D] Levels of IFNγ. Plasma samples were collected from the whole blood loop system at 0 (zero), 4 hours, 8 hours, 24 hours, and 48 hours. Vehicle is the negative control. The average of two donors is shown over time for AMG330 analog (Figure 28A), flotetuzumab analog (Figure 28B), DARPin® Protein #7 (Figure 28C), DARPin® Protein #8 (Figure 28D), DARPin® Protein #9 (Figure 28E), and DARPin® Protein 10 (Figure 28F). Zero values ​​are not shown. The dotted line represents the lower limit of quantification (LLOQ). All values ​​are below the upper limit of quantification (below ULOQ). [Figure 28E]Levels of IFNγ. Plasma samples were collected from the whole blood loop system at 0 (zero), 4 hours, 8 hours, 24 hours, and 48 hours. Vehicle is the negative control. The average of two donors is shown over time for AMG330 analog (Figure 28A), flotetuzumab analog (Figure 28B), DARPin® Protein #7 (Figure 28C), DARPin® Protein #8 (Figure 28D), DARPin® Protein #9 (Figure 28E), and DARPin® Protein 10 (Figure 28F). Zero values ​​are not shown. The dotted line represents the lower limit of quantification (LLOQ). All values ​​are below the upper limit of quantification (below ULOQ). [Figure 28F] Levels of IFNγ. Plasma samples were collected from the whole blood loop system at 0 (zero), 4 hours, 8 hours, 24 hours, and 48 hours. Vehicle is the negative control. The average of two donors is shown over time for AMG330 analog (Figure 28A), flotetuzumab analog (Figure 28B), DARPin® Protein #7 (Figure 28C), DARPin® Protein #8 (Figure 28D), DARPin® Protein #9 (Figure 28E), and DARPin® Protein 10 (Figure 28F). Zero values ​​are not shown. The dotted line represents the lower limit of quantification (LLOQ). All values ​​are below the upper limit of quantification (below ULOQ). [Figure 29A] Levels of TNFα. Plasma samples were collected from the whole blood loop system at 0 (zero), 4 hours, 8 hours, 24 hours, and 48 hours. Vehicle is the negative control. The average of two donors is shown over time for AMG330 analog (Figure 29A), flotetuzumab analog (Figure 29B), DARPin® Protein 7 (Figure 29C), DARPin® Protein 8 (Figure 29D), DARPin® Protein 9 (Figure 29E), and DARPin® Protein 10 (Figure 29F). Zero values ​​are not shown. The dotted line represents the LLOQ. All values ​​are below the ULOQ. [Figure 29B]Levels of TNFα. Plasma samples were collected from the whole blood loop system at 0 (zero), 4 hours, 8 hours, 24 hours, and 48 hours. Vehicle is the negative control. The average of two donors is shown over time for AMG330 analog (Figure 29A), flotetuzumab analog (Figure 29B), DARPin® Protein 7 (Figure 29C), DARPin® Protein 8 (Figure 29D), DARPin® Protein 9 (Figure 29E), and DARPin® Protein 10 (Figure 29F). Zero values ​​are not shown. The dotted line represents the LLOQ. All values ​​are below the ULOQ. [Figure 29C] Levels of TNFα. Plasma samples were collected from the whole blood loop system at 0 (zero), 4 hours, 8 hours, 24 hours, and 48 hours. Vehicle is the negative control. The average of two donors is shown over time for AMG330 analog (Figure 29A), flotetuzumab analog (Figure 29B), DARPin® Protein 7 (Figure 29C), DARPin® Protein 8 (Figure 29D), DARPin® Protein 9 (Figure 29E), and DARPin® Protein 10 (Figure 29F). Zero values ​​are not shown. The dotted line represents the LLOQ. All values ​​are below the ULOQ. [Figure 29D] Levels of TNFα. Plasma samples were collected from the whole blood loop system at 0 (zero), 4 hours, 8 hours, 24 hours, and 48 hours. Vehicle is the negative control. The average of two donors is shown over time for AMG330 analog (Figure 29A), flotetuzumab analog (Figure 29B), DARPin® Protein 7 (Figure 29C), DARPin® Protein 8 (Figure 29D), DARPin® Protein 9 (Figure 29E), and DARPin® Protein 10 (Figure 29F). Zero values ​​are not shown. The dotted line represents the LLOQ. All values ​​are below the ULOQ. [Figure 29E]Levels of TNFα. Plasma samples were collected from the whole blood loop system at 0 (zero), 4 hours, 8 hours, 24 hours, and 48 hours. Vehicle is the negative control. The average of two donors is shown over time for AMG330 analog (Figure 29A), flotetuzumab analog (Figure 29B), DARPin® Protein 7 (Figure 29C), DARPin® Protein 8 (Figure 29D), DARPin® Protein 9 (Figure 29E), and DARPin® Protein 10 (Figure 29F). Zero values ​​are not shown. The dotted line represents the LLOQ. All values ​​are below the ULOQ. [Figure 29F] Levels of TNFα. Plasma samples were collected from the whole blood loop system at 0 (zero), 4 hours, 8 hours, 24 hours, and 48 hours. Vehicle is the negative control. The average of two donors is shown over time for AMG330 analog (Figure 29A), flotetuzumab analog (Figure 29B), DARPin® Protein 7 (Figure 29C), DARPin® Protein 8 (Figure 29D), DARPin® Protein 9 (Figure 29E), and DARPin® Protein 10 (Figure 29F). Zero values ​​are not shown. The dotted line represents the LLOQ. All values ​​are below the ULOQ. [Figure 30A] T cell activation (% CD25 positive cells). Blood cells were collected from the whole blood loop at 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. % CD25 positive T cells are reported over time as the average of two donors for AMG330 analog (Figure 30A), flotetuzumab analog (Figure 30B), DARPin® Protein #7 (Figure 30C), DARPin® Protein #8 (Figure 30D), DARPin® Protein #9 (Figure 30E), and DARPin® Protein 10 (Figure 30F). Vehicle is the negative control. [Figure 30B]T cell activation (% CD25 positive cells). Blood cells were collected from the whole blood loop at 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. % CD25 positive T cells are reported over time as the average of two donors for AMG330 analog (Figure 30A), flotetuzumab analog (Figure 30B), DARPin® Protein #7 (Figure 30C), DARPin® Protein #8 (Figure 30D), DARPin® Protein #9 (Figure 30E), and DARPin® Protein 10 (Figure 30F). Vehicle is the negative control. [Figure 30C] T cell activation (% CD25 positive cells). Blood cells were collected from the whole blood loop at 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. % CD25 positive T cells are reported over time as the average of two donors for AMG330 analog (Figure 30A), flotetuzumab analog (Figure 30B), DARPin® Protein #7 (Figure 30C), DARPin® Protein #8 (Figure 30D), DARPin® Protein #9 (Figure 30E), and DARPin® Protein 10 (Figure 30F). Vehicle is the negative control. [Figure 30D] T cell activation (% CD25 positive cells). Blood cells were collected from the whole blood loop at 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. % CD25 positive T cells are reported over time as the average of two donors for AMG330 analog (Figure 30A), flotetuzumab analog (Figure 30B), DARPin® Protein #7 (Figure 30C), DARPin® Protein #8 (Figure 30D), DARPin® Protein #9 (Figure 30E), and DARPin® Protein 10 (Figure 30F). Vehicle is the negative control. [Figure 30E]T cell activation (% CD25 positive cells). Blood cells were collected from the whole blood loop at 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. % CD25 positive T cells are reported over time as the average of two donors for AMG330 analog (Figure 30A), flotetuzumab analog (Figure 30B), DARPin® Protein #7 (Figure 30C), DARPin® Protein #8 (Figure 30D), DARPin® Protein #9 (Figure 30E), and DARPin® Protein 10 (Figure 30F). Vehicle is the negative control. [Figure 30F] T cell activation (% CD25 positive cells). Blood cells were collected from the whole blood loop at 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. % CD25 positive T cells are reported over time as the average of two donors for AMG330 analog (Figure 30A), flotetuzumab analog (Figure 30B), DARPin® Protein #7 (Figure 30C), DARPin® Protein #8 (Figure 30D), DARPin® Protein #9 (Figure 30E), and DARPin® Protein 10 (Figure 30F). Vehicle is the negative control. [Figure 31A] T cell activation (% CD69-positive cells). Blood cells were collected from the whole blood loop at 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. The % CD69-positive T cells are reported over time as the average of two donors for AMG330 analog (Figure 31A), flotetuzumab analog (Figure 31B), DARPin® Protein #7 (Figure 31C), DARPin® Protein #8 (Figure 31D), DARPin® Protein #9 (Figure 31E), and DARPin® Protein 10 (Figure 31F). Vehicle is the negative control. [Figure 31B]T cell activation (% CD69-positive cells). Blood cells were collected from the whole blood loop at 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. The % CD69-positive T cells are reported over time as the average of two donors for AMG330 analog (Figure 31A), flotetuzumab analog (Figure 31B), DARPin® Protein #7 (Figure 31C), DARPin® Protein #8 (Figure 31D), DARPin® Protein #9 (Figure 31E), and DARPin® Protein 10 (Figure 31F). Vehicle is the negative control. [Figure 31C] T cell activation (% CD69-positive cells). Blood cells were collected from the whole blood loop at 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. The % CD69-positive T cells are reported over time as the average of two donors for AMG330 analog (Figure 31A), flotetuzumab analog (Figure 31B), DARPin® Protein #7 (Figure 31C), DARPin® Protein #8 (Figure 31D), DARPin® Protein #9 (Figure 31E), and DARPin® Protein 10 (Figure 31F). Vehicle is the negative control. [Figure 31D] T cell activation (% CD69-positive cells). Blood cells were collected from the whole blood loop at 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. The % CD69-positive T cells are reported over time as the average of two donors for AMG330 analog (Figure 31A), flotetuzumab analog (Figure 31B), DARPin® Protein #7 (Figure 31C), DARPin® Protein #8 (Figure 31D), DARPin® Protein #9 (Figure 31E), and DARPin® Protein 10 (Figure 31F). Vehicle is the negative control. [Figure 31E]T cell activation (% CD69-positive cells). Blood cells were collected from the whole blood loop at 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. The % CD69-positive T cells are reported over time as the average of two donors for AMG330 analog (Figure 31A), flotetuzumab analog (Figure 31B), DARPin® Protein #7 (Figure 31C), DARPin® Protein #8 (Figure 31D), DARPin® Protein #9 (Figure 31E), and DARPin® Protein 10 (Figure 31F). Vehicle is the negative control. [Figure 31F] T cell activation (% CD69-positive cells). Blood cells were collected from the whole blood loop at 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. The % CD69-positive T cells are reported over time as the average of two donors for AMG330 analog (Figure 31A), flotetuzumab analog (Figure 31B), DARPin® Protein #7 (Figure 31C), DARPin® Protein #8 (Figure 31D), DARPin® Protein #9 (Figure 31E), and DARPin® Protein 10 (Figure 31F). Vehicle is the negative control. [Figure 32A] T cell viability (% dead cells of total CD3+). Blood cells were collected from the whole blood loop system at 0 (zero), 4 hours, 8 hours, 24 hours, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. The % viable dye-positive T cells (i.e., % dead cells) are reported over time as the average of two donors for AMG330 analog (Figure 32A), flotetuzumab analog (Figure 32B), DARPin® Protein #7 (Figure 32C), DARPin® Protein #8 (Figure 32D), DARPin® Protein #9 (Figure 32E), and DARPin® Protein 10 (Figure 32F). Vehicle is the negative control. [Figure 32B]T cell viability (% dead cells of total CD3+). Blood cells were collected from the whole blood loop system at 0 (zero), 4 hours, 8 hours, 24 hours, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. The % viable dye-positive T cells (i.e., % dead cells) are reported over time as the average of two donors for AMG330 analog (Figure 32A), flotetuzumab analog (Figure 32B), DARPin® Protein #7 (Figure 32C), DARPin® Protein #8 (Figure 32D), DARPin® Protein #9 (Figure 32E), and DARPin® Protein 10 (Figure 32F). Vehicle is the negative control. [Figure 32C] T cell viability (% dead cells of total CD3+). Blood cells were collected from the whole blood loop system at 0 (zero), 4 hours, 8 hours, 24 hours, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. The % viable dye-positive T cells (i.e., % dead cells) are reported over time as the average of two donors for AMG330 analog (Figure 32A), flotetuzumab analog (Figure 32B), DARPin® Protein #7 (Figure 32C), DARPin® Protein #8 (Figure 32D), DARPin® Protein #9 (Figure 32E), and DARPin® Protein 10 (Figure 32F). Vehicle is the negative control. [Figure 32D] T cell viability (% dead cells of total CD3+). Blood cells were collected from the whole blood loop system at 0 (zero), 4 hours, 8 hours, 24 hours, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. The % viable dye-positive T cells (i.e., % dead cells) are reported over time as the average of two donors for AMG330 analog (Figure 32A), flotetuzumab analog (Figure 32B), DARPin® Protein #7 (Figure 32C), DARPin® Protein #8 (Figure 32D), DARPin® Protein #9 (Figure 32E), and DARPin® Protein 10 (Figure 32F). Vehicle is the negative control. [Figure 32E]T cell viability (% dead cells of total CD3+). Blood cells were collected from the whole blood loop system at 0 (zero), 4 hours, 8 hours, 24 hours, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. The % viable dye-positive T cells (i.e., % dead cells) are reported over time as the average of two donors for AMG330 analog (Figure 32A), flotetuzumab analog (Figure 32B), DARPin® Protein #7 (Figure 32C), DARPin® Protein #8 (Figure 32D), DARPin® Protein #9 (Figure 32E), and DARPin® Protein 10 (Figure 32F). Vehicle is the negative control. [Figure 32F] T cell viability (% dead cells of total CD3+). Blood cells were collected from the whole blood loop system at 0 (zero), 4 hours, 8 hours, 24 hours, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. The % viable dye-positive T cells (i.e., % dead cells) are reported over time as the average of two donors for AMG330 analog (Figure 32A), flotetuzumab analog (Figure 32B), DARPin® Protein #7 (Figure 32C), DARPin® Protein #8 (Figure 32D), DARPin® Protein #9 (Figure 32E), and DARPin® Protein 10 (Figure 32F). Vehicle is the negative control. [Figure 33A] CD33+ cell viability (% dead cells of total CD33+). Blood cells were collected from the whole blood loop system at 0 (zero), 4 hours, 8 hours, 24 hours, and 48 hours, stained with fluorophore-conjugated antibodies, and analyzed by flow cytometry. The % viable dye-positive CD33+ cells (i.e., % dead cells) are reported over time as the average of two donors for AMG330 analogs (Figure 33A), flotetuzumab analogs (Figure 33B), DARPin® Protein #7 (Figure 33C), DARPin® Protein #8 (Figure 33D), DARPin® Protein #9 (Figure 33E), and DARPin® Protein 10 (Figure 33F). Vehicle is the negative control. [Figure 33B]CD33+ cell viability (% dead cells of total CD33+). Blood cells were collected from the whole blood loop system at 0 (zero), 4 hours, 8 hours, 24 hours, and 48 hours, stained with fluorophore-conjugated antibodies, and analyzed by flow cytometry. The % viable dye-positive CD33+ cells (i.e., % dead cells) are reported over time as the average of two donors for AMG330 analogs (Figure 33A), flotetuzumab analogs (Figure 33B), DARPin® Protein #7 (Figure 33C), DARPin® Protein #8 (Figure 33D), DARPin® Protein #9 (Figure 33E), and DARPin® Protein 10 (Figure 33F). Vehicle is the negative control. [Figure 33C] CD33+ cell viability (% dead cells of total CD33+). Blood cells were collected from the whole blood loop system at 0 (zero), 4 hours, 8 hours, 24 hours, and 48 hours, stained with fluorophore-conjugated antibodies, and analyzed by flow cytometry. The % viable dye-positive CD33+ cells (i.e., % dead cells) are reported over time as the average of two donors for AMG330 analogs (Figure 33A), flotetuzumab analogs (Figure 33B), DARPin® Protein #7 (Figure 33C), DARPin® Protein #8 (Figure 33D), DARPin® Protein #9 (Figure 33E), and DARPin® Protein 10 (Figure 33F). Vehicle is the negative control. [Figure 33D] CD33+ cell viability (% dead cells of total CD33+). Blood cells were collected from the whole blood loop system at 0 (zero), 4 hours, 8 hours, 24 hours, and 48 hours, stained with fluorophore-conjugated antibodies, and analyzed by flow cytometry. The % viable dye-positive CD33+ cells (i.e., % dead cells) are reported over time as the average of two donors for AMG330 analogs (Figure 33A), flotetuzumab analogs (Figure 33B), DARPin® Protein #7 (Figure 33C), DARPin® Protein #8 (Figure 33D), DARPin® Protein #9 (Figure 33E), and DARPin® Protein 10 (Figure 33F). Vehicle is the negative control. [Figure 33E]CD33+ cell viability (% dead cells of total CD33+). Blood cells were collected from the whole blood loop system at 0 (zero), 4 hours, 8 hours, 24 hours, and 48 hours, stained with fluorophore-conjugated antibodies, and analyzed by flow cytometry. The % viable dye-positive CD33+ cells (i.e., % dead cells) are reported over time as the average of two donors for AMG330 analogs (Figure 33A), flotetuzumab analogs (Figure 33B), DARPin® Protein #7 (Figure 33C), DARPin® Protein #8 (Figure 33D), DARPin® Protein #9 (Figure 33E), and DARPin® Protein 10 (Figure 33F). Vehicle is the negative control. [Figure 33F] CD33+ cell viability (% dead cells of total CD33+). Blood cells were collected from the whole blood loop system at 0 (zero), 4 hours, 8 hours, 24 hours, and 48 hours, stained with fluorophore-conjugated antibodies, and analyzed by flow cytometry. The % viable dye-positive CD33+ cells (i.e., % dead cells) are reported over time as the average of two donors for AMG330 analogs (Figure 33A), flotetuzumab analogs (Figure 33B), DARPin® Protein #7 (Figure 33C), DARPin® Protein #8 (Figure 33D), DARPin® Protein #9 (Figure 33E), and DARPin® Protein 10 (Figure 33F). Vehicle is the negative control. [Figure 34A] CD123+ cell viability (% dead cells of total CD123+). Blood cells were collected from the whole blood loop system at time 0 (0), 4 hours, 8 hours, 24 hours, and 48 hours, stained with fluorophore-conjugated antibodies, and analyzed by flow cytometry. The % viable dye-positive CD123+ cells (i.e., % dead cells) are reported over time as the average of two donors for AMG330 analogs (Figure 34A), flotetuzumab analogs (Figure 34B), DARPin® Protein 7 (Figure 34C), DARPin® Protein #8 (Figure 34D), DARPin® Protein #9 (Figure 34E), and DARPin® Protein 10 (Figure 34F). Vehicle is the negative control. [Figure 34B]CD123+ cell viability (% dead cells of total CD123+). Blood cells were collected from the whole blood loop system at time 0 (0), 4 hours, 8 hours, 24 hours, and 48 hours, stained with fluorophore-conjugated antibodies, and analyzed by flow cytometry. The % viable dye-positive CD123+ cells (i.e., % dead cells) are reported over time as the average of two donors for AMG330 analogs (Figure 34A), flotetuzumab analogs (Figure 34B), DARPin® Protein 7 (Figure 34C), DARPin® Protein #8 (Figure 34D), DARPin® Protein #9 (Figure 34E), and DARPin® Protein 10 (Figure 34F). Vehicle is the negative control. [Figure 34C] CD123+ cell viability (% dead cells of total CD123+). Blood cells were collected from the whole blood loop system at time 0 (0), 4 hours, 8 hours, 24 hours, and 48 hours, stained with fluorophore-conjugated antibodies, and analyzed by flow cytometry. The % viable dye-positive CD123+ cells (i.e., % dead cells) are reported over time as the average of two donors for AMG330 analogs (Figure 34A), flotetuzumab analogs (Figure 34B), DARPin® Protein 7 (Figure 34C), DARPin® Protein #8 (Figure 34D), DARPin® Protein #9 (Figure 34E), and DARPin® Protein 10 (Figure 34F). Vehicle is the negative control. [Figure 34D] CD123+ cell viability (% dead cells of total CD123+). Blood cells were collected from the whole blood loop system at time 0 (0), 4 hours, 8 hours, 24 hours, and 48 hours, stained with fluorophore-conjugated antibodies, and analyzed by flow cytometry. The % viable dye-positive CD123+ cells (i.e., % dead cells) are reported over time as the average of two donors for AMG330 analogs (Figure 34A), flotetuzumab analogs (Figure 34B), DARPin® Protein 7 (Figure 34C), DARPin® Protein #8 (Figure 34D), DARPin® Protein #9 (Figure 34E), and DARPin® Protein 10 (Figure 34F). Vehicle is the negative control. [Figure 34E]CD123+ cell viability (% dead cells of total CD123+). Blood cells were collected from the whole blood loop system at time 0 (0), 4 hours, 8 hours, 24 hours, and 48 hours, stained with fluorophore-conjugated antibodies, and analyzed by flow cytometry. The % viable dye-positive CD123+ cells (i.e., % dead cells) are reported over time as the average of two donors for AMG330 analogs (Figure 34A), flotetuzumab analogs (Figure 34B), DARPin® Protein 7 (Figure 34C), DARPin® Protein #8 (Figure 34D), DARPin® Protein #9 (Figure 34E), and DARPin® Protein 10 (Figure 34F). Vehicle is the negative control. [Figure 34F] CD123+ cell viability (% dead cells of total CD123+). Blood cells were collected from the whole blood loop system at time 0 (0), 4 hours, 8 hours, 24 hours, and 48 hours, stained with fluorophore-conjugated antibodies, and analyzed by flow cytometry. The % viable dye-positive CD123+ cells (i.e., % dead cells) are reported over time as the average of two donors for AMG330 analogs (Figure 34A), flotetuzumab analogs (Figure 34B), DARPin® Protein 7 (Figure 34C), DARPin® Protein #8 (Figure 34D), DARPin® Protein #9 (Figure 34E), and DARPin® Protein 10 (Figure 34F). Vehicle is the negative control. [Figure 35A] T cell counts (cells per 10,000 beads). Blood cells were collected from the whole blood loop system at 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. Cell counts per 10,000 beads are reported over time as the average of two donors for AMG330 analogs (Figure 35A), flotetuzumab analogs (Figure 35B), DARPin® Protein #7 (Figure 35C), DARPin® Protein #8 (Figure 35D), DARPin® Protein #9 (Figure 35E), and DARPin® Protein 10 (Figure 35F). Vehicle is the negative control. [Figure 35B]T cell counts (cells per 10,000 beads). Blood cells were collected from the whole blood loop system at 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. Cell counts per 10,000 beads are reported over time as the average of two donors for AMG330 analogs (Figure 35A), flotetuzumab analogs (Figure 35B), DARPin® Protein #7 (Figure 35C), DARPin® Protein #8 (Figure 35D), DARPin® Protein #9 (Figure 35E), and DARPin® Protein 10 (Figure 35F). Vehicle is the negative control. [Figure 35C] T cell counts (cells per 10,000 beads). Blood cells were collected from the whole blood loop system at 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. Cell counts per 10,000 beads are reported over time as the average of two donors for AMG330 analogs (Figure 35A), flotetuzumab analogs (Figure 35B), DARPin® Protein #7 (Figure 35C), DARPin® Protein #8 (Figure 35D), DARPin® Protein #9 (Figure 35E), and DARPin® Protein 10 (Figure 35F). Vehicle is the negative control. [Figure 35D] T cell counts (cells per 10,000 beads). Blood cells were collected from the whole blood loop system at 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. Cell counts per 10,000 beads are reported over time as the average of two donors for AMG330 analogs (Figure 35A), flotetuzumab analogs (Figure 35B), DARPin® Protein #7 (Figure 35C), DARPin® Protein #8 (Figure 35D), DARPin® Protein #9 (Figure 35E), and DARPin® Protein 10 (Figure 35F). Vehicle is the negative control. [Figure 35E]T cell counts (cells per 10,000 beads). Blood cells were collected from the whole blood loop system at 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. Cell counts per 10,000 beads are reported over time as the average of two donors for AMG330 analogs (Figure 35A), flotetuzumab analogs (Figure 35B), DARPin® Protein #7 (Figure 35C), DARPin® Protein #8 (Figure 35D), DARPin® Protein #9 (Figure 35E), and DARPin® Protein 10 (Figure 35F). Vehicle is the negative control. [Figure 35F] T cell counts (cells per 10,000 beads). Blood cells were collected from the whole blood loop system at 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. Cell counts per 10,000 beads are reported over time as the average of two donors for AMG330 analogs (Figure 35A), flotetuzumab analogs (Figure 35B), DARPin® Protein #7 (Figure 35C), DARPin® Protein #8 (Figure 35D), DARPin® Protein #9 (Figure 35E), and DARPin® Protein 10 (Figure 35F). Vehicle is the negative control. [Figure 36A] CD33+ cell counts (cells per 10,000 beads). Blood cells were collected from the whole blood loop at 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. Cell counts per 10,000 beads are reported over time as the average of two donors for AMG330 analog (Figure 36A), flotetuzumab analog (Figure 36B), DARPin® Protein #7 (Figure 36C), DARPin® Protein #8 (Figure 36D), DARPin® Protein #9 (Figure 36E), and DARPin® Protein 10 (Figure 36F). Vehicle is the negative control. [Figure 36B]CD33+ cell counts (cells per 10,000 beads). Blood cells were collected from the whole blood loop at 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. Cell counts per 10,000 beads are reported over time as the average of two donors for AMG330 analog (Figure 36A), flotetuzumab analog (Figure 36B), DARPin® Protein #7 (Figure 36C), DARPin® Protein #8 (Figure 36D), DARPin® Protein #9 (Figure 36E), and DARPin® Protein 10 (Figure 36F). Vehicle is the negative control. [Figure 36C] CD33+ cell counts (cells per 10,000 beads). Blood cells were collected from the whole blood loop at 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. Cell counts per 10,000 beads are reported over time as the average of two donors for AMG330 analog (Figure 36A), flotetuzumab analog (Figure 36B), DARPin® Protein #7 (Figure 36C), DARPin® Protein #8 (Figure 36D), DARPin® Protein #9 (Figure 36E), and DARPin® Protein 10 (Figure 36F). Vehicle is the negative control. [Figure 36D] CD33+ cell counts (cells per 10,000 beads). Blood cells were collected from the whole blood loop at 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. Cell counts per 10,000 beads are reported over time as the average of two donors for AMG330 analog (Figure 36A), flotetuzumab analog (Figure 36B), DARPin® Protein #7 (Figure 36C), DARPin® Protein #8 (Figure 36D), DARPin® Protein #9 (Figure 36E), and DARPin® Protein 10 (Figure 36F). Vehicle is the negative control. [Figure 36E]CD33+ cell counts (cells per 10,000 beads). Blood cells were collected from the whole blood loop at 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. Cell counts per 10,000 beads are reported over time as the average of two donors for AMG330 analog (Figure 36A), flotetuzumab analog (Figure 36B), DARPin® Protein #7 (Figure 36C), DARPin® Protein #8 (Figure 36D), DARPin® Protein #9 (Figure 36E), and DARPin® Protein 10 (Figure 36F). Vehicle is the negative control. [Figure 36F] CD33+ cell counts (cells per 10,000 beads). Blood cells were collected from the whole blood loop at 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. Cell counts per 10,000 beads are reported over time as the average of two donors for AMG330 analog (Figure 36A), flotetuzumab analog (Figure 36B), DARPin® Protein #7 (Figure 36C), DARPin® Protein #8 (Figure 36D), DARPin® Protein #9 (Figure 36E), and DARPin® Protein 10 (Figure 36F). Vehicle is the negative control. [Figure 37A] Cell counts of CD123+ cells (cells per 10,000 beads). Blood cells were collected from the whole blood loop system at time 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. Cell counts per 10,000 beads are reported over time as the average of two donors for AMG330 analogs (Figure 37A), flotetuzumab analogs (Figure 37B), DARPin® Protein #7 (Figure 37C), DARPin® Protein #8 (Figure 37D), DARPin® Protein #9 (Figure 37E), and DARPin® Protein 10 (Figure 37F). Vehicle is the negative control. [Figure 37B]Cell counts of CD123+ cells (cells per 10,000 beads). Blood cells were collected from the whole blood loop system at time 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. Cell counts per 10,000 beads are reported over time as the average of two donors for AMG330 analogs (Figure 37A), flotetuzumab analogs (Figure 37B), DARPin® Protein #7 (Figure 37C), DARPin® Protein #8 (Figure 37D), DARPin® Protein #9 (Figure 37E), and DARPin® Protein 10 (Figure 37F). Vehicle is the negative control. [Figure 37C] Cell counts of CD123+ cells (cells per 10,000 beads). Blood cells were collected from the whole blood loop system at time 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. Cell counts per 10,000 beads are reported over time as the average of two donors for AMG330 analogs (Figure 37A), flotetuzumab analogs (Figure 37B), DARPin® Protein #7 (Figure 37C), DARPin® Protein #8 (Figure 37D), DARPin® Protein #9 (Figure 37E), and DARPin® Protein 10 (Figure 37F). Vehicle is the negative control. [Figure 37D] Cell counts of CD123+ cells (cells per 10,000 beads). Blood cells were collected from the whole blood loop system at time 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. Cell counts per 10,000 beads are reported over time as the average of two donors for AMG330 analogs (Figure 37A), flotetuzumab analogs (Figure 37B), DARPin® Protein #7 (Figure 37C), DARPin® Protein #8 (Figure 37D), DARPin® Protein #9 (Figure 37E), and DARPin® Protein 10 (Figure 37F). Vehicle is the negative control. [Figure 37E]Cell counts of CD123+ cells (cells per 10,000 beads). Blood cells were collected from the whole blood loop system at time 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. Cell counts per 10,000 beads are reported over time as the average of two donors for AMG330 analogs (Figure 37A), flotetuzumab analogs (Figure 37B), DARPin® Protein #7 (Figure 37C), DARPin® Protein #8 (Figure 37D), DARPin® Protein #9 (Figure 37E), and DARPin® Protein 10 (Figure 37F). Vehicle is the negative control. [Figure 37F] Cell counts of CD123+ cells (cells per 10,000 beads). Blood cells were collected from the whole blood loop system at time 0, 4, 8, 24, and 48 hours, stained with fluorophore-labeled antibodies, and analyzed by flow cytometry. Cell counts per 10,000 beads are reported over time as the average of two donors for AMG330 analogs (Figure 37A), flotetuzumab analogs (Figure 37B), DARPin® Protein #7 (Figure 37C), DARPin® Protein #8 (Figure 37D), DARPin® Protein #9 (Figure 37E), and DARPin® Protein 10 (Figure 37F). Vehicle is the negative control. [Figure 38A] On-rate (Figure 38A) and off-rate (Figure 38B) constants for the CD33 target measured for selected recombinant proteins DARPin® Protein 20, DARPin® Protein 21, and DARPin® Protein 22. [Figure 38B] On-rate (Figure 38A) and off-rate (Figure 38B) constants for the CD33 target measured for selected recombinant proteins DARPin® Protein 20, DARPin® Protein 21, and DARPin® Protein 22. [Figure 39A]On-rate (Figure 39A) and off-rate (Figure 39B) constants for the CD123 target measured for selected recombinant proteins DARPin® Protein 20, DARPin® Protein 21, and DARPin® Protein 22. [Figure 39B] On-rate (Figure 39A) and off-rate (Figure 39B) constants for the CD123 target measured for selected recombinant proteins DARPin® Protein 20, DARPin® Protein 21, and DARPin® Protein 22. [Figure 40] KD values ​​for the interaction of recombinant proteins with human CD33 [Figure 41] KD values ​​for the interaction of recombinant proteins with human CD123 [Figure 42A] Mean and SD of tumor volume. [Figure 42B] Mean and SD of tumor volume. [Figure 42C] Mean and SD of tumor volume. [Figure 43A] Single tumor growth curve. [Figure 43B] Single tumor growth curve. [Figure 43C] Single tumor growth curve. [Figure 43D] Single tumor growth curve. [Figure 43E] Single tumor growth curve. [Figure 43F] Single tumor growth curve. [Figure 43G] Single tumor growth curve. [Figure 43H] Single tumor growth curve. [Figure 43I] Single tumor growth curve. [Figure 43J] Single tumor growth curve. [Figure 43K] Single tumor growth curve. [Figure 43L] Single tumor growth curve. [Figure 44A]IFNγ levels. The mean IFNγ concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 44A), DARPin® Protein #58 (Figure 44B), DARPin® Protein #59 (Figure 44C), DARPin® Protein #57 (Figure 44D), DARPin® Protein #60 (Figure 44E), DARPin® Protein #56 (Figure 44F), DARPin® Protein #61 (Figure 44G), DARPin® Protein #74 (Figure 44H). The calculated LLOQ is indicated by a dotted line. [Figure 44B] IFNγ levels. The mean IFNγ concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 44A), DARPin® Protein #58 (Figure 44B), DARPin® Protein #59 (Figure 44C), DARPin® Protein #57 (Figure 44D), DARPin® Protein #60 (Figure 44E), DARPin® Protein #56 (Figure 44F), DARPin® Protein #61 (Figure 44G), DARPin® Protein #74 (Figure 44H). The calculated LLOQ is indicated by a dotted line. [Figure 44C]IFNγ levels. The mean IFNγ concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 44A), DARPin® Protein #58 (Figure 44B), DARPin® Protein #59 (Figure 44C), DARPin® Protein #57 (Figure 44D), DARPin® Protein #60 (Figure 44E), DARPin® Protein #56 (Figure 44F), DARPin® Protein #61 (Figure 44G), DARPin® Protein #74 (Figure 44H). The calculated LLOQ is indicated by a dotted line. [Figure 44D] IFNγ levels. The mean IFNγ concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 44A), DARPin® Protein #58 (Figure 44B), DARPin® Protein #59 (Figure 44C), DARPin® Protein #57 (Figure 44D), DARPin® Protein #60 (Figure 44E), DARPin® Protein #56 (Figure 44F), DARPin® Protein #61 (Figure 44G), DARPin® Protein #74 (Figure 44H). The calculated LLOQ is indicated by a dotted line. [Figure 44E]IFNγ levels. The mean IFNγ concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 44A), DARPin® Protein #58 (Figure 44B), DARPin® Protein #59 (Figure 44C), DARPin® Protein #57 (Figure 44D), DARPin® Protein #60 (Figure 44E), DARPin® Protein #56 (Figure 44F), DARPin® Protein #61 (Figure 44G), DARPin® Protein #74 (Figure 44H). The calculated LLOQ is indicated by a dotted line. [Figure 44F] IFNγ levels. The mean IFNγ concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 44A), DARPin® Protein #58 (Figure 44B), DARPin® Protein #59 (Figure 44C), DARPin® Protein #57 (Figure 44D), DARPin® Protein #60 (Figure 44E), DARPin® Protein #56 (Figure 44F), DARPin® Protein #61 (Figure 44G), DARPin® Protein #74 (Figure 44H). The calculated LLOQ is indicated by a dotted line. [Figure 44G]IFNγ levels. The mean IFNγ concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 44A), DARPin® Protein #58 (Figure 44B), DARPin® Protein #59 (Figure 44C), DARPin® Protein #57 (Figure 44D), DARPin® Protein #60 (Figure 44E), DARPin® Protein #56 (Figure 44F), DARPin® Protein #61 (Figure 44G), DARPin® Protein #74 (Figure 44H). The calculated LLOQ is indicated by a dotted line. [Figure 44H] IFNγ levels. The mean IFNγ concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 44A), DARPin® Protein #58 (Figure 44B), DARPin® Protein #59 (Figure 44C), DARPin® Protein #57 (Figure 44D), DARPin® Protein #60 (Figure 44E), DARPin® Protein #56 (Figure 44F), DARPin® Protein #61 (Figure 44G), DARPin® Protein #74 (Figure 44H). The calculated LLOQ is indicated by a dotted line. [Figure 45A]IL-2 levels. The mean IL-2 concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 45A), DARPin® Protein #58 (Figure 45B), DARPin® Protein #59 (Figure 45C), DARPin® Protein #57 (Figure 45D), DARPin® Protein #60 (Figure 45E), DARPin® Protein #56 (Figure 45F), DARPin® Protein #61 (Figure 45G), DARPin® Protein #74 (Figure 45H). The calculated LLOQ is indicated by a dotted line. [Figure 45B] IL-2 levels. The mean IL-2 concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 45A), DARPin® Protein #58 (Figure 45B), DARPin® Protein #59 (Figure 45C), DARPin® Protein #57 (Figure 45D), DARPin® Protein #60 (Figure 45E), DARPin® Protein #56 (Figure 45F), DARPin® Protein #61 (Figure 45G), DARPin® Protein #74 (Figure 45H). The calculated LLOQ is indicated by a dotted line. [Figure 45C]IL-2 levels. The mean IL-2 concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 45A), DARPin® Protein #58 (Figure 45B), DARPin® Protein #59 (Figure 45C), DARPin® Protein #57 (Figure 45D), DARPin® Protein #60 (Figure 45E), DARPin® Protein #56 (Figure 45F), DARPin® Protein #61 (Figure 45G), DARPin® Protein #74 (Figure 45H). The calculated LLOQ is indicated by a dotted line. [Figure 45D] IL-2 levels. The mean IL-2 concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 45A), DARPin® Protein #58 (Figure 45B), DARPin® Protein #59 (Figure 45C), DARPin® Protein #57 (Figure 45D), DARPin® Protein #60 (Figure 45E), DARPin® Protein #56 (Figure 45F), DARPin® Protein #61 (Figure 45G), DARPin® Protein #74 (Figure 45H). The calculated LLOQ is indicated by a dotted line. [Figure 45E]IL-2 levels. The mean IL-2 concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 45A), DARPin® Protein #58 (Figure 45B), DARPin® Protein #59 (Figure 45C), DARPin® Protein #57 (Figure 45D), DARPin® Protein #60 (Figure 45E), DARPin® Protein #56 (Figure 45F), DARPin® Protein #61 (Figure 45G), DARPin® Protein #74 (Figure 45H). The calculated LLOQ is indicated by a dotted line. [Figure 45F] IL-2 levels. The mean IL-2 concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 45A), DARPin® Protein #58 (Figure 45B), DARPin® Protein #59 (Figure 45C), DARPin® Protein #57 (Figure 45D), DARPin® Protein #60 (Figure 45E), DARPin® Protein #56 (Figure 45F), DARPin® Protein #61 (Figure 45G), DARPin® Protein #74 (Figure 45H). The calculated LLOQ is indicated by a dotted line. [Figure 45G]IL-2 levels. The mean IL-2 concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 45A), DARPin® Protein #58 (Figure 45B), DARPin® Protein #59 (Figure 45C), DARPin® Protein #57 (Figure 45D), DARPin® Protein #60 (Figure 45E), DARPin® Protein #56 (Figure 45F), DARPin® Protein #61 (Figure 45G), DARPin® Protein #74 (Figure 45H). The calculated LLOQ is indicated by a dotted line. [Figure 45H] IL-2 levels. The mean IL-2 concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 45A), DARPin® Protein #58 (Figure 45B), DARPin® Protein #59 (Figure 45C), DARPin® Protein #57 (Figure 45D), DARPin® Protein #60 (Figure 45E), DARPin® Protein #56 (Figure 45F), DARPin® Protein #61 (Figure 45G), DARPin® Protein #74 (Figure 45H). The calculated LLOQ is indicated by a dotted line. [Figure 46A]IL-6 levels. The mean IL-6 concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 46A), DARPin® Protein #58 (Figure 46B), DARPin® Protein #59 (Figure 46C), DARPin® Protein #57 (Figure 46D), DARPin® Protein #60 (Figure 46E), DARPin® Protein #56 (Figure 46F), DARPin® Protein #61 (Figure 46G), DARPin® Protein #74 (Figure 46H). The calculated LLOQ is indicated by a dotted line. [Figure 46B] IL-6 levels. The mean IL-6 concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 46A), DARPin® Protein #58 (Figure 46B), DARPin® Protein #59 (Figure 46C), DARPin® Protein #57 (Figure 46D), DARPin® Protein #60 (Figure 46E), DARPin® Protein #56 (Figure 46F), DARPin® Protein #61 (Figure 46G), DARPin® Protein #74 (Figure 46H). The calculated LLOQ is indicated by a dotted line. [Figure 46C]IL-6 levels. The mean IL-6 concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 46A), DARPin® Protein #58 (Figure 46B), DARPin® Protein #59 (Figure 46C), DARPin® Protein #57 (Figure 46D), DARPin® Protein #60 (Figure 46E), DARPin® Protein #56 (Figure 46F), DARPin® Protein #61 (Figure 46G), DARPin® Protein #74 (Figure 46H). The calculated LLOQ is indicated by a dotted line. [Figure 46D] IL-6 levels. The mean IL-6 concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 46A), DARPin® Protein #58 (Figure 46B), DARPin® Protein #59 (Figure 46C), DARPin® Protein #57 (Figure 46D), DARPin® Protein #60 (Figure 46E), DARPin® Protein #56 (Figure 46F), DARPin® Protein #61 (Figure 46G), DARPin® Protein #74 (Figure 46H). The calculated LLOQ is indicated by a dotted line. [Figure 46E]IL-6 levels. The mean IL-6 concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 46A), DARPin® Protein #58 (Figure 46B), DARPin® Protein #59 (Figure 46C), DARPin® Protein #57 (Figure 46D), DARPin® Protein #60 (Figure 46E), DARPin® Protein #56 (Figure 46F), DARPin® Protein #61 (Figure 46G), DARPin® Protein #74 (Figure 46H). The calculated LLOQ is indicated by a dotted line. [Figure 46F] IL-6 levels. The mean IL-6 concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 46A), DARPin® Protein #58 (Figure 46B), DARPin® Protein #59 (Figure 46C), DARPin® Protein #57 (Figure 46D), DARPin® Protein #60 (Figure 46E), DARPin® Protein #56 (Figure 46F), DARPin® Protein #61 (Figure 46G), DARPin® Protein #74 (Figure 46H). The calculated LLOQ is indicated by a dotted line. [Figure 46G]IL-6 levels. The mean IL-6 concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 46A), DARPin® Protein #58 (Figure 46B), DARPin® Protein #59 (Figure 46C), DARPin® Protein #57 (Figure 46D), DARPin® Protein #60 (Figure 46E), DARPin® Protein #56 (Figure 46F), DARPin® Protein #61 (Figure 46G), DARPin® Protein #74 (Figure 46H). The calculated LLOQ is indicated by a dotted line. [Figure 46H] IL-6 levels. The mean IL-6 concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 46A), DARPin® Protein #58 (Figure 46B), DARPin® Protein #59 (Figure 46C), DARPin® Protein #57 (Figure 46D), DARPin® Protein #60 (Figure 46E), DARPin® Protein #56 (Figure 46F), DARPin® Protein #61 (Figure 46G), DARPin® Protein #74 (Figure 46H). The calculated LLOQ is indicated by a dotted line. [Figure 47A]IL-8 levels. The mean IL-8 concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 47A), DARPin® Protein #58 (Figure 47B), DARPin® Protein #59 (Figure 47C), DARPin® Protein #57 (Figure 47D), DARPin® Protein #60 (Figure 47E), DARPin® Protein #56 (Figure 47F), DARPin® Protein #61 (Figure 47G), DARPin® Protein #74 (Figure 47H). The calculated LLOQ is indicated by the dotted line. [Figure 47B] IL-8 levels. The mean IL-8 concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 47A), DARPin® Protein #58 (Figure 47B), DARPin® Protein #59 (Figure 47C), DARPin® Protein #57 (Figure 47D), DARPin® Protein #60 (Figure 47E), DARPin® Protein #56 (Figure 47F), DARPin® Protein #61 (Figure 47G), DARPin® Protein #74 (Figure 47H). The calculated LLOQ is indicated by the dotted line. [Figure 47C]IL-8 levels. The mean IL-8 concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 47A), DARPin® Protein #58 (Figure 47B), DARPin® Protein #59 (Figure 47C), DARPin® Protein #57 (Figure 47D), DARPin® Protein #60 (Figure 47E), DARPin® Protein #56 (Figure 47F), DARPin® Protein #61 (Figure 47G), DARPin® Protein #74 (Figure 47H). The calculated LLOQ is indicated by the dotted line. [Figure 47D] IL-8 levels. The mean IL-8 concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 47A), DARPin® Protein #58 (Figure 47B), DARPin® Protein #59 (Figure 47C), DARPin® Protein #57 (Figure 47D), DARPin® Protein #60 (Figure 47E), DARPin® Protein #56 (Figure 47F), DARPin® Protein #61 (Figure 47G), DARPin® Protein #74 (Figure 47H). The calculated LLOQ is indicated by the dotted line. [Figure 47E]IL-8 levels. The mean IL-8 concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 47A), DARPin® Protein #58 (Figure 47B), DARPin® Protein #59 (Figure 47C), DARPin® Protein #57 (Figure 47D), DARPin® Protein #60 (Figure 47E), DARPin® Protein #56 (Figure 47F), DARPin® Protein #61 (Figure 47G), DARPin® Protein #74 (Figure 47H). The calculated LLOQ is indicated by the dotted line. [Figure 47F] IL-8 levels. The mean IL-8 concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 47A), DARPin® Protein #58 (Figure 47B), DARPin® Protein #59 (Figure 47C), DARPin® Protein #57 (Figure 47D), DARPin® Protein #60 (Figure 47E), DARPin® Protein #56 (Figure 47F), DARPin® Protein #61 (Figure 47G), DARPin® Protein #74 (Figure 47H). The calculated LLOQ is indicated by the dotted line. [Figure 47G]IL-8 levels. The mean IL-8 concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 47A), DARPin® Protein #58 (Figure 47B), DARPin® Protein #59 (Figure 47C), DARPin® Protein #57 (Figure 47D), DARPin® Protein #60 (Figure 47E), DARPin® Protein #56 (Figure 47F), DARPin® Protein #61 (Figure 47G), DARPin® Protein #74 (Figure 47H). The calculated LLOQ is indicated by the dotted line. [Figure 47H] IL-8 levels. The mean IL-8 concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 47A), DARPin® Protein #58 (Figure 47B), DARPin® Protein #59 (Figure 47C), DARPin® Protein #57 (Figure 47D), DARPin® Protein #60 (Figure 47E), DARPin® Protein #56 (Figure 47F), DARPin® Protein #61 (Figure 47G), DARPin® Protein #74 (Figure 47H). The calculated LLOQ is indicated by the dotted line. [Figure 48A]TNFα levels. The mean TNFα concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 48A), DARPin® Protein #58 (Figure 48B), DARPin® Protein #59 (Figure 48C), DARPin® Protein #57 (Figure 48D), DARPin® Protein #60 (Figure 48E), DARPin® Protein #56 (Figure 48F), DARPin® Protein #61 (Figure 48G), DARPin® Protein #74 (Figure 48H). The calculated LLOQ is indicated by a dotted line. [Figure 48B] TNFα levels. The mean TNFα concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 48A), DARPin® Protein #58 (Figure 48B), DARPin® Protein #59 (Figure 48C), DARPin® Protein #57 (Figure 48D), DARPin® Protein #60 (Figure 48E), DARPin® Protein #56 (Figure 48F), DARPin® Protein #61 (Figure 48G), DARPin® Protein #74 (Figure 48H). The calculated LLOQ is indicated by a dotted line. [Figure 48C]TNFα levels. The mean TNFα concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 48A), DARPin® Protein #58 (Figure 48B), DARPin® Protein #59 (Figure 48C), DARPin® Protein #57 (Figure 48D), DARPin® Protein #60 (Figure 48E), DARPin® Protein #56 (Figure 48F), DARPin® Protein #61 (Figure 48G), DARPin® Protein #74 (Figure 48H). The calculated LLOQ is indicated by a dotted line. [Figure 48D] TNFα levels. The mean TNFα concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 48A), DARPin® Protein #58 (Figure 48B), DARPin® Protein #59 (Figure 48C), DARPin® Protein #57 (Figure 48D), DARPin® Protein #60 (Figure 48E), DARPin® Protein #56 (Figure 48F), DARPin® Protein #61 (Figure 48G), DARPin® Protein #74 (Figure 48H). The calculated LLOQ is indicated by a dotted line. [Figure 48E]TNFα levels. The mean TNFα concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 48A), DARPin® Protein #58 (Figure 48B), DARPin® Protein #59 (Figure 48C), DARPin® Protein #57 (Figure 48D), DARPin® Protein #60 (Figure 48E), DARPin® Protein #56 (Figure 48F), DARPin® Protein #61 (Figure 48G), DARPin® Protein #74 (Figure 48H). The calculated LLOQ is indicated by a dotted line. [Figure 48F] TNFα levels. The mean TNFα concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 48A), DARPin® Protein #58 (Figure 48B), DARPin® Protein #59 (Figure 48C), DARPin® Protein #57 (Figure 48D), DARPin® Protein #60 (Figure 48E), DARPin® Protein #56 (Figure 48F), DARPin® Protein #61 (Figure 48G), DARPin® Protein #74 (Figure 48H). The calculated LLOQ is indicated by a dotted line. [Figure 48G]TNFα levels. The mean TNFα concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 48A), DARPin® Protein #58 (Figure 48B), DARPin® Protein #59 (Figure 48C), DARPin® Protein #57 (Figure 48D), DARPin® Protein #60 (Figure 48E), DARPin® Protein #56 (Figure 48F), DARPin® Protein #61 (Figure 48G), DARPin® Protein #74 (Figure 48H). The calculated LLOQ is indicated by a dotted line. [Figure 48H] TNFα levels. The mean TNFα concentration values ​​for all three donors in the blood loop system were calculated. Plasma samples were collected from the blood loop system at 0 (zero), 2, 4, 8, and 24 hours. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 48A), DARPin® Protein #58 (Figure 48B), DARPin® Protein #59 (Figure 48C), DARPin® Protein #57 (Figure 48D), DARPin® Protein #60 (Figure 48E), DARPin® Protein #56 (Figure 48F), DARPin® Protein #61 (Figure 48G), DARPin® Protein #74 (Figure 48H). The calculated LLOQ is indicated by a dotted line. [Figure 49A]Platelet counts. Blood samples were drawn from the blood loop system at time 0 (zero) and 2, 4, 8, and 24 hours, and platelets were automatically counted using a Sysmex XN-L350 Hematology (H). Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 49A), DARPin® Protein #58 (Figure 49B), DARPin® Protein #59 (Figure 49C), DARPin® Protein #57 (Figure 49D), DARPin® Protein #60 (Figure 49E), DARPin® Protein #56 (Figure 49F), DARPin® Protein #61 (Figure 49G), DARPin® Protein #74 (Figure 49H). The calculated LLOQ is indicated by a dotted line. [Figure 49B] Platelet counts. Blood samples were drawn from the blood loop system at time 0 (zero) and 2, 4, 8, and 24 hours, and platelets were automatically counted using a Sysmex XN-L350 Hematology (H). Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 49A), DARPin® Protein #58 (Figure 49B), DARPin® Protein #59 (Figure 49C), DARPin® Protein #57 (Figure 49D), DARPin® Protein #60 (Figure 49E), DARPin® Protein #56 (Figure 49F), DARPin® Protein #61 (Figure 49G), DARPin® Protein #74 (Figure 49H). The calculated LLOQ is indicated by a dotted line. [Figure 49C]Platelet counts. Blood samples were drawn from the blood loop system at time 0 (zero) and 2, 4, 8, and 24 hours, and platelets were automatically counted using a Sysmex XN-L350 Hematology (H). Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 49A), DARPin® Protein #58 (Figure 49B), DARPin® Protein #59 (Figure 49C), DARPin® Protein #57 (Figure 49D), DARPin® Protein #60 (Figure 49E), DARPin® Protein #56 (Figure 49F), DARPin® Protein #61 (Figure 49G), DARPin® Protein #74 (Figure 49H). The calculated LLOQ is indicated by a dotted line. [Figure 49D] Platelet counts. Blood samples were drawn from the blood loop system at time 0 (zero) and 2, 4, 8, and 24 hours, and platelets were automatically counted using a Sysmex XN-L350 Hematology (H). Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 49A), DARPin® Protein #58 (Figure 49B), DARPin® Protein #59 (Figure 49C), DARPin® Protein #57 (Figure 49D), DARPin® Protein #60 (Figure 49E), DARPin® Protein #56 (Figure 49F), DARPin® Protein #61 (Figure 49G), DARPin® Protein #74 (Figure 49H). The calculated LLOQ is indicated by a dotted line. [Figure 49E]Platelet counts. Blood samples were drawn from the blood loop system at time 0 (zero) and 2, 4, 8, and 24 hours, and platelets were automatically counted using a Sysmex XN-L350 Hematology (H). Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 49A), DARPin® Protein #58 (Figure 49B), DARPin® Protein #59 (Figure 49C), DARPin® Protein #57 (Figure 49D), DARPin® Protein #60 (Figure 49E), DARPin® Protein #56 (Figure 49F), DARPin® Protein #61 (Figure 49G), DARPin® Protein #74 (Figure 49H). The calculated LLOQ is indicated by a dotted line. [Figure 49F] Platelet counts. Blood samples were drawn from the blood loop system at time 0 (zero) and 2, 4, 8, and 24 hours, and platelets were automatically counted using a Sysmex XN-L350 Hematology (H). Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 49A), DARPin® Protein #58 (Figure 49B), DARPin® Protein #59 (Figure 49C), DARPin® Protein #57 (Figure 49D), DARPin® Protein #60 (Figure 49E), DARPin® Protein #56 (Figure 49F), DARPin® Protein #61 (Figure 49G), DARPin® Protein #74 (Figure 49H). The calculated LLOQ is indicated by a dotted line. [Figure 49G]Platelet counts. Blood samples were drawn from the blood loop system at time 0 (zero) and 2, 4, 8, and 24 hours, and platelets were automatically counted using a Sysmex XN-L350 Hematology (H). Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 49A), DARPin® Protein #58 (Figure 49B), DARPin® Protein #59 (Figure 49C), DARPin® Protein #57 (Figure 49D), DARPin® Protein #60 (Figure 49E), DARPin® Protein #56 (Figure 49F), DARPin® Protein #61 (Figure 49G), DARPin® Protein #74 (Figure 49H). The calculated LLOQ is indicated by a dotted line. [Figure 49H] Platelet counts. Blood samples were drawn from the blood loop system at time 0 (zero) and 2, 4, 8, and 24 hours, and platelets were automatically counted using a Sysmex XN-L350 Hematology (H). Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 49A), DARPin® Protein #58 (Figure 49B), DARPin® Protein #59 (Figure 49C), DARPin® Protein #57 (Figure 49D), DARPin® Protein #60 (Figure 49E), DARPin® Protein #56 (Figure 49F), DARPin® Protein #61 (Figure 49G), DARPin® Protein #74 (Figure 49H). The calculated LLOQ is indicated by a dotted line. [Figure 50A]White blood cell counts. Blood samples were drawn from the blood loop system at time 0 (0), and 2, 4, 8, and 24 hours, and white blood cells were automatically counted using a Sysmex XN-L350 Hematology Analyzer. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 50A), DARPin® Protein #58 (Figure 50B), DARPin® Protein #59 (Figure 50C), DARPin® Protein #57 (Figure 50D), DARPin® Protein #60 (Figure 50E), DARPin® Protein #56 (Figure 50F), DARPin® Protein #61 (Figure 50G), DARPin® Protein #74 (Figure 50H). The calculated LLOQ is indicated by a dotted line. [Figure 50B] White blood cell counts. Blood samples were drawn from the blood loop system at time 0 (0), and 2, 4, 8, and 24 hours, and white blood cells were automatically counted using a Sysmex XN-L350 Hematology Analyzer. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 50A), DARPin® Protein #58 (Figure 50B), DARPin® Protein #59 (Figure 50C), DARPin® Protein #57 (Figure 50D), DARPin® Protein #60 (Figure 50E), DARPin® Protein #56 (Figure 50F), DARPin® Protein #61 (Figure 50G), DARPin® Protein #74 (Figure 50H). The calculated LLOQ is indicated by a dotted line. [Figure 50C]White blood cell counts. Blood samples were drawn from the blood loop system at time 0 (0), and 2, 4, 8, and 24 hours, and white blood cells were automatically counted using a Sysmex XN-L350 Hematology Analyzer. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 50A), DARPin® Protein #58 (Figure 50B), DARPin® Protein #59 (Figure 50C), DARPin® Protein #57 (Figure 50D), DARPin® Protein #60 (Figure 50E), DARPin® Protein #56 (Figure 50F), DARPin® Protein #61 (Figure 50G), DARPin® Protein #74 (Figure 50H). The calculated LLOQ is indicated by a dotted line. [Figure 50D] White blood cell counts. Blood samples were drawn from the blood loop system at time 0 (0), and 2, 4, 8, and 24 hours, and white blood cells were automatically counted using a Sysmex XN-L350 Hematology Analyzer. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 50A), DARPin® Protein #58 (Figure 50B), DARPin® Protein #59 (Figure 50C), DARPin® Protein #57 (Figure 50D), DARPin® Protein #60 (Figure 50E), DARPin® Protein #56 (Figure 50F), DARPin® Protein #61 (Figure 50G), DARPin® Protein #74 (Figure 50H). The calculated LLOQ is indicated by a dotted line. [Figure 50E]White blood cell counts. Blood samples were drawn from the blood loop system at time 0 (0), and 2, 4, 8, and 24 hours, and white blood cells were automatically counted using a Sysmex XN-L350 Hematology Analyzer. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 50A), DARPin® Protein #58 (Figure 50B), DARPin® Protein #59 (Figure 50C), DARPin® Protein #57 (Figure 50D), DARPin® Protein #60 (Figure 50E), DARPin® Protein #56 (Figure 50F), DARPin® Protein #61 (Figure 50G), DARPin® Protein #74 (Figure 50H). The calculated LLOQ is indicated by a dotted line. [Figure 50F] White blood cell counts. Blood samples were drawn from the blood loop system at time 0 (0), and 2, 4, 8, and 24 hours, and white blood cells were automatically counted using a Sysmex XN-L350 Hematology Analyzer. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 50A), DARPin® Protein #58 (Figure 50B), DARPin® Protein #59 (Figure 50C), DARPin® Protein #57 (Figure 50D), DARPin® Protein #60 (Figure 50E), DARPin® Protein #56 (Figure 50F), DARPin® Protein #61 (Figure 50G), DARPin® Protein #74 (Figure 50H). The calculated LLOQ is indicated by a dotted line. [Figure 50G]White blood cell counts. Blood samples were drawn from the blood loop system at time 0 (0), and 2, 4, 8, and 24 hours, and white blood cells were automatically counted using a Sysmex XN-L350 Hematology Analyzer. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 50A), DARPin® Protein #58 (Figure 50B), DARPin® Protein #59 (Figure 50C), DARPin® Protein #57 (Figure 50D), DARPin® Protein #60 (Figure 50E), DARPin® Protein #56 (Figure 50F), DARPin® Protein #61 (Figure 50G), DARPin® Protein #74 (Figure 50H). The calculated LLOQ is indicated by a dotted line. [Figure 50H] White blood cell counts. Blood samples were drawn from the blood loop system at time 0 (0), and 2, 4, 8, and 24 hours, and white blood cells were automatically counted using a Sysmex XN-L350 Hematology Analyzer. Each point represents the mean of three donors (and error bars are SD). Flotetuzumab (Figure 50A), DARPin® Protein #58 (Figure 50B), DARPin® Protein #59 (Figure 50C), DARPin® Protein #57 (Figure 50D), DARPin® Protein #60 (Figure 50E), DARPin® Protein #56 (Figure 50F), DARPin® Protein #61 (Figure 50G), DARPin® Protein #74 (Figure 50H). The calculated LLOQ is indicated by a dotted line. [Figure 51A]Figure 51A. Potency titration curves (T cell activation) of DARPin® Protein #56, DARPin® Protein #57, DARPin® Protein #58, DARPin® Protein #59, DARPin® Protein #60, and DARPin® Protein #61 targeting CD123-CD33-CD70 on Molm13 tumor cells co-cultured with human pan-T cells. EC50 values ​​are shown in pM. Figure 51B. Potency titration curves (T cell activation) of DARPin® Protein #56, DARPin® Protein #57 targeting CD123-CD33-CD70 on Molm13 tumor cells co-cultured with human pan-T cells, as well as the corresponding negative controls DARPin® Protein #74, DARPin® Protein #75, DARPin® Protein #76, and DARPin® Protein #77. EC50 values ​​are shown in pM. Figure 51C. Potency titration curves (T cell activation) of DARPin® Protein #56 targeting CD123-CD33-CD70 and of the known benchmark control molecules AMG330 analog and Flotetuzumab analog on Molm13 tumor cells co-cultured with human pan-T cells. EC50 values ​​are shown in pM. Figure 51D. Potency titration curves (T cell activation) of DARPin® Protein #56 targeting CD123-CD33-CD70 on Molm13 wild-type or various TAA knockout tumor cells. EC50 values ​​are shown in pM. Figure 51E. Potency titration curves (T cell activation) of DARPin® Protein #57 targeting CD123-CD33-CD70 on Molm13 wild-type or various TAA knockout tumor cells. EC50 values ​​are shown in pM. [Figure 51B]Figure 51A. Potency titration curves (T cell activation) of DARPin® Protein #56, DARPin® Protein #57, DARPin® Protein #58, DARPin® Protein #59, DARPin® Protein #60, and DARPin® Protein #61 targeting CD123-CD33-CD70 on Molm13 tumor cells co-cultured with human pan-T cells. EC50 values ​​are shown in pM. Figure 51B. Potency titration curves (T cell activation) of DARPin® Protein #56, DARPin® Protein #57 targeting CD123-CD33-CD70 on Molm13 tumor cells co-cultured with human pan-T cells, as well as the corresponding negative controls DARPin® Protein #74, DARPin® Protein #75, DARPin® Protein #76, and DARPin® Protein #77. EC50 values ​​are shown in pM. Figure 51C. Potency titration curves (T cell activation) of DARPin® Protein #56 targeting CD123-CD33-CD70 and of the known benchmark control molecules AMG330 analog and Flotetuzumab analog on Molm13 tumor cells co-cultured with human pan-T cells. EC50 values ​​are shown in pM. Figure 51D. Potency titration curves (T cell activation) of DARPin® Protein #56 targeting CD123-CD33-CD70 on Molm13 wild-type or various TAA knockout tumor cells. EC50 values ​​are shown in pM. Figure 51E. Potency titration curves (T cell activation) of DARPin® Protein #57 targeting CD123-CD33-CD70 on Molm13 wild-type or various TAA knockout tumor cells. EC50 values ​​are shown in pM. [Figure 51C]Figure 51A. Potency titration curves (T cell activation) of DARPin® Protein #56, DARPin® Protein #57, DARPin® Protein #58, DARPin® Protein #59, DARPin® Protein #60, and DARPin® Protein #61 targeting CD123-CD33-CD70 on Molm13 tumor cells co-cultured with human pan-T cells. EC50 values ​​are shown in pM. Figure 51B. Potency titration curves (T cell activation) of DARPin® Protein #56, DARPin® Protein #57 targeting CD123-CD33-CD70 on Molm13 tumor cells co-cultured with human pan-T cells, as well as the corresponding negative controls DARPin® Protein #74, DARPin® Protein #75, DARPin® Protein #76, and DARPin® Protein #77. EC50 values ​​are shown in pM. Figure 51C. Potency titration curves (T cell activation) of DARPin® Protein #56 targeting CD123-CD33-CD70 and of the known benchmark control molecules AMG330 analog and Flotetuzumab analog on Molm13 tumor cells co-cultured with human pan-T cells. EC50 values ​​are shown in pM. Figure 51D. Potency titration curves (T cell activation) of DARPin® Protein #56 targeting CD123-CD33-CD70 on Molm13 wild-type or various TAA knockout tumor cells. EC50 values ​​are shown in pM. Figure 51E. Potency titration curves (T cell activation) of DARPin® Protein #57 targeting CD123-CD33-CD70 on Molm13 wild-type or various TAA knockout tumor cells. EC50 values ​​are shown in pM. [Figure 51D]Figure 51A. Potency titration curves (T cell activation) of DARPin® Protein #56, DARPin® Protein #57, DARPin® Protein #58, DARPin® Protein #59, DARPin® Protein #60, and DARPin® Protein #61 targeting CD123-CD33-CD70 on Molm13 tumor cells co-cultured with human pan-T cells. EC50 values ​​are shown in pM. Figure 51B. Potency titration curves (T cell activation) of DARPin® Protein #56, DARPin® Protein #57 targeting CD123-CD33-CD70 on Molm13 tumor cells co-cultured with human pan-T cells, as well as the corresponding negative controls DARPin® Protein #74, DARPin® Protein #75, DARPin® Protein #76, and DARPin® Protein #77. EC50 values ​​are shown in pM. Figure 51C. Potency titration curves (T cell activation) of DARPin® Protein #56 targeting CD123-CD33-CD70 and of the known benchmark control molecules AMG330 analog and Flotetuzumab analog on Molm13 tumor cells co-cultured with human pan-T cells. EC50 values ​​are shown in pM. Figure 51D. Potency titration curves (T cell activation) of DARPin® Protein #56 targeting CD123-CD33-CD70 on Molm13 wild-type or various TAA knockout tumor cells. EC50 values ​​are shown in pM. Figure 51E. Potency titration curves (T cell activation) of DARPin® Protein #57 targeting CD123-CD33-CD70 on Molm13 wild-type or various TAA knockout tumor cells. EC50 values ​​are shown in pM. [Figure 51E]Figure 51A. Potency titration curves (T cell activation) of DARPin® Protein #56, DARPin® Protein #57, DARPin® Protein #58, DARPin® Protein #59, DARPin® Protein #60, and DARPin® Protein #61 targeting CD123-CD33-CD70 on Molm13 tumor cells co-cultured with human pan-T cells. EC50 values ​​are shown in pM. Figure 51B. Potency titration curves (T cell activation) of DARPin® Protein #56, DARPin® Protein #57 targeting CD123-CD33-CD70 on Molm13 tumor cells co-cultured with human pan-T cells, as well as the corresponding negative controls DARPin® Protein #74, DARPin® Protein #75, DARPin® Protein #76, and DARPin® Protein #77. EC50 values ​​are shown in pM. Figure 51C. Potency titration curves (T cell activation) of DARPin® Protein #56 targeting CD123-CD33-CD70 and of the known benchmark control molecules AMG330 analog and Flotetuzumab analog on Molm13 tumor cells co-cultured with human pan-T cells. EC50 values ​​are shown in pM. Figure 51D. Potency titration curves (T cell activation) of DARPin® Protein #56 targeting CD123-CD33-CD70 on Molm13 wild-type or various TAA knockout tumor cells. EC50 values ​​are shown in pM. Figure 51E. Potency titration curves (T cell activation) of DARPin® Protein #57 targeting CD123-CD33-CD70 on Molm13 wild-type or various TAA knockout tumor cells. EC50 values ​​are shown in pM. [Figure 52A]Figure 52A. Potency titration curves (cell killing) of DARPin® Protein #56, DARPin® Protein #57, DARPin® Protein #58, DARPin® Protein #59, DARPin® Protein #60, and DARPin® Protein #61 targeting CD123-CD33-CD70 on Molm13 tumor cells co-cultured with human pan-T cells. EC50 values ​​are shown in pM. Figure 52B. Potency titration curves (cell killing) of DARPin® Protein #56, DARPin® Protein #57 targeting CD123-CD33-CD70 on Molm13 tumor cells co-cultured with human pan-T cells, as well as the corresponding negative controls DARPin® Protein #74, DARPin® Protein #75, DARPin® Protein #76, DARPin® Protein #77. EC50 values ​​are shown in pM. Figure 52C. Potency titration curves (cell killing) of DARPin® Protein #56 targeting CD123-CD33-CD70 and of the known benchmark control molecules AMG330-analogue and flotetuzumab-analogue on Molm13 tumor cells co-cultured with human pan-T cells. EC50 values ​​are shown in pM. Figure 52D. Potency titration curves (cell killing) of DARPin® Protein #56 targeting CD123-CD33-CD70 against Molm13 wild-type or various TAA knockout tumor cells. EC50 values ​​are shown in pM. [Figure 52B]Figure 52A. Potency titration curves (cell killing) of DARPin® Protein #56, DARPin® Protein #57, DARPin® Protein #58, DARPin® Protein #59, DARPin® Protein #60, and DARPin® Protein #61 targeting CD123-CD33-CD70 on Molm13 tumor cells co-cultured with human pan-T cells. EC50 values ​​are shown in pM. Figure 52B. Potency titration curves (cell killing) of DARPin® Protein #56, DARPin® Protein #57 targeting CD123-CD33-CD70 on Molm13 tumor cells co-cultured with human pan-T cells, as well as the corresponding negative controls DARPin® Protein #74, DARPin® Protein #75, DARPin® Protein #76, DARPin® Protein #77. EC50 values ​​are shown in pM. Figure 52C. Potency titration curves (cell killing) of DARPin® Protein #56 targeting CD123-CD33-CD70 and of the known benchmark control molecules AMG330-analogue and flotetuzumab-analogue on Molm13 tumor cells co-cultured with human pan-T cells. EC50 values ​​are shown in pM. Figure 52D. Potency titration curves (cell killing) of DARPin® Protein #56 targeting CD123-CD33-CD70 against Molm13 wild-type or various TAA knockout tumor cells. EC50 values ​​are shown in pM. [Figure 52C]Figure 52A. Potency titration curves (cell killing) of DARPin® Protein #56, DARPin® Protein #57, DARPin® Protein #58, DARPin® Protein #59, DARPin® Protein #60, and DARPin® Protein #61 targeting CD123-CD33-CD70 on Molm13 tumor cells co-cultured with human pan-T cells. EC50 values ​​are shown in pM. Figure 52B. Potency titration curves (cell killing) of DARPin® Protein #56, DARPin® Protein #57 targeting CD123-CD33-CD70 on Molm13 tumor cells co-cultured with human pan-T cells, as well as the corresponding negative controls DARPin® Protein #74, DARPin® Protein #75, DARPin® Protein #76, DARPin® Protein #77. EC50 values ​​are shown in pM. Figure 52C. Potency titration curves (cell killing) of DARPin® Protein #56 targeting CD123-CD33-CD70 and of the known benchmark control molecules AMG330-analogue and flotetuzumab-analogue on Molm13 tumor cells co-cultured with human pan-T cells. EC50 values ​​are shown in pM. Figure 52D. Potency titration curves (cell killing) of DARPin® Protein #56 targeting CD123-CD33-CD70 against Molm13 wild-type or various TAA knockout tumor cells. EC50 values ​​are shown in pM. [Figure 52D]Figure 52A. Potency titration curves (cell killing) of DARPin® Protein #56, DARPin® Protein #57, DARPin® Protein #58, DARPin® Protein #59, DARPin® Protein #60, and DARPin® Protein #61 targeting CD123-CD33-CD70 on Molm13 tumor cells co-cultured with human pan-T cells. EC50 values ​​are shown in pM. Figure 52B. Potency titration curves (cell killing) of DARPin® Protein #56, DARPin® Protein #57 targeting CD123-CD33-CD70 on Molm13 tumor cells co-cultured with human pan-T cells, as well as the corresponding negative controls DARPin® Protein #74, DARPin® Protein #75, DARPin® Protein #76, DARPin® Protein #77. EC50 values ​​are shown in pM. Figure 52C. Potency titration curves (cell killing) of DARPin® Protein #56 targeting CD123-CD33-CD70 and of the known benchmark control molecules AMG330-analogue and flotetuzumab-analogue on Molm13 tumor cells co-cultured with human pan-T cells. EC50 values ​​are shown in pM. Figure 52D. Potency titration curves (cell killing) of DARPin® Protein #56 targeting CD123-CD33-CD70 against Molm13 wild-type or various TAA knockout tumor cells. EC50 values ​​are shown in pM. [Figure 52E]Figure 52A. Potency titration curves (cell killing) of DARPin® Protein #56, DARPin® Protein #57, DARPin® Protein #58, DARPin® Protein #59, DARPin® Protein #60, and DARPin® Protein #61 targeting CD123-CD33-CD70 on Molm13 tumor cells co-cultured with human pan-T cells. EC50 values ​​are shown in pM. Figure 52B. Potency titration curves (cell killing) of DARPin® Protein #56, DARPin® Protein #57 targeting CD123-CD33-CD70 on Molm13 tumor cells co-cultured with human pan-T cells, as well as the corresponding negative controls DARPin® Protein #74, DARPin® Protein #75, DARPin® Protein #76, DARPin® Protein #77. EC50 values ​​are shown in pM. Figure 52C. Potency titration curves (cell killing) of DARPin® Protein #56 targeting CD123-CD33-CD70 and of the known benchmark control molecules AMG330-analogue and flotetuzumab-analogue on Molm13 tumor cells co-cultured with human pan-T cells. EC50 values ​​are shown in pM. Figure 52D. Potency titration curves (cell killing) of DARPin® Protein #56 targeting CD123-CD33-CD70 against Molm13 wild-type or various TAA knockout tumor cells. EC50 values ​​are shown in pM. [Figure 53A] Figure 53A. Potency titration curve of DARPin® protein #56 targeting CD123-CD33-CD70 on AML patient BMMC tumor cells (allogeneic and autologous T cell activation). EC50 values ​​are shown in pM. Figure 53B. Potency titration curve of DARPin® protein #56 targeting CD123-CD33-CD70 on AML patient BMMC tumor cells (cell killing) compared to flotetuzumab and AMG330. [Figure 53B]Figure 53A. Potency titration curve of DARPin® protein #56 targeting CD123-CD33-CD70 on AML patient BMMC tumor cells (allogeneic and autologous T cell activation). EC50 values ​​are shown in pM. Figure 53B. Potency titration curve of DARPin® protein #56 targeting CD123-CD33-CD70 on AML patient BMMC tumor cells (cell killing) compared to flotetuzumab and AMG330. [Figure 54A] Figure 5A. Potency titration curves of selected multispecific DARPin® proteins targeting CD123-CD33-CD70 on AML patient BMMC tumor cells compared to flotetuzumab (T cell activation). Figure 54B. Potency titration curves of selected multispecific DARPin® proteins targeting CD123-CD33-CD70 on AML patient BMMC tumor cells compared to flotetuzumab (tumor cell killing). [Figure 54B] Figure 5A. Potency titration curves of selected multispecific DARPin® proteins targeting CD123-CD33-CD70 on AML patient BMMC tumor cells compared to flotetuzumab (T cell activation). Figure 54B. Potency titration curves of selected multispecific DARPin® proteins targeting CD123-CD33-CD70 on AML patient BMMC tumor cells compared to flotetuzumab (tumor cell killing). [Figure 55] Median expression across different LSC and HSC samples expressed as delta MFI. [Figure 56] The preferential killing of LSCs (filled bars) over HSCs (open bars) by DARPin® protein #56 and DARPin® protein #57 confirms the existence of a therapeutic window between LSCs and HSCs. [Figure 57A]Molm-13 cell killing versus IFNγ release. Molm-13 cell killing is shown as a filled square / curve, and the concentration of IFNγ is shown as a filled circle / curve. Figure 57A. Effect of DARPin® Protein #56 on cell killing and cytokine release. Figure 57B. Effect of DARPin® Protein #57 on cell killing and cytokine release. Figure 57C. Effect of DARPin® Protein #59 on cell killing and cytokine release. Figure 57D. Effect of DARPin® Protein #62 on cell killing and cytokine release. Figure 57E. Effect of flotetuzumab on cell killing and cytokine release. Figure 57F. Effect of AMG330 on cell killing and cytokine release. [Figure 57B] Molm-13 cell killing versus IFNγ release. Molm-13 cell killing is shown as a filled square / curve, and the concentration of IFNγ is shown as a filled circle / curve. Figure 57A. Effect of DARPin® Protein #56 on cell killing and cytokine release. Figure 57B. Effect of DARPin® Protein #57 on cell killing and cytokine release. Figure 57C. Effect of DARPin® Protein #59 on cell killing and cytokine release. Figure 57D. Effect of DARPin® Protein #62 on cell killing and cytokine release. Figure 57E. Effect of flotetuzumab on cell killing and cytokine release. Figure 57F. Effect of AMG330 on cell killing and cytokine release. [Figure 57C] Molm-13 cell killing versus IFNγ release. Molm-13 cell killing is shown as a filled square / curve, and the concentration of IFNγ is shown as a filled circle / curve. Figure 57A. Effect of DARPin® Protein #56 on cell killing and cytokine release. Figure 57B. Effect of DARPin® Protein #57 on cell killing and cytokine release. Figure 57C. Effect of DARPin® Protein #59 on cell killing and cytokine release. Figure 57D. Effect of DARPin® Protein #62 on cell killing and cytokine release. Figure 57E. Effect of flotetuzumab on cell killing and cytokine release. Figure 57F. Effect of AMG330 on cell killing and cytokine release. [Figure 57D] Molm-13 cell killing versus IFNγ release. Molm-13 cell killing is shown as a filled square / curve, and the concentration of IFNγ is shown as a filled circle / curve. Figure 57A. Effect of DARPin® Protein #56 on cell killing and cytokine release. Figure 57B. Effect of DARPin® Protein #57 on cell killing and cytokine release. Figure 57C. Effect of DARPin® Protein #59 on cell killing and cytokine release. Figure 57D. Effect of DARPin® Protein #62 on cell killing and cytokine release. Figure 57E. Effect of flotetuzumab on cell killing and cytokine release. Figure 57F. Effect of AMG330 on cell killing and cytokine release. [Figure 57E] Molm-13 cell killing versus IFNγ release. Molm-13 cell killing is shown as a filled square / curve, and the concentration of IFNγ is shown as a filled circle / curve. Figure 57A. Effect of DARPin® Protein #56 on cell killing and cytokine release. Figure 57B. Effect of DARPin® Protein #57 on cell killing and cytokine release. Figure 57C. Effect of DARPin® Protein #59 on cell killing and cytokine release. Figure 57D. Effect of DARPin® Protein #62 on cell killing and cytokine release. Figure 57E. Effect of flotetuzumab on cell killing and cytokine release. Figure 57F. Effect of AMG330 on cell killing and cytokine release. [Figure 57F]Molm-13 cell killing versus IFNγ release. Molm-13 cell killing is shown as a filled square / curve, and the concentration of IFNγ is shown as a filled circle / curve. Figure 57A. Effect of DARPin® Protein #56 on cell killing and cytokine release. Figure 57B. Effect of DARPin® Protein #57 on cell killing and cytokine release. Figure 57C. Effect of DARPin® Protein #59 on cell killing and cytokine release. Figure 57D. Effect of DARPin® Protein #62 on cell killing and cytokine release. Figure 57E. Effect of flotetuzumab on cell killing and cytokine release. Figure 57F. Effect of AMG330 on cell killing and cytokine release. [Figure 58A] Figure 58A. Tumor growth over time in mice (n=5 mice per donor / 2 hPBMC donors used) that were injected intraperitoneally with hPBMCs and xenografted subcutaneously with MOLM-13 tumor cells 2 days after hPBMC injection and treated with PBS 1x (closed circles), 0.5 mg / kg DARPin® Protein #56 (closed squares), or 0.5 mg / kg AMG330 (closed triangles). Treatment began 4 days after tumor cell xenografting. Data are presented as mean + SEM. Figure 58B. Assessment of tumor volume 17 days after tumor cell xenografting in the mice described in Figure 58A. [Figure 58B] Figure 58A. Tumor growth over time in mice (n=5 mice per donor / 2 hPBMC donors used) that were injected intraperitoneally with hPBMCs and xenografted subcutaneously with MOLM-13 tumor cells 2 days after hPBMC injection and treated with PBS 1x (closed circles), 0.5 mg / kg DARPin® Protein #56 (closed squares), or 0.5 mg / kg AMG330 (closed triangles). Treatment began 4 days after tumor cell xenografting. Data are presented as mean + SEM. Figure 58B. Assessment of tumor volume 17 days after tumor cell xenografting in the mice described in Figure 58A. [Figure 59A]Self-killing versus cytokine (IFN-γ, TNFα, IL-2) release of patient-derived AML cells after 2 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 59A. The effect of flotetuzumab on cell death and IFNγ release is similar. Figure 59B. The effect of flotetuzumab on cell death and TNFα release is similar. Figure 59C. The effect of flotetuzumab on cell death and IL-2 release is similar. Figure 59D. The effect of AMG330 on cell killing and IFNγ release is similar. Figure 59E. The effect of AMG330 on cell killing and TNFα release is similar. Figure 59F. The effect of AMG330 on cell killing and IL-2 release is similar. Figure 59G. The effect of DARPin® Protein #56 on cell killing and IFNγ release. Figure 59H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 591. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 59J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 59K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 59L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 59B]Self-killing versus cytokine (IFN-γ, TNFα, IL-2) release of patient-derived AML cells after 2 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 59A. The effect of flotetuzumab on cell death and IFNγ release is similar. Figure 59B. The effect of flotetuzumab on cell death and TNFα release is similar. Figure 59C. The effect of flotetuzumab on cell death and IL-2 release is similar. Figure 59D. The effect of AMG330 on cell killing and IFNγ release is similar. Figure 59E. The effect of AMG330 on cell killing and TNFα release is similar. Figure 59F. The effect of AMG330 on cell killing and IL-2 release is similar. Figure 59G. The effect of DARPin® Protein #56 on cell killing and IFNγ release. Figure 59H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 591. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 59J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 59K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 59L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 59C]Self-killing versus cytokine (IFN-γ, TNFα, IL-2) release of patient-derived AML cells after 2 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 59A. The effect of flotetuzumab on cell death and IFNγ release is similar. Figure 59B. The effect of flotetuzumab on cell death and TNFα release is similar. Figure 59C. The effect of flotetuzumab on cell death and IL-2 release is similar. Figure 59D. The effect of AMG330 on cell killing and IFNγ release is similar. Figure 59E. The effect of AMG330 on cell killing and TNFα release is similar. Figure 59F. The effect of AMG330 on cell killing and IL-2 release is similar. Figure 59G. The effect of DARPin® Protein #56 on cell killing and IFNγ release. Figure 59H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 591. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 59J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 59K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 59L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 59D]Self-killing versus cytokine (IFN-γ, TNFα, IL-2) release of patient-derived AML cells after 2 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 59A. The effect of flotetuzumab on cell death and IFNγ release is similar. Figure 59B. The effect of flotetuzumab on cell death and TNFα release is similar. Figure 59C. The effect of flotetuzumab on cell death and IL-2 release is similar. Figure 59D. The effect of AMG330 on cell killing and IFNγ release is similar. Figure 59E. The effect of AMG330 on cell killing and TNFα release is similar. Figure 59F. The effect of AMG330 on cell killing and IL-2 release is similar. Figure 59G. The effect of DARPin® Protein #56 on cell killing and IFNγ release. Figure 59H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 591. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 59J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 59K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 59L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 59E]Self-killing versus cytokine (IFN-γ, TNFα, IL-2) release of patient-derived AML cells after 2 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 59A. The effect of flotetuzumab on cell death and IFNγ release is similar. Figure 59B. The effect of flotetuzumab on cell death and TNFα release is similar. Figure 59C. The effect of flotetuzumab on cell death and IL-2 release is similar. Figure 59D. The effect of AMG330 on cell killing and IFNγ release is similar. Figure 59E. The effect of AMG330 on cell killing and TNFα release is similar. Figure 59F. The effect of AMG330 on cell killing and IL-2 release is similar. Figure 59G. The effect of DARPin® Protein #56 on cell killing and IFNγ release. Figure 59H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 591. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 59J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 59K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 59L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 59F]Self-killing versus cytokine (IFN-γ, TNFα, IL-2) release of patient-derived AML cells after 2 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 59A. The effect of flotetuzumab on cell death and IFNγ release is similar. Figure 59B. The effect of flotetuzumab on cell death and TNFα release is similar. Figure 59C. The effect of flotetuzumab on cell death and IL-2 release is similar. Figure 59D. The effect of AMG330 on cell killing and IFNγ release is similar. Figure 59E. The effect of AMG330 on cell killing and TNFα release is similar. Figure 59F. The effect of AMG330 on cell killing and IL-2 release is similar. Figure 59G. The effect of DARPin® Protein #56 on cell killing and IFNγ release. Figure 59H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 591. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 59J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 59K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 59L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 59G]Self-killing versus cytokine (IFN-γ, TNFα, IL-2) release of patient-derived AML cells after 2 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 59A. The effect of flotetuzumab on cell death and IFNγ release is similar. Figure 59B. The effect of flotetuzumab on cell death and TNFα release is similar. Figure 59C. The effect of flotetuzumab on cell death and IL-2 release is similar. Figure 59D. The effect of AMG330 on cell killing and IFNγ release is similar. Figure 59E. The effect of AMG330 on cell killing and TNFα release is similar. Figure 59F. The effect of AMG330 on cell killing and IL-2 release is similar. Figure 59G. The effect of DARPin® Protein #56 on cell killing and IFNγ release. Figure 59H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 591. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 59J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 59K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 59L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 59H]Self-killing versus cytokine (IFN-γ, TNFα, IL-2) release of patient-derived AML cells after 2 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 59A. The effect of flotetuzumab on cell death and IFNγ release is similar. Figure 59B. The effect of flotetuzumab on cell death and TNFα release is similar. Figure 59C. The effect of flotetuzumab on cell death and IL-2 release is similar. Figure 59D. The effect of AMG330 on cell killing and IFNγ release is similar. Figure 59E. The effect of AMG330 on cell killing and TNFα release is similar. Figure 59F. The effect of AMG330 on cell killing and IL-2 release is similar. Figure 59G. The effect of DARPin® Protein #56 on cell killing and IFNγ release. Figure 59H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 591. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 59J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 59K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 59L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 59I]Self-killing versus cytokine (IFN-γ, TNFα, IL-2) release of patient-derived AML cells after 2 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 59A. The effect of flotetuzumab on cell death and IFNγ release is similar. Figure 59B. The effect of flotetuzumab on cell death and TNFα release is similar. Figure 59C. The effect of flotetuzumab on cell death and IL-2 release is similar. Figure 59D. The effect of AMG330 on cell killing and IFNγ release is similar. Figure 59E. The effect of AMG330 on cell killing and TNFα release is similar. Figure 59F. The effect of AMG330 on cell killing and IL-2 release is similar. Figure 59G. The effect of DARPin® Protein #56 on cell killing and IFNγ release. Figure 59H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 591. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 59J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 59K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 59L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 59J]Self-killing versus cytokine (IFN-γ, TNFα, IL-2) release of patient-derived AML cells after 2 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 59A. The effect of flotetuzumab on cell death and IFNγ release is similar. Figure 59B. The effect of flotetuzumab on cell death and TNFα release is similar. Figure 59C. The effect of flotetuzumab on cell death and IL-2 release is similar. Figure 59D. The effect of AMG330 on cell killing and IFNγ release is similar. Figure 59E. The effect of AMG330 on cell killing and TNFα release is similar. Figure 59F. The effect of AMG330 on cell killing and IL-2 release is similar. Figure 59G. The effect of DARPin® Protein #56 on cell killing and IFNγ release. Figure 59H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 591. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 59J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 59K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 59L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 59K]Self-killing versus cytokine (IFN-γ, TNFα, IL-2) release of patient-derived AML cells after 2 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 59A. The effect of flotetuzumab on cell death and IFNγ release is similar. Figure 59B. The effect of flotetuzumab on cell death and TNFα release is similar. Figure 59C. The effect of flotetuzumab on cell death and IL-2 release is similar. Figure 59D. The effect of AMG330 on cell killing and IFNγ release is similar. Figure 59E. The effect of AMG330 on cell killing and TNFα release is similar. Figure 59F. The effect of AMG330 on cell killing and IL-2 release is similar. Figure 59G. The effect of DARPin® Protein #56 on cell killing and IFNγ release. Figure 59H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 591. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 59J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 59K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 59L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 59L]Self-killing versus cytokine (IFN-γ, TNFα, IL-2) release of patient-derived AML cells after 2 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 59A. The effect of flotetuzumab on cell death and IFNγ release is similar. Figure 59B. The effect of flotetuzumab on cell death and TNFα release is similar. Figure 59C. The effect of flotetuzumab on cell death and IL-2 release is similar. Figure 59D. The effect of AMG330 on cell killing and IFNγ release is similar. Figure 59E. The effect of AMG330 on cell killing and TNFα release is similar. Figure 59F. The effect of AMG330 on cell killing and IL-2 release is similar. Figure 59G. The effect of DARPin® Protein #56 on cell killing and IFNγ release. Figure 59H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 591. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 59J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 59K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 59L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 60A]Self-killing versus cytokine (IFN-γ, TNFα, IL-2) release of patient-derived AML cells after 5 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 60A. The effect of flotetuzumab on cell death and IFNγ release is similar. Figure 60B. The effect of flotetuzumab on cell death and TNFα release is similar. Figure 60C. The effect of flotetuzumab on cell killing and IL-2 release is similar. FIG. 60D. The effect of AMG330 on cell killing and IFNγ release is similar. Figure 60E. The effect of AMG330 on cell killing and TNFα release is similar. Figure 60F. The effect of AMG330 on cell killing and IL-2 release is similar. Figure 60G. The effect of DARPin® Protein #56 on cell killing and IFNγ release. Figure 60H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 601. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 60J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 60K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 60L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 60B]Self-killing versus cytokine (IFN-γ, TNFα, IL-2) release of patient-derived AML cells after 5 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 60A. The effect of flotetuzumab on cell death and IFNγ release is similar. Figure 60B. The effect of flotetuzumab on cell death and TNFα release is similar. Figure 60C. The effect of flotetuzumab on cell killing and IL-2 release is similar. FIG. 60D. The effect of AMG330 on cell killing and IFNγ release is similar. Figure 60E. The effect of AMG330 on cell killing and TNFα release is similar. Figure 60F. The effect of AMG330 on cell killing and IL-2 release is similar. Figure 60G. The effect of DARPin® Protein #56 on cell killing and IFNγ release. Figure 60H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 601. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 60J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 60K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 60L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 60C]Self-killing versus cytokine (IFN-γ, TNFα, IL-2) release of patient-derived AML cells after 5 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 60A. The effect of flotetuzumab on cell death and IFNγ release is similar. Figure 60B. The effect of flotetuzumab on cell death and TNFα release is similar. Figure 60C. The effect of flotetuzumab on cell killing and IL-2 release is similar. FIG. 60D. The effect of AMG330 on cell killing and IFNγ release is similar. Figure 60E. The effect of AMG330 on cell killing and TNFα release is similar. Figure 60F. The effect of AMG330 on cell killing and IL-2 release is similar. Figure 60G. The effect of DARPin® Protein #56 on cell killing and IFNγ release. Figure 60H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 601. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 60J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 60K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 60L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 60D]Self-killing versus cytokine (IFN-γ, TNFα, IL-2) release of patient-derived AML cells after 5 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 60A. The effect of flotetuzumab on cell death and IFNγ release is similar. Figure 60B. The effect of flotetuzumab on cell death and TNFα release is similar. Figure 60C. The effect of flotetuzumab on cell killing and IL-2 release is similar. FIG. 60D. The effect of AMG330 on cell killing and IFNγ release is similar. Figure 60E. The effect of AMG330 on cell killing and TNFα release is similar. Figure 60F. The effect of AMG330 on cell killing and IL-2 release is similar. Figure 60G. The effect of DARPin® Protein #56 on cell killing and IFNγ release. Figure 60H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 601. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 60J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 60K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 60L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 60E]Self-killing versus cytokine (IFN-γ, TNFα, IL-2) release of patient-derived AML cells after 5 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 60A. The effect of flotetuzumab on cell death and IFNγ release is similar. Figure 60B. The effect of flotetuzumab on cell death and TNFα release is similar. Figure 60C. The effect of flotetuzumab on cell killing and IL-2 release is similar. FIG. 60D. The effect of AMG330 on cell killing and IFNγ release is similar. Figure 60E. The effect of AMG330 on cell killing and TNFα release is similar. Figure 60F. The effect of AMG330 on cell killing and IL-2 release is similar. Figure 60G. The effect of DARPin® Protein #56 on cell killing and IFNγ release. Figure 60H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 601. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 60J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 60K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 60L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 60F]Self-killing versus cytokine (IFN-γ, TNFα, IL-2) release of patient-derived AML cells after 5 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 60A. The effect of flotetuzumab on cell death and IFNγ release is similar. Figure 60B. The effect of flotetuzumab on cell death and TNFα release is similar. Figure 60C. The effect of flotetuzumab on cell killing and IL-2 release is similar. FIG. 60D. The effect of AMG330 on cell killing and IFNγ release is similar. Figure 60E. The effect of AMG330 on cell killing and TNFα release is similar. Figure 60F. The effect of AMG330 on cell killing and IL-2 release is similar. Figure 60G. The effect of DARPin® Protein #56 on cell killing and IFNγ release. Figure 60H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 601. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 60J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 60K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 60L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 60G]Self-killing versus cytokine (IFN-γ, TNFα, IL-2) release of patient-derived AML cells after 5 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 60A. The effect of flotetuzumab on cell death and IFNγ release is similar. Figure 60B. The effect of flotetuzumab on cell death and TNFα release is similar. Figure 60C. The effect of flotetuzumab on cell killing and IL-2 release is similar. FIG. 60D. The effect of AMG330 on cell killing and IFNγ release is similar. Figure 60E. The effect of AMG330 on cell killing and TNFα release is similar. Figure 60F. The effect of AMG330 on cell killing and IL-2 release is similar. Figure 60G. The effect of DARPin® Protein #56 on cell killing and IFNγ release. Figure 60H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 601. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 60J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 60K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 60L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 60H]Self-killing versus cytokine (IFN-γ, TNFα, IL-2) release of patient-derived AML cells after 5 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 60A. The effect of flotetuzumab on cell death and IFNγ release is similar. Figure 60B. The effect of flotetuzumab on cell death and TNFα release is similar. Figure 60C. The effect of flotetuzumab on cell killing and IL-2 release is similar. FIG. 60D. The effect of AMG330 on cell killing and IFNγ release is similar. Figure 60E. The effect of AMG330 on cell killing and TNFα release is similar. Figure 60F. The effect of AMG330 on cell killing and IL-2 release is similar. Figure 60G. The effect of DARPin® Protein #56 on cell killing and IFNγ release. Figure 60H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 601. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 60J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 60K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 60L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 60I]Self-killing versus cytokine (IFN-γ, TNFα, IL-2) release of patient-derived AML cells after 5 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 60A. The effect of flotetuzumab on cell death and IFNγ release is similar. Figure 60B. The effect of flotetuzumab on cell death and TNFα release is similar. Figure 60C. The effect of flotetuzumab on cell killing and IL-2 release is similar. FIG. 60D. The effect of AMG330 on cell killing and IFNγ release is similar. Figure 60E. The effect of AMG330 on cell killing and TNFα release is similar. Figure 60F. The effect of AMG330 on cell killing and IL-2 release is similar. Figure 60G. The effect of DARPin® Protein #56 on cell killing and IFNγ release. Figure 60H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 601. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 60J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 60K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 60L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 60J]Self-killing versus cytokine (IFN-γ, TNFα, IL-2) release of patient-derived AML cells after 5 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 60A. The effect of flotetuzumab on cell death and IFNγ release is similar. Figure 60B. The effect of flotetuzumab on cell death and TNFα release is similar. Figure 60C. The effect of flotetuzumab on cell killing and IL-2 release is similar. FIG. 60D. The effect of AMG330 on cell killing and IFNγ release is similar. Figure 60E. The effect of AMG330 on cell killing and TNFα release is similar. Figure 60F. The effect of AMG330 on cell killing and IL-2 release is similar. Figure 60G. The effect of DARPin® Protein #56 on cell killing and IFNγ release. Figure 60H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 601. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 60J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 60K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 60L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 60K]Self-killing versus cytokine (IFN-γ, TNFα, IL-2) release of patient-derived AML cells after 5 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 60A. The effect of flotetuzumab on cell death and IFNγ release is similar. Figure 60B. The effect of flotetuzumab on cell death and TNFα release is similar. Figure 60C. The effect of flotetuzumab on cell killing and IL-2 release is similar. FIG. 60D. The effect of AMG330 on cell killing and IFNγ release is similar. Figure 60E. The effect of AMG330 on cell killing and TNFα release is similar. Figure 60F. The effect of AMG330 on cell killing and IL-2 release is similar. Figure 60G. The effect of DARPin® Protein #56 on cell killing and IFNγ release. Figure 60H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 601. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 60J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 60K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 60L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 60L)]Self-killing versus cytokine (IFN-γ, TNFα, IL-2) release of patient-derived AML cells after 5 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 60A. The effect of flotetuzumab on cell death and IFNγ release is similar. Figure 60B. The effect of flotetuzumab on cell death and TNFα release is similar. Figure 60C. The effect of flotetuzumab on cell killing and IL-2 release is similar. FIG. 60D. The effect of AMG330 on cell killing and IFNγ release is similar. Figure 60E. The effect of AMG330 on cell killing and TNFα release is similar. Figure 60F. The effect of AMG330 on cell killing and IL-2 release is similar. Figure 60G. The effect of DARPin® Protein #56 on cell killing and IFNγ release. Figure 60H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 601. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 60J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 60K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 60L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 61A]Autologous killing (E:T ratio 4:1) of patient-derived AML cells in the presence of human pan-T cells versus cytokine (IFN-γ, TNFα, IL-2) release after 2 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 61A. Effect of flotetuzumab analogs on cell killing and IFNγ release. Figure 61B. Effect of flotetuzumab analogs on cell killing and TNFα release. Figure 61C. Effect of flotetuzumab analogs on cell killing and IL-2 release. Figure 61D. Effect of AMG330 analogs on cell killing and IFNγ release. Figure 61E. Effect of AMG330 analogs on cell killing and TNFα release. Figure 61F. Effect of AMG330 analogs on cell killing and IL-2 release. Figure 61G. Effect of DARPin® protein #56 on cell killing and IFNγ release. Figure 61H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 61I. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 61J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 61K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 61L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 61B]Autologous killing (E:T ratio 4:1) of patient-derived AML cells in the presence of human pan-T cells versus cytokine (IFN-γ, TNFα, IL-2) release after 2 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 61A. Effect of flotetuzumab analogs on cell killing and IFNγ release. Figure 61B. Effect of flotetuzumab analogs on cell killing and TNFα release. Figure 61C. Effect of flotetuzumab analogs on cell killing and IL-2 release. Figure 61D. Effect of AMG330 analogs on cell killing and IFNγ release. Figure 61E. Effect of AMG330 analogs on cell killing and TNFα release. Figure 61F. Effect of AMG330 analogs on cell killing and IL-2 release. Figure 61G. Effect of DARPin® protein #56 on cell killing and IFNγ release. Figure 61H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 61I. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 61J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 61K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 61L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 61C]Autologous killing (E:T ratio 4:1) of patient-derived AML cells in the presence of human pan-T cells versus cytokine (IFN-γ, TNFα, IL-2) release after 2 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 61A. Effect of flotetuzumab analogs on cell killing and IFNγ release. Figure 61B. Effect of flotetuzumab analogs on cell killing and TNFα release. Figure 61C. Effect of flotetuzumab analogs on cell killing and IL-2 release. Figure 61D. Effect of AMG330 analogs on cell killing and IFNγ release. Figure 61E. Effect of AMG330 analogs on cell killing and TNFα release. Figure 61F. Effect of AMG330 analogs on cell killing and IL-2 release. Figure 61G. Effect of DARPin® protein #56 on cell killing and IFNγ release. Figure 61H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 61I. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 61J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 61K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 61L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 61D]Autologous killing (E:T ratio 4:1) of patient-derived AML cells in the presence of human pan-T cells versus cytokine (IFN-γ, TNFα, IL-2) release after 2 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 61A. Effect of flotetuzumab analogs on cell killing and IFNγ release. Figure 61B. Effect of flotetuzumab analogs on cell killing and TNFα release. Figure 61C. Effect of flotetuzumab analogs on cell killing and IL-2 release. Figure 61D. Effect of AMG330 analogs on cell killing and IFNγ release. Figure 61E. Effect of AMG330 analogs on cell killing and TNFα release. Figure 61F. Effect of AMG330 analogs on cell killing and IL-2 release. Figure 61G. Effect of DARPin® protein #56 on cell killing and IFNγ release. Figure 61H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 61I. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 61J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 61K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 61L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 61E]Autologous killing (E:T ratio 4:1) of patient-derived AML cells in the presence of human pan-T cells versus cytokine (IFN-γ, TNFα, IL-2) release after 2 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 61A. Effect of flotetuzumab analogs on cell killing and IFNγ release. Figure 61B. Effect of flotetuzumab analogs on cell killing and TNFα release. Figure 61C. Effect of flotetuzumab analogs on cell killing and IL-2 release. Figure 61D. Effect of AMG330 analogs on cell killing and IFNγ release. Figure 61E. Effect of AMG330 analogs on cell killing and TNFα release. Figure 61F. Effect of AMG330 analogs on cell killing and IL-2 release. Figure 61G. Effect of DARPin® protein #56 on cell killing and IFNγ release. Figure 61H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 61I. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 61J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 61K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 61L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 61F]Autologous killing (E:T ratio 4:1) of patient-derived AML cells in the presence of human pan-T cells versus cytokine (IFN-γ, TNFα, IL-2) release after 2 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 61A. Effect of flotetuzumab analogs on cell killing and IFNγ release. Figure 61B. Effect of flotetuzumab analogs on cell killing and TNFα release. Figure 61C. Effect of flotetuzumab analogs on cell killing and IL-2 release. Figure 61D. Effect of AMG330 analogs on cell killing and IFNγ release. Figure 61E. Effect of AMG330 analogs on cell killing and TNFα release. Figure 61F. Effect of AMG330 analogs on cell killing and IL-2 release. Figure 61G. Effect of DARPin® protein #56 on cell killing and IFNγ release. Figure 61H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 61I. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 61J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 61K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 61L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 61G]Autologous killing (E:T ratio 4:1) of patient-derived AML cells in the presence of human pan-T cells versus cytokine (IFN-γ, TNFα, IL-2) release after 2 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 61A. Effect of flotetuzumab analogs on cell killing and IFNγ release. Figure 61B. Effect of flotetuzumab analogs on cell killing and TNFα release. Figure 61C. Effect of flotetuzumab analogs on cell killing and IL-2 release. Figure 61D. Effect of AMG330 analogs on cell killing and IFNγ release. Figure 61E. Effect of AMG330 analogs on cell killing and TNFα release. Figure 61F. Effect of AMG330 analogs on cell killing and IL-2 release. Figure 61G. Effect of DARPin® protein #56 on cell killing and IFNγ release. Figure 61H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 61I. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 61J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 61K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 61L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 61H]Autologous killing (E:T ratio 4:1) of patient-derived AML cells in the presence of human pan-T cells versus cytokine (IFN-γ, TNFα, IL-2) release after 2 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 61A. Effect of flotetuzumab analogs on cell killing and IFNγ release. Figure 61B. Effect of flotetuzumab analogs on cell killing and TNFα release. Figure 61C. Effect of flotetuzumab analogs on cell killing and IL-2 release. Figure 61D. Effect of AMG330 analogs on cell killing and IFNγ release. Figure 61E. Effect of AMG330 analogs on cell killing and TNFα release. Figure 61F. Effect of AMG330 analogs on cell killing and IL-2 release. Figure 61G. Effect of DARPin® protein #56 on cell killing and IFNγ release. Figure 61H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 61I. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 61J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 61K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 61L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 61I]Autologous killing (E:T ratio 4:1) of patient-derived AML cells in the presence of human pan-T cells versus cytokine (IFN-γ, TNFα, IL-2) release after 2 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 61A. Effect of flotetuzumab analogs on cell killing and IFNγ release. Figure 61B. Effect of flotetuzumab analogs on cell killing and TNFα release. Figure 61C. Effect of flotetuzumab analogs on cell killing and IL-2 release. Figure 61D. Effect of AMG330 analogs on cell killing and IFNγ release. Figure 61E. Effect of AMG330 analogs on cell killing and TNFα release. Figure 61F. Effect of AMG330 analogs on cell killing and IL-2 release. Figure 61G. Effect of DARPin® protein #56 on cell killing and IFNγ release. Figure 61H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 61I. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 61J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 61K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 61L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 61J]Autologous killing (E:T ratio 4:1) of patient-derived AML cells in the presence of human pan-T cells versus cytokine (IFN-γ, TNFα, IL-2) release after 2 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 61A. Effect of flotetuzumab analogs on cell killing and IFNγ release. Figure 61B. Effect of flotetuzumab analogs on cell killing and TNFα release. Figure 61C. Effect of flotetuzumab analogs on cell killing and IL-2 release. Figure 61D. Effect of AMG330 analogs on cell killing and IFNγ release. Figure 61E. Effect of AMG330 analogs on cell killing and TNFα release. Figure 61F. Effect of AMG330 analogs on cell killing and IL-2 release. Figure 61G. Effect of DARPin® protein #56 on cell killing and IFNγ release. Figure 61H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 61I. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 61J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 61K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 61L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 61K]Autologous killing (E:T ratio 4:1) of patient-derived AML cells in the presence of human pan-T cells versus cytokine (IFN-γ, TNFα, IL-2) release after 2 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 61A. Effect of flotetuzumab analogs on cell killing and IFNγ release. Figure 61B. Effect of flotetuzumab analogs on cell killing and TNFα release. Figure 61C. Effect of flotetuzumab analogs on cell killing and IL-2 release. Figure 61D. Effect of AMG330 analogs on cell killing and IFNγ release. Figure 61E. Effect of AMG330 analogs on cell killing and TNFα release. Figure 61F. Effect of AMG330 analogs on cell killing and IL-2 release. Figure 61G. Effect of DARPin® protein #56 on cell killing and IFNγ release. Figure 61H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 61I. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 61J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 61K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 61L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 61L]Autologous killing (E:T ratio 4:1) of patient-derived AML cells in the presence of human pan-T cells versus cytokine (IFN-γ, TNFα, IL-2) release after 2 days of incubation. Cell killing is shown as % viable cells by filled squares / curve and as cytokine concentration by filled circles / curve. Figure 61A. Effect of flotetuzumab analogs on cell killing and IFNγ release. Figure 61B. Effect of flotetuzumab analogs on cell killing and TNFα release. Figure 61C. Effect of flotetuzumab analogs on cell killing and IL-2 release. Figure 61D. Effect of AMG330 analogs on cell killing and IFNγ release. Figure 61E. Effect of AMG330 analogs on cell killing and TNFα release. Figure 61F. Effect of AMG330 analogs on cell killing and IL-2 release. Figure 61G. Effect of DARPin® protein #56 on cell killing and IFNγ release. Figure 61H. Effect of DARPin® protein #56 on cell killing and TNFα release. Figure 61I. Effect of DARPin® protein #56 on cell killing and IL-2 release. Figure 61J. Effect of DARPin® protein #57 on cell killing and IFNγ release. Figure 61K. Effect of DARPin® protein #57 on cell killing and TNFα release. Figure 61L. Effect of DARPin® protein #57 on cell killing and IL-2 release. [Figure 62A] Cytokine release in whole blood from two healthy donors spiked with Molm13 cells. Figure 62A. IFN release in blood of donor 1; Figure 62B. IFN release in blood of donor 2; Figure 62C. IL-2 release in blood of donor 1; Figure 62D. IL-2 release in blood of donor 2; Figure 62E. IL-6 release in blood of donor 1; Figure 62F. IL-6 release in blood of donor 2; Figure 62G. TNFα release in blood of donor 1; Figure 62H. TNFα release in blood of donor 2. [Figure 62B]Cytokine release in whole blood from two healthy donors spiked with Molm13 cells. Figure 62A. IFN release in blood of donor 1; Figure 62B. IFN release in blood of donor 2; Figure 62C. IL-2 release in blood of donor 1; Figure 62D. IL-2 release in blood of donor 2; Figure 62E. IL-6 release in blood of donor 1; Figure 62F. IL-6 release in blood of donor 2; Figure 62G. TNFα release in blood of donor 1; Figure 62H. TNFα release in blood of donor 2. [Figure 62C] Cytokine release in whole blood from two healthy donors spiked with Molm13 cells. Figure 62A. IFN release in blood of donor 1; Figure 62B. IFN release in blood of donor 2; Figure 62C. IL-2 release in blood of donor 1; Figure 62D. IL-2 release in blood of donor 2; Figure 62E. IL-6 release in blood of donor 1; Figure 62F. IL-6 release in blood of donor 2; Figure 62G. TNFα release in blood of donor 1; Figure 62H. TNFα release in blood of donor 2. [Figure 62D] Cytokine release in whole blood from two healthy donors spiked with Molm13 cells. Figure 62A. IFN release in blood of donor 1; Figure 62B. IFN release in blood of donor 2; Figure 62C. IL-2 release in blood of donor 1; Figure 62D. IL-2 release in blood of donor 2; Figure 62E. IL-6 release in blood of donor 1; Figure 62F. IL-6 release in blood of donor 2; Figure 62G. TNFα release in blood of donor 1; Figure 62H. TNFα release in blood of donor 2. [Figure 62E] Cytokine release in whole blood from two healthy donors spiked with Molm13 cells. Figure 62A. IFN release in blood of donor 1; Figure 62B. IFN release in blood of donor 2; Figure 62C. IL-2 release in blood of donor 1; Figure 62D. IL-2 release in blood of donor 2; Figure 62E. IL-6 release in blood of donor 1; Figure 62F. IL-6 release in blood of donor 2; Figure 62G. TNFα release in blood of donor 1; Figure 62H. TNFα release in blood of donor 2. [Figure 62F]Cytokine release in whole blood from two healthy donors spiked with Molm13 cells. Figure 62A. IFN release in blood of donor 1; Figure 62B. IFN release in blood of donor 2; Figure 62C. IL-2 release in blood of donor 1; Figure 62D. IL-2 release in blood of donor 2; Figure 62E. IL-6 release in blood of donor 1; Figure 62F. IL-6 release in blood of donor 2; Figure 62G. TNFα release in blood of donor 1; Figure 62H. TNFα release in blood of donor 2. [Figure 62G] Cytokine release in whole blood from two healthy donors spiked with Molm13 cells. Figure 62A. IFN release in blood of donor 1; Figure 62B. IFN release in blood of donor 2; Figure 62C. IL-2 release in blood of donor 1; Figure 62D. IL-2 release in blood of donor 2; Figure 62E. IL-6 release in blood of donor 1; Figure 62F. IL-6 release in blood of donor 2; Figure 62G. TNFα release in blood of donor 1; Figure 62H. TNFα release in blood of donor 2. [Figure 62H] Cytokine release in whole blood from two healthy donors spiked with Molm13 cells. Figure 62A. IFN release in blood of donor 1; Figure 62B. IFN release in blood of donor 2; Figure 62C. IL-2 release in blood of donor 1; Figure 62D. IL-2 release in blood of donor 2; Figure 62E. IL-6 release in blood of donor 1; Figure 62F. IL-6 release in blood of donor 2; Figure 62G. TNFα release in blood of donor 1; Figure 62H. TNFα release in blood of donor 2. [Figure 63]SPR traces of simultaneous binding of DARPin® Protein #56 to serum albumin, CD70, CD123, CD33, and CD3. (a) Binding of DARPin® Protein #56 to immobilized HSA. (b) Association of hCD70 to the HSA / DARPin® Protein #56 complex (* / ●) or PBST injection. (c) Binding of hCD123 to the HSA / DARPin® Protein #56 / hCD70 complex (*) or the HSA / DARPin® Protein #56 complex (■) or PBST injection. (d) Binding of hCD33 to the HSA / DARPin® Protein #56 / hCD70 / hCD123 complex (*) or the HSA / DARPin® Protein #56 complex (▼) or PBST injection. (e) Binding of scCD3 to the HSA / DARPin® Protein #56 / hCD70 / hCD123 / hCD33 complex (*) or the HSA / DARPin® Protein #56 complex (▲), or PBST injection followed by a 180 s dissociation phase. The injection scheme is described in Table 11. [Figure 64] Comparison of "measured" vs. "calculated" SPR traces of simultaneous binding of DARPin® protein #56 to all targets. "Measured" binding trace obtained from Figure 63(*). "Calculated" traces were generated by combining the individual single control injection SPR traces of DARPin® protein #56 binding to serum albumin, CD70, CD123, CD33, or CD3, respectively. [Figure 65] Tumor growth over time in mice (n=5 mice per donor / 2-6 hPBMC donors used depending on treatment) injected intraperitoneally with hPBMCs and xenografted subcutaneously with MOLM-13 cells and treated 8 days after tumor cell injection with PBS 1× (vehicle), 0.5 mg / kg DARPin® Protein #74, 0.5 mg / kg DARPin® Protein #75, 0.5 mg / kg DARPin® Protein #56, 0.5 mg / kg DARPin® Protein #57, 0.5 mg / kg AMG330 analog, and 0.5 mg / kg flotetuzumab analog. [Figure 66] Tumor growth over time in mice injected intraperitoneally with hPBMCs (n=5 mice per donor / 2-6 hPBMC donors used depending on treatment), xenografted subcutaneously with MOLM-13 cells, and treated with PBS 1X (vehicle), 8 days after tumor cell injection, 2 mg / kg DARPin® Protein #56, 0.5 mg / kg DARPin® Protein #56, 0.2 mg / kg DARPin® Protein #56, and 0.02 mg / kg DARPin® Protein #56. [Figure 67A] Frequency of CD45-expressing human immune cells (i.e., CD45+) (as measured by T cell activation markers CD25 and CD69) (Figure 67A) and activated human T cells (CD4-expressing, Figure 67B) or CD8-expressing, Figure 67C) in dissociated MOLM-13 tumors after treatment. Activated T cells were gated as viable CD4+ / CD25+ / CD69+ cells (Figure 67B) and viable CD8+ / CD25+ / CD69+ cells (Figure 67C). [Figure 67B] Frequency of CD45-expressing human immune cells (i.e., CD45+) (as measured by T cell activation markers CD25 and CD69) (Figure 67A) and activated human T cells (CD4-expressing, Figure 67B) or CD8-expressing, Figure 67C) in dissociated MOLM-13 tumors after treatment. Activated T cells were gated as viable CD4+ / CD25+ / CD69+ cells (Figure 67B) and viable CD8+ / CD25+ / CD69+ cells (Figure 67C). [Figure 67C] Frequency of CD45-expressing human immune cells (i.e., CD45+) (as measured by T cell activation markers CD25 and CD69) (Figure 67A) and activated human T cells (CD4-expressing, Figure 67B) or CD8-expressing, Figure 67C) in dissociated MOLM-13 tumors after treatment. Activated T cells were gated as viable CD4+ / CD25+ / CD69+ cells (Figure 67B) and viable CD8+ / CD25+ / CD69+ cells (Figure 67C). [Figure 68A]Cytokine and chemokine release in mouse serum after treatment. Figure 68A. Levels of IFNγ, Figure 68B. Levels of IL-6, Figure 68C. Levels of IL-2, and Figure 68D. Levels of TNFα. [Figure 68B] Cytokine and chemokine release in mouse serum after treatment. Figure 68A. Levels of IFNγ, Figure 68B. Levels of IL-6, Figure 68C. Levels of IL-2, and Figure 68D. Levels of TNFα. [Figure 68C] Cytokine and chemokine release in mouse serum after treatment. Figure 68A. Levels of IFNγ, Figure 68B. Levels of IL-6, Figure 68C. Levels of IL-2, and Figure 68D. Levels of TNFα. [Figure 68D] Cytokine and chemokine release in mouse serum after treatment. Figure 68A. Levels of IFNγ, Figure 68B. Levels of IL-6, Figure 68C. Levels of IL-2, and Figure 68D. Levels of TNFα. [Figure 69A] Cytokine and chemokine release in the mouse tumor environment after treatment. Figure 69A. IFNγ levels; Figure 69B. IL-6 levels. [Figure 69B] Cytokine and chemokine release in the mouse tumor environment after treatment. Figure 69A. IFNγ levels; Figure 69B. IL-6 levels. DETAILED DESCRIPTION OF THE INVENTION

[0057] Disclosed herein is 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.

[0058] More specifically, recombinant proteins containing designed ankyrin repeat domains with binding specificity for different targets, such as CD3, CD33, CD123, and CD70, are disclosed herein. Nucleic acids encoding the binding proteins, pharmaceutical compositions containing the binding proteins or nucleic acids, and methods for using the binding proteins, nucleic acids, or pharmaceutical compositions are also disclosed. Designed ankyrin repeat protein libraries (WO 2002 / 020565; Binz et al., Nat. Biotechnol. 22:575-582, 2004; Stumpp et al., Drug Discov. Today 13:695-701 (2008)) can be used to select target-specific designed ankyrin repeat domains that bind to their targets with high affinity. Such target-specific designed ankyrin repeat domains can then be used as valuable components of recombinant binding proteins for treating diseases. Designed ankyrin repeat proteins are a class of binding molecules that have the potential to overcome the limitations of monoclonal antibodies and thus enable novel therapeutic approaches. Such ankyrin repeat proteins may contain a single designed ankyrin repeat domain or a combination of two or more designed ankyrin repeat domains with the same or different target specificities (Stumpp et al., Drug Discov. Today, Vol. 13, pp. 695-701, 2008; U.S. Patent No. 9,458,211). Ankyrin repeat proteins containing only a single designed ankyrin repeat domain are small proteins (14 kDa) that can be selected to bind to a given target protein with high affinity and specificity. These features, along with the possibility of combining two or more designed ankyrin repeat domains in one protein, make designed ankyrin repeat proteins ideal agonist, antagonist, and / or inhibitor candidates. Furthermore, such ankyrin repeat proteins can be engineered to carry various effector functions, such as cytotoxic agents or half-life extenders, enabling entirely new drug formats. Taken together, the designed ankyrin repeat proteins are examples of the next generation of protein therapeutics with the potential to exceed existing antibody drugs.

[0059] DARPin® is a trademark owned by Molecular Partners AG, Switzerland.

[0060] Molecules expressed on the surface of tumor cells, such as CD33, CD123, and CD70, are potential targets for anticancer therapy, especially when they are expressed on tumor cells but not or much less on healthy cells. As an example, CD33, CD123, and CD70 are expressed on the surface of AML blasts and leukemia stem cells, but are not or barely expressed at the same time on the surface of healthy cells, including hematopoietic stem cells.

[0061] In one aspect, the present 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.

[0062] In one aspect, the present 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 the recombinant protein is capable of simultaneously binding to the protein expressed on the surface of an immune cell and the two different tumor-associated antigens.

[0063] In one aspect, the present 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.

[0064] In one aspect, the present 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 the recombinant protein is capable of simultaneously binding to the protein expressed on the surface of an immune cell and the three different tumor-associated antigens.

[0065] In one embodiment, the tumor-associated antigens are co-expressed in tumor cells. In one embodiment, the tumor cells are tumor cells derived from a liquid tumor. In another embodiment, the tumor cells are liquid tumor cells derived from leukemia. In one embodiment, the leukemia is acute myeloid leukemia (AML).

[0066] In one aspect, the present invention provides a recombinant protein, which binds to a surface that displays a tumor-associated antigen with a lower dissociation constant (K) when compared to a recombinant protein comprising only one of the binding agents that specifically binds to the tumor-associated antigen. D ) can be used to join them.

[0067] In one aspect, the invention provides a recombinant protein, wherein the surface presenting the tumor-associated antigen is the surface of the tumor cell.

[0068] In one aspect, the present invention provides a recombinant protein, wherein the lower dissociation constant (K D ) is at least about 2-fold, at least about 4-fold, at least about 10-fold, at least about 20-fold, at least about 40-fold, or at least about 100-fold smaller than the corresponding dissociation constant of the recombinant protein comprising only one of the binding agents that specifically binds to a tumor-associated antigen.

[0069] In one aspect, the present invention provides a recombinant protein, wherein the immune cell is a T cell.

[0070] In one aspect, the invention provides a recombinant protein wherein said T cells are CD8+ cytotoxic T cells.

[0071] In one aspect, the invention provides a recombinant protein, wherein the protein expressed on the surface of said immune cell is a protein that is part of said T cell receptor complex.

[0072] In one aspect, the present invention provides a recombinant protein, wherein the protein that is part of the T cell receptor complex is CD3.

[0073] In one aspect, the present invention provides a recombinant protein, wherein said first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or an engineered repeat domain.

[0074] In one aspect, the present invention provides a recombinant protein, wherein said first binding agent is an engineered ankyrin repeat domain that has binding specificity for said protein expressed on the surface of an immune cell.

[0075] In one aspect, the present invention provides a recombinant protein, wherein said first binding agent comprises a designed ankyrin repeat domain that has binding specificity for CD3.

[0076] In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence that is at least 85% identical, 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 one of the amino acid sequences of SEQ ID NOs: 1 to 5, preferably SEQ ID NO: 2 or 3, in which A in the penultimate position of SEQ ID NOs: 1 to 4 is optionally replaced by L and / or A in the last position of SEQ ID NOs: 1 to 4 is optionally replaced by N, or L in the penultimate position of SEQ ID NO: 5 is optionally replaced by A and / or N in the last position of SEQ ID NO: 5 is optionally replaced by A.

[0077] In one aspect, the invention provides a recombinant protein, wherein said ankyrin repeat domain with binding specificity for CD3 comprises the amino acid sequence of SEQ ID NO: 1 to 5, preferably SEQ ID NO: 2 or 3, wherein A in the penultimate position of SEQ ID NO: 1 to 4 is optionally replaced by L and / or A in the last position of SEQ ID NO: 1 to 4 is optionally replaced by N, or L in the penultimate position of SEQ ID NO: 5 is optionally replaced by A and / or N in the last position of SEQ ID NO: 5 is optionally replaced by A.

[0078] In one aspect, the invention provides a recombinant protein wherein said ankyrin repeat domain with binding specificity for CD3 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 2, wherein A in the penultimate position of SEQ ID NO: 2 is optionally replaced by L and / or A in the last position of SEQ ID NO: 2 is optionally replaced by N.

[0079] In one aspect, the invention provides a recombinant protein, wherein said ankyrin repeat domain with binding specificity for CD3 comprises the amino acid sequence of SEQ ID NO: 2, wherein A in the penultimate position of SEQ ID NO: 2 is optionally replaced by L and / or A in the last position of SEQ ID NO: 2 is optionally replaced by N.

[0080] In one aspect, the invention provides a recombinant protein wherein said ankyrin repeat domain with binding specificity for CD3 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 3, wherein A in the penultimate position of SEQ ID NO: 3 is optionally replaced by L and / or A in the last position of SEQ ID NO: 3 is optionally replaced by N.

[0081] In one aspect, the invention provides a recombinant protein, wherein said ankyrin repeat domain with binding specificity for CD3 comprises the amino acid sequence of SEQ ID NO: 3, wherein A in the penultimate position of SEQ ID NO: 3 is optionally replaced by L and / or A in the last position of SEQ ID NO: 3 is optionally replaced by N.

[0082] In one aspect, the present 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 (TAA1), (ii) a third binding agent that specifically binds to a second tumor-associated antigen (TAA2), and (iii) a fourth binding agent that specifically binds to a third tumor-associated antigen (TAA3).

[0083] In one aspect, the present invention provides a recombinant protein, wherein the binding agents that specifically bind to tumor-associated antigens are selected from the group consisting of: (i) a second binding agent that specifically binds to a first tumor-associated antigen (TAA1), (ii) a third binding agent that specifically binds to a second tumor-associated antigen (TAA2), and (iii) a fourth binding agent that specifically binds to a third tumor-associated antigen (TAA3), and the selected binding agents are capable of simultaneously binding to each tumor-associated antigen target.

[0084] In one aspect, the present invention provides a recombinant protein, wherein said second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, hi one aspect, the present invention provides a recombinant protein, wherein said second binding agent is a designed ankyrin repeat domain having binding specificity for said TAA1.

[0085] In one aspect, the present invention provides a recombinant protein, wherein said TAA1 is CD33.

[0086] In one aspect, the present invention provides a recombinant protein, wherein said second binding agent comprises a designed ankyrin repeat domain that has binding specificity for CD33.

[0087] In one aspect, the invention provides a recombinant protein, wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence that is at least 85% identical, 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 one of the amino acid sequences of SEQ ID NOs: 15, 67-70, and 111-112, wherein A in the penultimate position of SEQ ID NOs: 15, 67-70, and 111-112 is optionally replaced by L, and / or A in the last position of SEQ ID NOs: 15, 67-70, and 111-112 is optionally replaced by N.

[0088] In one aspect, the invention provides a recombinant protein, wherein the ankyrin repeat domain with binding specificity for CD33 comprises any one of the amino acid sequences of SEQ ID NOs: 15, 67-70 and 111-112, wherein A at the penultimate position of SEQ ID NOs: 15, 67-70 and 111-112 is optionally replaced by L and / or A at the last position of SEQ ID NOs: 15, 67-70 and 111-112 is optionally replaced by N.

[0089] In one aspect, the invention provides a recombinant protein wherein said ankyrin repeat domain with binding specificity for CD33 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 15, wherein A in the penultimate position of SEQ ID NO: 15 is optionally replaced by L and / or A in the last position of SEQ ID NO: 15 is optionally replaced by N.

[0090] In one aspect, the invention provides a recombinant protein, wherein said ankyrin repeat domain with binding specificity for CD33 comprises the amino acid sequence of SEQ ID NO: 15, wherein A in the penultimate position of SEQ ID NO: 15 is optionally replaced by L and / or A in the last position of SEQ ID NO: 15 is optionally replaced by N.

[0091] In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence that is at least 85% identical, e.g., at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical to the amino acid sequence of SEQ ID NO: 67. In one aspect, the invention provides a recombinant protein wherein A in the penultimate position of SEQ ID NO: 67 is optionally replaced by L and / or A in the last position of SEQ ID NO: 67 is optionally replaced by N.

[0092] In one aspect, the invention provides a recombinant protein, wherein said ankyrin repeat domain with binding specificity for CD33 comprises the amino acid sequence of SEQ ID NO: 67, wherein A in the penultimate position of SEQ ID NO: 67 is optionally replaced by L and / or A in the last position of SEQ ID NO: 67 is optionally replaced by N.

[0093] In one aspect, the invention provides a recombinant protein wherein said ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 68, wherein A in the penultimate position of SEQ ID NO: 68 is optionally replaced by L and / or A in the last position of SEQ ID NO: 68 is optionally replaced by N.

[0094] In one aspect, the invention provides a recombinant protein, wherein said ankyrin repeat domain with binding specificity for CD33 comprises the amino acid sequence of SEQ ID NO: 68, wherein A in the penultimate position of SEQ ID NO: 68 is optionally replaced by L and / or A in the last position of SEQ ID NO: 68 is optionally replaced by N.

[0095] In one aspect, the invention provides a recombinant protein wherein said ankyrin repeat domain with binding specificity for CD33 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 69, wherein A in the penultimate position of SEQ ID NO: 69 is optionally replaced by L and / or A in the last position of SEQ ID NO: 69 is optionally replaced by N.

[0096] In one aspect, the invention provides a recombinant protein, wherein said ankyrin repeat domain with binding specificity for CD33 comprises the amino acid sequence of SEQ ID NO: 69, wherein A in the penultimate position of SEQ ID NO: 69 is optionally replaced by L and / or A in the last position of SEQ ID NO: 70 is optionally replaced by N.

[0097] In one aspect, the invention provides a recombinant protein wherein said ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 13, wherein A in the penultimate position of SEQ ID NO: 70 is optionally replaced by L and / or A in the last position of SEQ ID NO: 70 is optionally replaced by N.

[0098] In one aspect, the invention provides a recombinant protein, wherein said ankyrin repeat domain with binding specificity for CD33 comprises the amino acid sequence of SEQ ID NO: 70, wherein A in the penultimate position of SEQ ID NO: 70 is optionally replaced by L and / or A in the last position of SEQ ID NO: 70 is optionally replaced by N.

[0099] In one aspect, the invention provides a recombinant protein wherein the ankyrin repeat domain with binding specificity for CD33 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 111, wherein A in the penultimate position of SEQ ID NO: 111 is optionally replaced by L and / or A in the last position of SEQ ID NO: 111 is optionally replaced by N.

[0100] In one aspect, the invention provides a recombinant protein, wherein said ankyrin repeat domain with binding specificity for CD33 comprises the amino acid sequence of SEQ ID NO: 111, wherein A in the penultimate position of SEQ ID NO: 111 is optionally replaced by L and / or A in the last position of SEQ ID NO: 111 is optionally replaced by N.

[0101] In one aspect, the invention provides a recombinant protein wherein said ankyrin repeat domain with binding specificity for CD33 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 112, wherein A in the penultimate position of SEQ ID NO: 112 is optionally replaced by L and / or A in the last position of SEQ ID NO: 112 is optionally replaced by N.

[0102] In one aspect, the invention provides a recombinant protein, wherein said ankyrin repeat domain with binding specificity for CD33 comprises the amino acid sequence of SEQ ID NO: 112, wherein A in the penultimate position of SEQ ID NO: 112 is optionally replaced by L and / or A in the last position of SEQ ID NO: 112 is optionally replaced by N.

[0103] In one aspect, the present invention provides a recombinant protein wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and said second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain.

[0104] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is a designed ankyrin repeat domain that has binding specificity for said protein expressed on the surface of an immune cell, and said second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

[0105] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is an engineered ankyrin repeat domain with binding specificity for CD3 and said second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or an engineered repeat domain.

[0106] In one aspect, the present invention provides a recombinant protein in which the ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 1 to 5, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical, wherein the A at the second last position of SEQ ID NOs: 1 to 4 is optionally substituted by L, and / or the A at the last position of SEQ ID NOs: 1 to 4 is optionally substituted by N, or the L at the second last position of SEQ ID NO: 5 is optionally substituted by A, and / or the N at the last position of SEQ ID NO: 5 is optionally substituted by A, and the second binder is an antibody, antibody mimetic, scaffold protein, repetitive protein, or designed repeat domain.

[0107] In one aspect, the present invention provides a recombinant protein in which the ankyrin repeat domain having binding specificity for CD3 comprises the amino acid sequences of SEQ ID NOs: 1 to 5, wherein the A at the second last position of SEQ ID NOs: 1 to 4 is optionally substituted by L, and / or the A at the last position of SEQ ID NOs: 1 to 4 is optionally substituted by N, or the L at the second last position of SEQ ID NO: 5 is optionally substituted by A, and / or the N at the last position of SEQ ID NO: 5 is optionally substituted by A, and the second binder is an antibody, antibody mimetic, scaffold protein, repetitive protein, or designed repeat domain.

[0108] In one aspect, the present invention provides a recombinant protein in which the first binder is an antibody, antibody mimetic, scaffold protein, repetitive protein, or designed repeat domain, and the second binder is a designed ankyrin repeat domain having binding specificity for CD33.

[0109] In one aspect, the invention provides a recombinant protein wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein the ankyrin repeat domain with binding specificity for CD33 comprises an amino acid sequence that is at least 85% identical, 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: 15, 67-70, and 111-112.

[0110] In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and the ankyrin repeat domain having binding specificity for CD33 comprises any one of the amino acid sequences of SEQ ID NOs: 15, 67-70, and 111-112.

[0111] In one aspect, the present invention provides a recombinant protein wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and said third binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain.

[0112] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is a designed ankyrin repeat domain that has binding specificity for said protein expressed on the surface of an immune cell, and said third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

[0113] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is an engineered ankyrin repeat domain with binding specificity for CD3 and said third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or an engineered repeat domain.

[0114] In one aspect, the present invention provides a recombinant protein in which the ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence that is at least 85% identical to any one of the amino acid sequences of SEQ ID NOs: 1 to 5, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% identical, the A at the second last position of SEQ ID NOs: 1 to 4 is optionally replaced by L, and / or the A at the last position of SEQ ID NOs: 1 to 4 is optionally replaced by N, or the L at the second last position of SEQ ID NO: 5 is optionally replaced by A, and / or the N at the last position of SEQ ID NO: 5 is optionally replaced by A, and the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repetitive protein, or a designed repeat domain.

[0115] In one aspect, the present invention provides a recombinant protein in which the ankyrin repeat domain having binding specificity for CD3 comprises the amino acid sequences of SEQ ID NOs: 1 to 5, the A at the second last position of SEQ ID NOs: 1 to 4 is optionally replaced by L, and / or the A at the last position of SEQ ID NOs: 1 to 4 is optionally replaced by N, or the L at the second last position of SEQ ID NO: 5 is optionally replaced by A, and / or the N at the last position of SEQ ID NO: 5 is optionally replaced by A, and the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repetitive protein, or a designed repeat domain.

[0116] In one aspect, the present invention provides a recombinant protein in which the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repetitive protein, or a designed repeat domain, and the third binding agent is a designed ankyrin repeat domain having binding specificity for CD123.

[0117] In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein the ankyrin repeat domain with binding specificity for CD123 comprises an amino acid sequence that is at least 85% identical, 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 one of the amino acid sequences of SEQ ID NOs: 6, 65-66, and 102-106, wherein A at the penultimate position of SEQ ID NOs: 6, 65-66, and 102-106 is optionally replaced by L, and / or A at the last position of SEQ ID NOs: 6, 65-66, and 102-106 is optionally replaced by N.

[0118] In one aspect, the present invention provides a recombinant protein, wherein said first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or an engineered repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD123 comprises any one of the amino acid sequences of SEQ ID NOs: 6, 65-66, and 102-106, wherein A at the penultimate position of SEQ ID NOs: 6, 65-66, and 102-106 is optionally replaced by L and / or A at the last position of SEQ ID NOs: 6, 65-66, and 102-106 is optionally replaced by N.

[0119] In one aspect, the invention provides a recombinant protein wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD123 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 6, wherein A in the penultimate position of SEQ ID NO: 6 is optionally replaced by L and / or A in the last position of SEQ ID NO: 6 is optionally replaced by N.

[0120] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or an engineered repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD123 comprises the amino acid sequence of SEQ ID NO: 6, wherein A in the penultimate position of SEQ ID NO: 6 is optionally replaced by L and / or A in the last position of SEQ ID NO: 6 is optionally replaced by N.

[0121] In one aspect, the invention provides a recombinant protein wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD123 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 65, wherein A in the penultimate position of SEQ ID NO: 65 is optionally replaced by L and / or A in the last position of SEQ ID NO: 65 is optionally replaced by N.

[0122] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or an engineered repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD123 comprises the amino acid sequence of SEQ ID NO: 65, wherein A in the penultimate position of SEQ ID NO: 65 is optionally replaced by L and / or A in the last position of SEQ ID NO: 65 is optionally replaced by N.

[0123] In one aspect, the invention provides a recombinant protein wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD123 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 66, wherein A in the penultimate position of SEQ ID NO: 66 is optionally replaced by L and / or A in the last position of SEQ ID NO: 66 is optionally replaced by N.

[0124] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or an engineered repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD123 comprises the amino acid sequence of SEQ ID NO: 66, wherein A in the penultimate position of SEQ ID NO: 66 is optionally replaced by L and / or A in the last position of SEQ ID NO: 66 is optionally replaced by N.

[0125] In one aspect, the invention provides a recombinant protein wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD123 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 102, wherein A in the penultimate position of SEQ ID NO: 102 is optionally replaced by L and / or A in the last position of SEQ ID NO: 102 is optionally replaced by N.

[0126] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or an engineered repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD123 comprises the amino acid sequence of SEQ ID NO: 102, wherein A in the penultimate position of SEQ ID NO: 102 is optionally replaced by L and / or A in the last position of SEQ ID NO: 102 is optionally replaced by N.

[0127] In one aspect, the invention provides a recombinant protein wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD123 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 103, wherein A in the penultimate position of SEQ ID NO: 103 is optionally replaced by L and / or A in the last position of SEQ ID NO: 103 is optionally replaced by N.

[0128] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or an engineered repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD123 comprises the amino acid sequence of SEQ ID NO: 103, wherein A in the penultimate position of SEQ ID NO: 103 is optionally replaced by L and / or A in the last position of SEQ ID NO: 103 is optionally replaced by N.

[0129] In one aspect, the invention provides a recombinant protein wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD123 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 104, wherein A in the penultimate position of SEQ ID NO: 104 is optionally replaced by L and / or A in the last position of SEQ ID NO: 104 is optionally replaced by N.

[0130] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or an engineered repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD123 comprises the amino acid sequence of SEQ ID NO: 104, wherein A in the penultimate position of SEQ ID NO: 104 is optionally replaced by L and / or A in the last position of SEQ ID NO: 104 is optionally replaced by N.

[0131] In one aspect, the invention provides a recombinant protein wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD123 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 105, wherein A in the penultimate position of SEQ ID NO: 105 is optionally replaced by L and / or A in the last position of SEQ ID NO: 105 is optionally replaced by N.

[0132] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or an engineered repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD123 comprises the amino acid sequence of SEQ ID NO: 105, wherein A in the penultimate position of SEQ ID NO: 105 is optionally replaced by L and / or A in the last position of SEQ ID NO: 105 is optionally replaced by N.

[0133] In one aspect, the invention provides a recombinant protein wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD123 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 106, wherein A in the penultimate position of SEQ ID NO: 106 is optionally replaced by L and / or A in the last position of SEQ ID NO: 106 is optionally replaced by N.

[0134] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or an engineered repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD123 comprises the amino acid sequence of SEQ ID NO: 106, wherein A in the penultimate position of SEQ ID NO: 106 is optionally replaced by L and / or A in the last position of SEQ ID NO: 106 is optionally replaced by N.

[0135] In one aspect, the present invention provides a recombinant protein wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, the second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and the third binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain.

[0136] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is a designed ankyrin repeat domain having binding specificity for said protein expressed on the surface of an immune cell, said second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein or designed repeat domain, and said third binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein or designed repeat domain.

[0137] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is a designed ankyrin repeat domain with binding specificity for CD3, said second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and said third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain.

[0138] In one aspect, the invention provides an ankyrin repeat domain having binding specificity for CD3 comprising an amino acid sequence that is at least 85% identical, 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 one of the amino acid sequences of SEQ ID NOs: 1 to 5, and wherein A is optionally replaced by L and / or A in the last position of SEQ ID NO: 1-4 is optionally replaced by N, or L in the penultimate position of SEQ ID NO: 5 is optionally replaced by A and / or N in the last position of SEQ ID NO: 5 is optionally replaced by A; the second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain; and the third binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain.

[0139] In one aspect, the invention provides a recombinant protein, wherein said ankyrin repeat domain with binding specificity for CD3 comprises the amino acid sequence of SEQ ID NOs: 1 to 5, wherein A in the penultimate position of SEQ ID NOs: 1 to 4 is optionally substituted by L, and / or A in the last position of SEQ ID NO: 6 is optionally substituted by N, and / or L in the penultimate position of SEQ ID NO: 5 is optionally substituted by A, and / or N in the last position of SEQ ID NO: 5 is optionally substituted by A; said second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain; and said third binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain.

[0140] In one aspect, the invention provides a recombinant protein wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, said second binding agent is a designed ankyrin repeat domain with binding specificity for CD33, and said third binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain.

[0141] In one aspect, the invention provides a recombinant protein wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein the ankyrin repeat domain with binding specificity for CD33 comprises an amino acid sequence that is at least 85% identical, 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 one of the amino acid sequences of SEQ ID NOs: 15, 67-70, and 111-112; and the third binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain.

[0142] In one aspect, the present invention provides a recombinant protein, wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, wherein said ankyrin repeat domain with binding specificity for CD33 comprises any one of the amino acid sequences of SEQ ID NOs: 15, 67-70, and 111-112, and said third binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain.

[0143] In one aspect, the invention provides a recombinant protein wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and said ankyrin repeat domain with binding specificity for CD33 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 15; and said third binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain.

[0144] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, wherein said ankyrin repeat domain with binding specificity for CD33 comprises the amino acid sequence of SEQ ID NO: 15, and said third binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain.

[0145] In one aspect, the invention provides a recombinant protein wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD33 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 67; and said third binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain.

[0146] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, wherein said ankyrin repeat domain with binding specificity for CD33 comprises the amino acid sequence of SEQ ID NO: 67, and said third binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain.

[0147] In one aspect, the invention provides a recombinant protein wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD33 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 68; and said third binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain.

[0148] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, wherein said ankyrin repeat domain with binding specificity for CD33 comprises the amino acid sequence of SEQ ID NO: 68, and said third binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain.

[0149] In one aspect, the invention provides a recombinant protein wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and said ankyrin repeat domain with binding specificity for CD33 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 69; and said third binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain.

[0150] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, wherein said ankyrin repeat domain with binding specificity for CD33 comprises the amino acid sequence of SEQ ID NO: 69, and said third binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain.

[0151] In one aspect, the invention provides a recombinant protein wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD33 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 70; and said third binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain.

[0152] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, wherein said ankyrin repeat domain with binding specificity for CD33 comprises the amino acid sequence of SEQ ID NO: 70, and said third binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain.

[0153] In one aspect, the invention provides a recombinant protein wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD33 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 111; and said third binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain.

[0154] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, wherein said ankyrin repeat domain with binding specificity for CD33 comprises the amino acid sequence of SEQ ID NO: 111, and said third binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain.

[0155] In one aspect, the invention provides a recombinant protein wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and said ankyrin repeat domain with binding specificity for CD33 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 112; and said third binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain.

[0156] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, wherein said ankyrin repeat domain with binding specificity for CD33 comprises the amino acid sequence of SEQ ID NO: 112, and said third binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain.

[0157] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, said second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and said third binding agent is a designed ankyrin repeat domain with binding specificity for CD123.

[0158] In one aspect, the invention provides a recombinant protein wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein or designed repeat domain and the second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein or designed repeat domain, and wherein the ankyrin repeat domain with binding specificity for CD123 comprises an amino acid sequence that is at least 85% identical, 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 one of the amino acid sequences of SEQ ID NOs: 6, 65-66, and 102-106.

[0159] In one aspect, the present invention provides a recombinant protein wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and the second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and the ankyrin repeat domain with binding specificity for CD123 comprises any one of the amino acid sequences of SEQ ID NOs: 6, 65-66, and 102-106.

[0160] In one aspect, the invention provides a recombinant protein wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain and the second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein the ankyrin repeat domain with binding specificity for CD123 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 6.

[0161] In one aspect, the invention provides a recombinant protein wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and said second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD123 comprises the amino acid sequence of SEQ ID NO: 6.

[0162] In one aspect, the invention provides a recombinant protein wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain and the second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein the ankyrin repeat domain with binding specificity for CD123 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 65.

[0163] In one aspect, the invention provides a recombinant protein wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and said second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD123 comprises the amino acid sequence of SEQ ID NO: 65.

[0164] In one aspect, the invention provides a recombinant protein wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain and the second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein the ankyrin repeat domain with binding specificity for CD123 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 66.

[0165] In one aspect, the invention provides a recombinant protein wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and said second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD123 comprises the amino acid sequence of SEQ ID NO: 66.

[0166] In one aspect, the invention provides a recombinant protein wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain and the second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein the ankyrin repeat domain with binding specificity for CD123 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 102.

[0167] In one aspect, the invention provides a recombinant protein wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and said second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD123 comprises the amino acid sequence of SEQ ID NO: 102.

[0168] In one aspect, the invention provides a recombinant protein wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain and the second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein the ankyrin repeat domain with binding specificity for CD123 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 103.

[0169] In one aspect, the invention provides a recombinant protein wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and said second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD123 comprises the amino acid sequence of SEQ ID NO: 103.

[0170] In one aspect, the invention provides a recombinant protein wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain and the second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein the ankyrin repeat domain with binding specificity for CD123 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 104.

[0171] In one aspect, the invention provides a recombinant protein wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and said second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD123 comprises the amino acid sequence of SEQ ID NO: 104.

[0172] In one aspect, the invention provides a recombinant protein wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain and the second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein the ankyrin repeat domain with binding specificity for CD123 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 105.

[0173] In one aspect, the invention provides a recombinant protein wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and said second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD123 comprises the amino acid sequence of SEQ ID NO: 105.

[0174] In one aspect, the invention provides a recombinant protein wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain and the second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein the ankyrin repeat domain with binding specificity for CD123 comprises an amino acid sequence at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 106.

[0175] In one aspect, the invention provides a recombinant protein wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and said second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD123 comprises the amino acid sequence of SEQ ID NO: 106.

[0176] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, said second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and said fourth binding agent is a designed ankyrin repeat domain with binding specificity for CD70.

[0177] In one aspect, the invention provides a recombinant protein wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein or designed repeat domain and the second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein or designed repeat domain, and wherein the ankyrin repeat domain with binding specificity for CD70 comprises an amino acid sequence that is at least 85% identical, 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 one of the amino acid sequences of SEQ ID NOs: 64 and 107-110.

[0178] In one aspect, the invention provides a recombinant protein, wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and the second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and the ankyrin repeat domain having binding specificity for CD70 comprises any one of the amino acid sequences of SEQ ID NOs: 64 and 107-110.

[0179] In one aspect, the invention provides a recombinant protein wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain and the second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein the ankyrin repeat domain with binding specificity for CD70 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 64.

[0180] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and said second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD70 comprises the amino acid sequence of SEQ ID NO: 64.

[0181] In one aspect, the invention provides a recombinant protein wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain and the second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein the ankyrin repeat domain with binding specificity for CD70 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 107.

[0182] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and said second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD70 comprises the amino acid sequence of SEQ ID NO: 107.

[0183] In one aspect, the invention provides a recombinant protein wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain and the second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein the ankyrin repeat domain with binding specificity for CD70 comprises an amino acid sequence at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 108.

[0184] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and said second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD70 comprises the amino acid sequence of SEQ ID NO: 108.

[0185] In one aspect, the invention provides a recombinant protein wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain and the second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein the ankyrin repeat domain with binding specificity for CD70 comprises an amino acid sequence at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 109.

[0186] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and said second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD70 comprises the amino acid sequence of SEQ ID NO: 109.

[0187] In one aspect, the invention provides a recombinant protein wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain and the second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein the ankyrin repeat domain with binding specificity for CD70 comprises an amino acid sequence that is at least 85% identical, 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 the amino acid sequence of SEQ ID NO: 110.

[0188] In one aspect, the invention provides a recombinant protein, wherein said first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and said second binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein said ankyrin repeat domain with binding specificity for CD70 comprises the amino acid sequence of SEQ ID NO: 110.

[0189] In one aspect, the present invention provides a recombinant protein, wherein the first binding agent is an antibody, antibody mimetic, scaffold protein, repeat protein, or designed repeat domain, and wherein the ankyrin repeat domain with binding specificity for CD33 is at least 85% identical, 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%, to any one of the amino acid sequences of SEQ ID NOs: 15, 67-70, and 111-112. %, or at least about 99% identical to any one of the amino acid sequences of SEQ ID NOs: 6, 65-66, and 102-106, and the ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence that is at least 85% identical, for example, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least ...

Claims

1. (1) a first binding agent that specifically binds to a protein expressed on the surface of an immune cell, wherein the protein expressed on the surface of an immune cell is CD3; and (2) at least three binding agents that specifically bind to tumor-associated antigens; A recombinant protein comprising: The three binding agents specifically bind to different tumor-associated antigens, and the binding agents that specifically bind to tumor-associated antigens are (i) a second binding agent that specifically binds to a first tumor-associated antigen (TAA1), said TAA1 being CD33; (ii) a third binding agent that specifically binds to a second tumor-associated antigen (TAA2), wherein the TAA2 is CD123; and (iii) a fourth binding agent that specifically binds to a third tumor-associated antigen (TAA3), said TAA3 being CD70, said recombinant protein.

2. The recombinant protein has a lower dissociation constant (K D ) and 2. The recombinant protein of claim 1, wherein the surface that optionally presents the tumor-associated antigen is the surface of a tumor cell.

3. The lower dissociation constant (K D 3. The recombinant protein according to claim 2, wherein the dissociation constant of the binding domain of the binding agent of the recombinant protein is at least about 2-fold, at least about 4-fold, at least about 10-fold, at least about 20-fold, at least about 40-fold, or at least about 100-fold lower than that of the corresponding dissociation constant of the recombinant protein comprising only one of the binding agents that specifically binds to a tumor-associated antigen.

4. 2. The recombinant protein of claim 1, wherein the first binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and / or the second binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and / or the third binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain, and / or the fourth binding agent is an antibody, an antibody mimetic, a scaffold protein, a repeat protein, or a designed repeat domain. (i) the first binding agent is a designed ankyrin repeat domain having binding specificity for CD3; Optionally, said ankyrin repeat domain having binding specificity for CD3 comprises an amino acid sequence that is at least 95% identical to any one of the amino acid sequences of SEQ ID NOs: 1 to 5, and / or (ii) the second binding agent is a designed ankyrin repeat domain that has binding specificity for CD33; Optionally, said ankyrin repeat domain having binding specificity for CD33 comprises an amino acid sequence that is at least 95% identical to an amino acid sequence of any one of SEQ ID NOs: 15, 67-70, and 111-112; and / or (iii) the third binding agent is a designed ankyrin repeat domain that has binding specificity for CD123, Optionally, said ankyrin repeat domain having binding specificity for CD123 comprises an amino acid sequence that is at least 95% identical to any one of the amino acid sequences of SEQ ID NOs: 6, 65-66, and 102-106; and / or (iv) the fourth binding agent is a designed ankyrin repeat domain that has binding specificity for CD70, The recombinant protein according to any of claims 1 to 4, wherein the ankyrin repeat domain, optionally with binding specificity for CD70, comprises an amino acid sequence that is at least 95% identical to an amino acid sequence of any one of SEQ ID NOs: 64 and 107 to 110.

6. the recombinant protein comprises the first, second, third, and fourth binding agents, the first, second, third, and fourth binding agents being arranged from N-terminus to C-terminus according to the following formula: (fourth binding agent)-(third binding agent)-(second binding agent)-(first binding agent); the first, second, third, and fourth binding agents are covalently attached to a peptide linker; 6. The recombinant protein of claim 5, optionally wherein the peptide linker is a proline-threonine rich peptide linker.

7. The recombinant protein is -6 Dissociation constant (K D ) binds to human CD3 in PBS, and / or the recombinant protein binds to human CD33 in PBS with a dissociation constant (K D ) of less than 10 −7 M; and / or said recombinant protein binds to human CD123 in PBS with a dissociation constant (K D ) of less than 10 −7 M; and / or The recombinant protein of claim 1, wherein the recombinant protein binds to human CD70 in PBS with a dissociation constant (K D ) of less than 10 −7 M.

8. The recombinant protein has an EC 50 The recombinant protein of claim 1 , which binds to human CD3 at

9. The recombinant protein comprises the second, third, and fourth binding agents, and the recombinant protein comprises -6 M to 10 -12 The dissociation constant of M (K D ) to the TAA1 in PBS, and the recombinant protein is bound to the TAA1 in PBS at 10 -6 M to 10 -12 The dissociation constant of M (K D ) to bind to the TAA2 in PBS, and the recombinant protein is -6 M to 10 -12 The dissociation constant of M (K D 6. The recombinant protein of claim 5, which binds to TAA3 in PBS with

10. the recombinant protein further comprises a half-life extending moiety; Optionally, the half-life extending moiety is a binding agent that specifically binds to human serum albumin; Optionally, said binding agent that specifically binds to human serum albumin is a designed ankyrin repeat domain that has binding specificity for human serum albumin, Optionally, said ankyrin repeat domain having binding specificity for human serum albumin comprises an amino acid sequence that is at least 95% identical to the amino acid sequence of any one of SEQ ID NOs: 34 to 36, 2. The recombinant protein of claim 1, optionally wherein the recombinant protein binds to human serum albumin in PBS with a dissociation constant (KD) of less than 10-7 M, and the half-life extending moiety is located N-terminal to the first binding agent and the binding agent that specifically binds to a tumor-associated antigen.

11. the recombinant protein comprises a polypeptide having an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:95; or 2. The recombinant protein of claim 1, wherein the recombinant protein comprises a polypeptide having an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO:

96.

12. A nucleic acid encoding the recombinant protein of claim 1.

13. A pharmaceutical composition comprising a recombinant protein according to claim 1 or a nucleic acid according to claim 12, and a pharma- ceutically acceptable carrier and / or diluent.

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

15. 14. A pharmaceutical composition according to claim 13 for use in the treatment of cancer, comprising: Optionally, said cancer is leukemia, and optionally said cancer is acute myeloid leukemia.

Images