Synthetic CD123 binding proteins, manufacture, and uses thereof

Abstract

The present disclosure provides synthetic CD123 binding proteins, compositions containing such binding proteins, and methods of making and using such binding proteins.

Description

SYNTHETIC CD123 BINDING PROTEINS, MANUFACTURE, AND USES THEREOFCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims benefit of and priority to U.S. Provisional Patent Application No. 63 / 656,568, filed June 5, 2024, the entire contents of which are hereby incorporated by reference for all purposes.SEQUENCE LISTING

[0002] The present specification makes reference to a Sequence Listing, which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML file, created on June 2, 2025, is named AIP-009WO_SL.xml and is 98,304 bytes in size.FIELD

[0003] The disclosure relates generally to synthetic CD123 binding proteins, their manufacture and use in the treatment of various disorders, including cancer.BACKGROUND

[0004] According to the National Cancer Institute (“NCI”), in 2022 there were 18.1 million cases of cancer in the United States, over 1.9 million of which were new. See, e.g., Siegel et al. (2022) CA CANCER J. CLIN. 72:7-33. NCI estimates that direct cancer-related medical costs in the U.S. were $183 billion in 2015 and are projected to increase to $246 billion by 2030. See, e.g., The American Cancer Society Cancer Action Network, The Costs of Cancer 2020 Edition. Cancer is caused by abnormal proliferation of cells and can occur in any tissue in the body. These abnormal cells can evade the body’s natural defenses, making it difficult for the immune system to fight cancer cells.

[0005] Hematologic malignancies such as leukemia, multiple myeloma, non-Hodgkin lymphoma, and Hodgkin lymphoma are caused by proliferation of abnormal blood cells. These abnormal cells can originate from either myeloid or lymphoid cell types and outcompete and disrupt the function of normal blood cells. Incidence of these cancers has increased globally. See, e.g., Zhang et al. (2023) BLOOD C NCER J. 13(1): 82, citing to a 2019 Global Burden of Disease Study. Blood cancers can also be harder to treat with traditional cancer therapeutics such as chemotherapy and / or radiation.

[0006] CD123, which is also known as Interleukin-3 receptor alpha chain (IL-3Ra), has been shown to be overexpressed in several hematological malignancies. See, e.g., Achi et al. (2020) CANCERS 12(11): 3087; Testa et al. (2019) C NCERS 11(9): 1358. Current therapeutic approaches targeting hematological cancers expressing CD 123 include, for example, cytokines modified with toxic payloads (e.g., recombinant IL-3 fused to diphtheria toxin), and antibodies or antibody-drug conjugates targeting CD123. See, e.g., Achi et al. (2020), supra; Testa et al. (2019), supra. Newer therapies such as cell therapies such as chimeric antigen receptor (CAR)-cells are promising in hematologic cancers, but remain challenging because of, for example, lack of specificity / off-target effects, toxicity, and reduced efficacy over time. Accordingly, a need remains for improving efficacy and specificity of chimeric antigen receptor therapies, such as through more specific and effective targeting to tumor cells.SUMMARY

[0007] The disclosure is based, in part, upon the discovery of synthetic CD123 binding proteins that specifically and competitively bind CD 123 (by blocking IL-3 from binding to CD123). When IL-3 binds to CD123 and CD123 dimerizes with CD131, intracellular hematopoietic cell signaling can be activated and promote features such as proliferation and survival. It is believed that IL-3 binding to CD123 on a cancer cell can increase malignancy by promoting cancer cell survival and proliferation. The disclosure contemplates that blocking IL-3 signaling can be used to prevent proliferation and survival of malignant cells. For example, a synthetic CD123 binding protein that antagonizes IL-3 activity by binding to CD 123 and blocking IL-3 -mediated signaling can modulate downstream factors that would otherwise increase malignancy. In another approach, a synthetic CD123 binding protein can “engage” cancer cells with another modality (e.g., molecule, cell, etc. that can target the cancer cell for destruction. For example, in one approach, a CD 123 binding protein provided herein can be attached to another entity, such as an effector molecule (e.g., comprising a cytotoxic agent) that can kill the cancer cell. An alternative approach can include engineering a therapeutic cell (e.g., a cell engineered with a chimeric antigen receptor (CAR)) to recognize a CD 123 binding protein, and to use the synthetic CD 123 binding protein to bring the CAR (e.g., expressed in a CAR-T cell) into proximity with the cancer cell to which the CD123 binding protein has bound. Using this approach, the therapeutic cell can target the cancer cell for destruction. Yet anotherapproach can be to attach a CD123 binding protein to an effector (e.g., a small molecule, e.g., fluorescein or a derivative thereof, etc.) that binds to another protein e.g., an effector binding protein) that is expressed on a second, different cell (e.g., a therapeutic cell, e.g., a cell engineered with a chimeric antigen receptor (CAR), e.g., a CAR-T cell, etc.) that can target the cancer cell for destruction. Currently, there is a long felt and unmet need for improvements in compositions and methods for targeting cancer cells including those of hematopoietic origin. Accordingly, the disclosure provides, among other things, synthetic CD 123 binding proteins with improved safety, selectivity, efficacy, potency, manufacturability, scalability, and stability can meet such needs on their own or in combination with other components as provided herein, as well as methods of making and using such binding proteins.

[0008] In one aspect, the disclosure provides a synthetic CD123 binding protein, the binding protein comprising: (a) an amino acid sequence from 35 amino acids to 100 amino acids in length; (b) a net negative charge in phosphate buffered saline (PBS); (c) a binding affinity for CD 123 stronger than 1 pM; and (d) a stability profile such that the protein (i) retains at least 90% binding affinity to CD 123 upon cooling to room temperature after thermal denaturation at 95°C in PBS for at least about five minutes relative to the protein prior to thermal denaturation; (ii) retains at least 90% binding affinity to CD 123 after incubation for 16 hours at 37°C of incubation in PBS relative to the protein under the same conditions prior to incubating; and / or (iii) retains at least 90% binding affinity to CD123 in PBS following chemical denaturation in 4 M urea for 1 hour at room temperature relative to the protein prior to chemical denaturation.

[0009] In another aspect, the disclosure provides a synthetic CD123 binding protein, the binding protein comprising: (a) an amino acid sequence from 30 amino acids to 95 amino acids in length; (b) a net negative charge in PBS; (c) a binding affinity for CD123 stronger than 10 pM; (d) at least three alpha helices; (e) at least two amino acid loops, where a first loop having a first amino acid sequence connects a terminal amino acid (e.g., a C-terminal amino acid) of a first alpha helix to a terminal amino acid (e.g., a N-terminal amino acid) of a second alpha helix, and a second loop having a second amino acid sequence connects a second, terminal amino acid (e.g., a C-terminal amino acid) of the second alpha helix to a terminal amino acid (e.g., an N-terminal amino acid) of a third alpha helix; and (f) a hydrophobic core defined by at least two hydrophobic amino acids present in at least one, two or three of the three alpha helices.

[0010] In certain embodiments, the synthetic CD 123 binding protein binds to CD 123 through a paratope of the CD123 binding protein, which paratope is defined by amino acids X6 - X7 - X9 - X10 - XI 1 - X39 - X43 (SEQ ID NO: 87), wherein X6 is selected from Y or F, X7 is selected from A, S, or G, X9 is E, XI 0 is selected from Y, F, or W, XI 1 is selected from L or I, X39 is selected from L, M, I, or V, and X43 is selected from H or Y, and wherein the amino acid numbering corresponds to the numbering of SEQ ID NOs: 25 or 29.

[0011] In one aspect, the disclosure provides a synthetic CD123 binding protein comprising a conformational paratope defined by amino acids X6 - X7 - X9 - XI 0 - XI 1 - X39 - X43, wherein X6 is selected from Y or F, X7 is selected from A, S, or G, X9 is E, X10 is selected from Y, F, or W, XI 1 is selected from L or I, X39 is selected from L, M, I, or V, and X43 is selected from H or Y, and wherein the amino acid numbering corresponds to the numbering of SEQ ID NOs: 25 or 29.

[0012] In certain embodiments, the synthetic CD123 binding protein comprises a conformational paratope defined by amino acids X6 - X7 - X9 - X10 - XI 1 - X39 - X43, wherein X6 is Y, X7 is A, X9 is E, X10 is Y, XI 1 is L, X39 is L, and X43 is H.

[0013] Depending on circumstances, in some embodiments, the synthetic CD123 binding protein comprises one or more of the following features: (a) free of tryptophan amino acids; (b) free of methionine amino acids; (c) free of lysine amino acids; (d) does not comprise an unpaired cysteine amino acid when cysteine amino acids are present in the protein; (e) free of glycosylation sites; (f) free of protease cleavage sites; and (g) soluble up to at least 1 mM in PBS at 4°C for one month.

[0014] In certain embodiments, the synthetic CD 123 binding protein has a binding affinity between about 10 pM to about 0.1 nM; about 7.5 pM to about 0.75 nM; about 5 pM to about 0.5 nM; about 2.5 pM to about 0.25 nM; about 1 pM to about 1 nM; about 0.75 pM to about 1 nM, about 0.5 pM to about 1 nM; about 0.25 pM to about 1 nM; about 0.10 pM to about 1 nM; about 75 nM to about 1 nM; about 50 nM to about 1 nM; about 25 nM to about 1 nM; about 10 nM to about 1 nM; and about 5 nM to about 1 nM.

[0015] In certain embodiments, synthetic CD 123 binding protein has a binding affinity stronger than about 1 pM, about 0.75 pM, about 0.5 pM, about 0.25 pM, about 0.1 pM, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, about 0.1 nM, about 0.01 nM, and about 0.001 nM.

[0016] In certain embodiments, the N-terminus of the first alpha helix is preceded by one or more (e.g., 2, 3, 4, 5, 10, 20, 30, or 40 amino acids) N-terminal amino acids. In certain embodiments, the C-terminus of the third alpha helix is followed by one or more (e.g., 2, 3, 4, 5, 10, 20, 30, or 40 amino acids) C-terminal amino acids.

[0017] In some embodiments, a synthetic CD123 binding protein of the disclosure comprises one or more of the following: (a) the first, second, and / or third alpha helix each containing at least one hydrophobic amino acid, wherein, optionally, one or more of the at least one hydrophobic amino acids is not solvent accessible; (b) the first, second, and / or third alpha helix each contains at least two or three hydrophobic amino acids, wherein, optionally, one or more of the at least two or three hydrophobic amino acids is not solvent accessible; (c) the first, second, and / or third alpha helix each contain at least one or two solvent accessible amino acids; (d) the first and / or second loop contains at least one hydrophobic amino acid, or (e) the binding protein comprises any combination of elements selected from (a), (b), (c), (d), and (e) . In certain embodiments, (a) the second and third alpha helix each contains at least two hydrophobic amino acids; (b) the first, second, and third alpha helix each contains at least one solvent accessible amino acid; (c) the first, second, and third alpha helix each contain at least two hydrophobic and one solvent accessible amino acids; (d) the second and third alpha helix each contains at least four solvent accessible amino acids; and / or (e) the first loop contains at least one hydrophobic amino acid.

[0018] In certain embodiments, the N-terminus of the first alpha helix of the synthetic CD123 binding protein is preceded by one or more N-terminal amino acids. Depending on circumstances, the N-terminus of the first alpha helix comprises an N-terminal extension. For example, in some embodiments, the N-terminal extension has an amino acid sequence comprising that of SEQ ID NO: 34.

[0019] In certain embodiments, the C-terminus of the third alpha helix of the synthetic CD123 binding protein is followed by one or more C-terminal amino acids. Depending on circumstances, the C-terminus of the third alpha helix comprises a C-terminal extension. For example, in some embodiments, the C-terminal extension has an amino acid sequence comprising that of SEQ ID NO: 37.

[0020] As provided herein, in certain embodiments, the synthetic CD 123 binding protein comprises from 35 amino acids to 70 amino acids in length, from 35 amino acids to 75 amino acids in length, from 35 amino acids to 65 amino acids in length, from 35 amino acids to 60 amino acids in length, from 35 amino acids to 55 amino acids in length, from 35 amino acids to 50 amino acids in length, from 35 amino acids to 45 amino acids in length, from 40 amino acids to 75 amino acids in length, from 40 amino acids to 80 amino acids in length, from 40 amino acids to 70 amino acids in length, from 40 amino acids to 65 amino acids in length, from 40 amino acids to 60 amino acids in length, from 40 amino acids to 55 amino acids in length, from 45 amino acids to 70 amino acids in length, from 45 amino acids to 65 amino acids in length, from 45 amino acids to 60 amino acids in length, from 50 amino acids in length to 60 amino acids in length, from 45 amino acids to 55 amino acids in length, from 50 amino acids to 70 amino acids in length, from 50 amino acids to 65 amino acids in length, or from 40 amino acids to 50 amino acids in length. For example, in certain embodiments, the synthetic CD123 binding protein comprises 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids.

[0021] In one aspect, the disclosure provides a synthetic CD123 binding protein comprising:(i) an amino acid sequence arranged in a primary structure of Z1-D1-L1-D2-L2-D3-Z2 (Formula I), wherein DI, D2, and D3 are domains 1, 2, and 3, respectively; LI, and L2, are loops 1, and 2, respectively; and Z1 and Z2 are N- and C-terminal regions, respectively; and (ii) an amino acid of SEQ ID NO: 25, wherein DI, D2, and D3, independently comprise any of the following combinations: (a) DI comprises an amino acid sequence of SEQ ID NO: 26, wherein X4 is A or V; X5 is Y, E, H, Q, F, or V; and X14 is E, L, or I; (b) D2 comprises an amino acid sequence of SEQ ID NO: 27, wherein X20 is E or T; X25 is L, Y, Q, S, G, or N; X26 is R, K, V, or I; X27 is H, V, or A; X30 is E, L, or D; and X31 is R, I, or Q; and (c) D3 comprises an amino acid sequence of SEQ ID NO: 28, wherein X35 is V, K, R, E, H, G, Q, or S; X37 is Q, or S; X41 is D, S, or E; and X43 is H or Y. In certain embodiments, the synthetic CD123 binding protein comprises LI, which comprises an amino acid sequence of GX16IS, wherein XI 6 is A or F; and / or L2, which comprises an amino acid sequence of GDD. Depending on circumstances, in some embodiments, the synthetic CD123 binding protein comprises Zl, which comprises an amino acid sequence of SGY and / or Z2, which comprises an amino acid sequence of RX45GS, wherein X45 is Y, K, H, R, or E.

[0022] In certain embodiments, the disclosure provides a synthetic CD123 binding protein comprising an amino acid sequence of SEQ ID NO: 25, wherein X4 is A or V; X5 is Y, E, H, Q, F or V; X14 is E, L, or I; X16 is A or F; X20 is E or T; X25 is L, Y, Q, S, G, or N; X26 is R, K, V, or I; X27 is H, V, or A; X30 is E, L, or D; X31 is R, I, or Q; X35 is V, K, R, E, H, G, Q, or S; X37 is Q, or S; X41 is D, S, or E; X43 is H or Y; and X45 is Y, K, H, R, or E. For example, in some embodiments, the synthetic CD123 binding protein comprises an amino acid sequence selected from any of SEQ ID NOs: 1-24 or 41-64.

[0023] In another aspect, the disclosure provides a synthetic CD123 binding protein comprising: (i) an amino acid sequence arranged in a primary structure of Z1-D1-L1-D2- L2-D3-Z2 (Formula I), wherein DI, D2, and D3 are domains 1, 2, and 3, respectively; LI, and L2, are loops 1, and 2, respectively; and Z1 and Z2 are N- and C-terminal regions, respectively; and (ii) an amino acid of SEQ ID NO: 29, wherein DI, D2, and D3, independently comprise any of the following combinations: (a) DI comprises an amino acid sequence of SEQ ID NO: 30, wherein X5 is Y, Q, or F; (b) D2 comprises an amino acid sequence of SEQ ID NO: 31, wherein X25 is L, Q, S, G, or N; and X26 is R, K, or I; and (c) D3 comprises an amino acid sequence of SEQ ID NO: 32, wherein X35 is V, K, E, H, G, or S; and X43 is H or Y. In certain embodiments, the synthetic CD123 binding protein comprises LI, which comprises an amino acid sequence of SEQ ID NO: 33; and / or L2, which comprises an amino acid sequence of GDD. Depending on circumstances, in some embodiments, the synthetic CD123 binding protein comprises Zl, which comprises an amino acid sequence of SGY and / or Z2, which comprises an amino acid sequence of RX45GS, wherein X45 is Y, K, or E.

[0024] In certain embodiments, the disclosure provides a synthetic CD123 binding protein comprising an amino acid sequence of SEQ ID NO: 29, wherein X5 is Y, Q, or F; X25 is L, Q, S, G, or N; X26 is R, K, or I; X35 is V, K, E, H, G, or S; X43 is H or Y; and X45 is Y, K, or E. For example, in some embodiments, the synthetic CD123 binding protein comprises an amino acid sequence selected from any of SEQ ID NOs: 1-9 or 41-49.

[0025] The disclosure also provides synthetic CD123 binding proteins with one or more lysine residues rationally positioned at certain positions along the length of the binding protein, which may act as a conjugation site for modification (e.g., conjugation of an effector). For example, in certain embodiments, the synthetic CD 123 binding protein provided herein has a lysine residue at any of positions X26, X35, or X45, relative to SEQ ID NOs: 25 or 29.

[0026] In certain embodiments, an effector is conjugated to a lysine at position X26, X35, or X45. In certain embodiments, the conjugation is directly to the lysine (e.g., via NHS- based chemistry). As provided herein, in some embodiments, the effector comprises a cytotoxic molecule (e.g., diphtheria toxin or a portion thereof) or a small molecule or chelator. For example, in some embodiments, the small molecule comprises fluorescein or a derivative thereof. In some embodiments, the fluorescein derivative comprises fluorescein isothiocyanate (FITC). In certain embodiments, the chelator comprises tetraxetan (DOTA).

[0027] Also provided herein are synthetic CD123 binding proteins with binding affinities for CD 123 that are stronger than 1 pM.

[0028] In certain embodiments, the binding affinity of a synthetic CD 123 binding protein of the disclosure is between about 1 pM to about 0.001 nM; about 1 pM to about 0.01 nM; about 1 pM to about 0.75 nM; about 1 pM to about 0.5 nM; about 1 pM to about 0.25 nM; about 1 pM to about 0.01 nM; about 1 pM to about 1 nM; about 0.5 pM to about 1 nM; about 0.25 pM to about 1 nM; about 0.10 pM to about 1 nM; about 0.10 pM to about 0.01 nM; about 75 nM to about 1 nM; about 50 nM to about 1 nM; about 25 nM to about 1 nM; about 10 nM to about 1 nM; and about 5 nM to about 1 nM. In some embodiments, the binding affinity is stronger than about 1 pM, about 0.75 pM, about 0.5 pM, about 0.25 pM, about 0.1 pM, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, about 0.1 nM, about 0.01 nM, and about 0.001 nM.

[0029] In certain embodiments, the disclosure provides synthetic CD123 binding proteins with amino acid sequences of 80 (e.g., 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 90.5, 91, 91.5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, 99.5, 99.6, 99.7, 99.8, 99.9) percent identity to the amino acid sequence of any of SEQ ID NOs: 1-24 or 41-64.

[0030] In some embodiments, a synthetic CD 123 binding protein provided herein has an amino acid sequence comprising or according to one or more sequences set forth in Table 9. In certain embodiments, the synthetic CD123 binding protein has an amino acid sequence comprising or according to one or more sequences as set forth in Table 1, with one or more amino acid substitutions as set forth in Table 2B.

[0031] In certain embodiments, the synthetic CD123 binding protein comprises a paratope defined by a paratope represented by X28 - X29 - X32 - X39 - X41 - X42, where X28 is Vor L, X29 is D, X32 is D, X39 is T or V, X41 is I or L, and X42 is R or Q, wherein the amino acid positions correspond to those of SEQ ID NOs: 25 or 29 from N-terminus to C- terminus. For example, in some embodiments, the CD123 binding protein comprises a paratope defined by any combination of positions as set forth in Table 6.

[0032] In one aspect, the disclosure provides a synthetic CD123 binding protein comprising an amino acid sequence with reference to any of SEQ ID NOs: 1-24 or 41-64, but having one or more changes to one or more amino acid residues as set forth in Table 6.

[0033] Also provided herein are pharmaceutical compositions comprising a synthetic CD 123 binding protein as provided herein and a pharmaceutically acceptable carrier. In certain embodiments, the synthetic CD123 binding protein further comprises an effector molecule (e.g., fluorescein, FITC, FITC-5-FL, DOTA). In some embodiments, the effector is attached, directly or indirectly, to the synthetic CD123 binding protein at a lysine residue. For example, in some embodiments, the lysine residue is at position 26, 35, or 45 relative to SEQ ID NO: 25 or SEQ ID NO: 29. In certain embodiments, the effector is attached indirectly to the synthetic CD 123 binding protein via a linker. Depending on the circumstances, the linker can comprise an amino acid sequence selected from Table 3B. In certain embodiments, the linker comprises a lysine. In some embodiments, the lysine is on the C-terminal end of the linker. In some embodiments, the lysine is on the N-terminal end of the linker.

[0034] In another aspect, the disclosure provides a method of targeting CD123, the method comprising contacting a cell that expresses CD123 on its cell surface with a composition comprising a synthetic CD123 binding protein or pharmaceutical composition, each as provided herein. In certain embodiments, the synthetic CD 123 binding protein decreases IL-3 -mediated activity (e.g., proliferation, survival) in a cancer cell expressing CD 123 relative to cell-mediated activity in the cancer cell in the absence of the CD 123 binding protein. In certain embodiments, the contacting step comprises contacting a population of cells expressing CD123 with a composition comprising a synthetic CD123 binding protein, wherein the synthetic binding protein further comprises an effector that binds to an antigen on a second, different cell, which second different cell promotes cytotoxicity of the population, wherein after the contacting, a greater portion of the population is dead as compared to contacting without the effector and / or without the synthetic CD 123 binding protein.

[0035] In one aspect, the disclosure provides a method of antagonizing IL-3 activity in a cancer cell, the method comprising contacting a cancer cell with a synthetic CD123 binding protein or a pharmaceutical composition as provided herein.

[0036] In another aspect, the disclosure provides a method of treating cancer by administering to a subject in need thereof a conjugate comprising a synthetic CD123 binding protein as provided herein and an effector, or a pharmaceutical composition as provided herein, comprising an effector, wherein the effector targets a second different cell. Upon administration of the conjugate, the CD 123 -expressing cancer cell is brought into proximity with the second different cell expressing an effector antigen that promotes or increases cytotoxicity mediated by the second, different cell in the CD 123 -expressing cancer cell.

[0037] In one aspect, the disclosure provides a method of treating cancer by administering to a subject in need thereof a conjugate comprising a synthetic CD123 binding protein as provided herein and an effector, or a pharmaceutical composition as provided herein, comprising an effector, wherein the effector comprises a cytotoxic molecule. Following administration, the cytotoxic molecule is brought into proximity with the CD123- expressing cancer cell.

[0038] In another aspect, the disclosure provides a method of treating one or more cancer - related conditions in a subject in need thereof, the method comprising administering to the subject an effective amount of a synthetic CD 123 binding protein as provided herein, or a pharmaceutical composition as provided herein. In some embodiments, the administration is before, during, or after administration or use of one or more other treatments. In certain embodiments, the one or more other treatments is or comprises a biological agent (e.g., biologies, gene therapy, peptides), a small molecule (e.g., chemotherapy, corticosteroids, antivirals, antibiotics, anti-inflammatory agents, etc.), one or more cells (e.g., immunotherapy), and / or one or more mechanical interventions (e.g., surgery, cryotherapy). In some embodiments, the subject is diagnosed as having or at risk of having a cancer or population of cancerous cells (e.g., myeloma, lymphoma, leukemia, a solid tumor). In some embodiments, the subject has been diagnosed as having cancer and / or a population of cancerous cells.

[0039] These and other aspects and features of the disclosure are described in the following detailed description and claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0040] FIGs. 1A and IB are schematic representations of certain exemplary aspects of CD123 binding complexes and exemplary synthetic CD123 binding proteins. FIG. 1A is a schematic representation of a ligand-bound IL-3R complex comprising a CD123 monomer (a subunit of the IL-3R complex) and a CD131 monomer (0 subunit of IL-3R complex). IL-3 is thought to bind to a binding pocket near the D2 / D3 domains of CD 123 and the NTD domain of CD123 is believed to join CD123 to CD131. Binding of IL-3 to CD123 and heterodimerization of CD 123 to CD131 activates intracellular signaling (through the CD131 intracellular domain) in hematopoietic lineage cells including phosphorylation of JAK (e.g., JAK2) and activation of STAT (e.g., STAT5) leading to expression of genes for growth, differentiation, and survival in healthy or malignant hematopoietic cells. CD123 comprises several domains, as follows: NTD = N-terminal domain thought to be important for dimerization with the 0 common subunit; D2 and D3 = fibronectin type Ill-like domains that form the IL-3 binding site; a single pass transmembrane domain; and a short intracellular domain indicated by an arrow. FIG. IB is a schematic representation of CD123 bound by an exemplary synthetic CD123 binding miniprotein. In this schematic, the miniprotein blocks IL-3 binding and dimerization of CD 123 with the 0 common subunit, and, thus blocks downstream signaling.

[0041] FIG. 2A and 2B are circular dichroism (CD) spectra showing the stability profiles of an exemplary ly sine-containing synthetic CD 123 binding protein (Reference Miniprotein 4; SEQ ID NO: 4) over a wavelength range of 200-250 nm. FIG. 2A shows stability profiles of Reference Miniprotein 4 during heating at 10°C intervals between temperatures of about 25°C - 95°C. FIG. 2B shows a stability profile of Reference Miniprotein 4 during heating and cooling from about 25°C to about 91 °C and then back down to about 25°C. The CD spectra indicate that the protein showed proper folding, unfolding, and refolding, and was thermostable at about 25°C (open circle) going up to 91 °C (solid inverted triangle) and properly folding as cooling back down to 25°C (open triangle) occurred.

[0042] FIG. 3 is a line graph showing affinity measurements by surface plasmon resonance SPR) depicting competitive binding to CD 123 by exemplary synthetic CD 123 binding proteins as compared to IL-3. The graph shows binding activity in response units (RU) on the y-axis and time (seconds) on the x-axis. Surface-immobilized CD123 monomers werecontacted with solutions of: (i) one of three exemplary synthetic CD 123 binding proteins (Reference Miniproteins 2, 3, and 4; SEQ ID NOs: 2, 3, 4) or (ii) running buffer alone. Upon reaching equilibrium, addition of IL-3 to each of (i) and (ii) showed no signal increase in the CD 123 -binding protein containing samples (indicating saturation of CD 123 by the Reference Miniproteins), but sharp signal increase (indicating binding to CD 123 by IL-3) in the buffer only samples.

[0043] FIGs. 4A and 4B show representative predicted structures of CD 123 bound by interleukin-3 (IL-3) or an exemplary synthetic CD123 binding protein (Reference Miniprotein 4; SEQ ID NO: 4). FIG. 4A shows a representative, known, model structure of IL-3 bound to CD 123. FIG. 4B shows a representative model structure of an exemplary synthetic CD 123 binding protein (Reference Miniprotein 4; SEQ ID NO: 4) bound to CD 123, which predicts binding of the CD 123 binding protein at the same site as IL-3.DETAILED DESCRIPTION

[0044] The disclosure is based, in part, upon the discovery of synthetic CD123 binding proteins that specifically and preferentially bind CD 123 and can prevent IL-3 from binding to CD 123. As a result, the synthetic CD 123 binding proteins can bind to and modulate the activity of IL-3, thereby modulating cancer cell proliferation and survival. Without wishing to be bound by theory, it is believed that synthetic CD123 binding proteins provided herein can be used to bind to CD 123 and antagonize IL-3 binding to malignant hematopoietic cells, such as by decreasing IL-3R-mediated intracellular signaling. A decrease in such signaling can be used in conjunction with one or more additional therapies or one or more additional components attached to the synthetic CD 123 binding protein.

[0045] Further, in certain embodiments, synthetic CD 123 binding proteins can be used to engage therapeutic cells (e.g., to bring a therapeutic cell into close proximity with a malignant cell). Such engagement can be accomplished by, for example, attaching a CD 123 binding protein to another molecule on the surface of a therapeutic cell whereby binding of the therapeutic cell to a target cell (e.g., hematopoietic cell) via the CD123 binding protein can target the target cell (e.g., hematopoietic cell) for destruction.Accordingly, engaging cancer cells using a synthetic CD123 binding protein to bind CD123 expressed on a cell (e.g., on a cancer cell) may allow a therapeutic cell, when administered to a subject, to successfully treat certain cells (e.g, cancer cells), thus, improving treatment outcomes.

[0046] Accordingly, the present disclosure provides, among other things, synthetic CD123 binding proteins, methods of making such binding proteins, and methods of using such proteins to treat related disorders.I. DEFINITIONS

[0047] Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. For example, nomenclatures utilized in connection with, and techniques of, e.g., polypeptide and polynucleotide chemistry and synthesis, molecular and cellular biology, protein biology and biochemistry, immunology, etc. as described herein are those well-known and commonly used in the art.

[0048] As used herein, the singular forms “a,” “an” and “the” include plural referents unless context clearly dictates otherwise. Thus, for example, in some embodiments, reference to, e.g., a synthetic CD 123 binding protein includes a single binding protein, a plurality of synthetic binding proteins, etc.

[0049] As used herein, the expression “and / or” in connection with two or more recited objects includes individually each of the recited objects and the various combinations of two or more of the recited objects, unless otherwise understood from the context and use.

[0050] Where the use of the term “about” is before a quantitative value, the present disclosure also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred. Ranges can be expressed in this disclosure as from “about” one particular value, and / or to “about” another particular value. When values are expressed as approximations by use of the antecedent “about,” it is understood that the disclosure also contemplates embodiments that specify the particular values and ranges of values without the approximations.

[0051] As used herein, the phrases “solvent accessible residue” and “solvent accessible amino acid” refer to an amino acid that, when disposed in a folded molecule (e.g., in its a tertiary conformation) and in a solvent, is characterized in that the amino acid is at least partially accessible or exposed to the solvent. Solvent accessible amino acids can be determined using a variety of approaches including, e.g., Rosetta software suite, Neighbor Count, and Neighbor vector algorithms (Durham et. al. (2009) J. MOL. MODE . 15(9): 1093-108).

[0052] As used herein, the phrase “conservative substitution” refers to a substitution with a structurally and / or functionally similar amino acid. The following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine (S) and Threonine (T); 2) Aspartic Acid (D) and Glutamic Acid (E); 3) Asparagine (N) and Glutamine (Q); 4) Arginine (R) and Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), and Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), and Tryptophan (W). Conservative substitutions may also be defined by the BLAST (Basic Local Alignment Search Tool) algorithm, the BLOSUM substitution matrix (e.g., BLOSUM 62 matrix), or the PAM substitution^ matrix (e.g., the PAM 250 matrix). In certain embodiments, a binding protein of the disclosure comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 conservative substitutions relative to a reference amino acid sequence.

[0053] As used herein, the phrase “corresponding to” designates a position / identity of an amino acid or a nucleic acid in a polymeric molecule such as an amino acid in an amino acid sequence or a nucleic acid in a nucleic acid sequence. It is understood by the skilled artisan that such amino acids or nucleic acids in such a polymer are often designated using a canonical numbering system based on a reference related polymer, so that, for example, an amino acid in a first polymer “corresponding to” position seven in the reference amino acid, for example, need not actually be the seventh amino acid in the first polymer. Those of ordinary skill in the art are aware of methodology to identify “corresponding” amino acids or nucleic acids between two molecules (e.g., a polymer and a reference polymer), including, such as, commercially available algorithms, databases, or other information given context regarding particular polymers.

[0054] As used herein, the term “domain” refers to a region or segment of a given synthetic binding protein disclosed herein, and can include one or more structural features (e.g., amino acid, primary, structure or secondary structure features) and / or one or more functional features (e.g., binding properties). In the context of secondary structure, a “structural domain” can be an uninterrupted linear sequence that adopts a single type of secondary structure, for example, ten continuous amino acid residues that are all part of the same alpha helix structure or beta sheet. In the context of binding, a “binding domain” can be a discontinuous portion of the overall amino acid sequence that facilitates chemical interactions with a target molecule or indirectly stabilizes such interactions.

[0055] As used herein, the phrase “effective amount” refers to the amount of an active agent (e.g., a synthetic CD123 binding protein disclosed herein) sufficient to effectbeneficial or desired results. An effective amount can be administered in one or more administrations, applications, or dosages and is not intended to be limited to a particular formulation or administration route.

[0056] As used herein, the term “effector” refers to a molecule or molecular entity that confers one or more particular characteristics on itself or another molecule or molecular entity with or to which it is associated. For example, an effector may include a synthetic binding protein such as a miniprotein, or something other than a miniprotein that is associated with a synthetic CD123 binding protein disclosed herein (e.g., via a covalent linkage), such as a detectable label (e.g., visualizable or otherwise measurable such as by fluorescence or radiolabel detection), small molecule (e.g., a fluorophore (e.g., fluorescein and / or derivatives such as fluorescein isothiocyanate (FITC), etc.), nanoparticle (e.g., a lipid nanoparticle, a polymer nanoparticle, etc.), polynucleotide (e.g., an aptamer, an siRNA, an shRNA, an oligonucleotide, etc.), a chelator (e.g., tetraxetan), a radionuclide, etc. An effector may be a synthetic binding protein (e.g., a monovalent synthetic binding protein linked to a CD123 binding protein disclosed herein to create a bivalent synthetic protein where one or both of the proteins causes a change, e.g., in a cellular function, e.g., in a disease state, etc.). An effector may be used as a moiety that is recognized or binds to another component, for example, an antibody or other binding protein. That is, an antibody or other binding protein can bind to an effector, which would then bring the antibody or other binding protein and anything associated with it, such as a therapeutic cell (e.g, a cell expressing a CAR) into proximity with the CD 123 miniprotein and the effector.

[0057] As used herein, the term “loop” refers to (i) a structure (e.g, polypeptide) that connects two structural domains (e.g., a loop may be disposed between two alpha helices, between an alpha helix and a beta sheet, or between two beta sheets in a given synthetic CD123 binding protein) and / or (ii) a structure (e.g., peptide) present at the N- and / or C- terminal end of a given monovalent synthetic binding protein.

[0058] As used herein, the term “linker” refers to a structure (e.g., a polypeptide linker, or a chemical crosslinker (e.g., a homobifunctional or a heterobifunctional cross linking agent) between two molecules (e.g., two synthetic CD123 binding proteins disclosed herein) or between, e.g., a synthetic CD 123 binding protein and an effector, wherein each of the entities that are linked is covalently linked to one another.

[0059] As used herein, the terms / phrases “synthetic binding protein,” “synthetic miniprotein,” and “miniprotein” are used interchangeably, and refer to a polypeptidebetween about 35 to about 100 amino acids in length, e.g., from about 30 to about 90 amino acids in length, from about 30 to about 70 amino acids in length, from about 35 to about 75 amino acids in length, from about 35 to about 65 amino acids, from about 35 to about 60 amino acids in length, from about 35 to about 55 amino acids in length, from about, from about 35 to about 50 amino acids in length, from about 35 to about 45 amino acids in length, from about 40 to about 80 amino acids in length, from about 40 to about 75 amino acids in length, from about 40 to about 70 amino acids in length, from about 40 to about 65 amino acids in length, from about 40 to about 60 amino acids in length, from about 40 to about 55 amino acids in length, from about 40 to about 50 amino acids in length, from about 45 to about 70 amino acids in length, from about 45 to about 65 amino acids in length, from about 45 to about 60 amino acids in length, from about 50 to about 60 amino acids in length, from about 45 to about 55 amino acids in length, from 50 amino acids to 70 amino acids in length, from 50 amino acids to 65 amino acids in length, or 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids in length, that are capable of binding to a given target, e.g., CD 123 with a desired binding affinity (e.g., stronger than 1 pM).

[0060] As used herein, the phrase “percent identity” and “% identity” refers to the extent to which two sequences (e.g., a polypeptide) have the same amino acid or nucleotide at the same positions in an alignment. The percent identity between a polypeptide sequence and a reference sequence is defined as the percentage of amino acid residues in the polypeptide sequence that are identical to the amino acid residues in the reference sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. It is contemplated that a reference sequence can be an amino acid sequence corresponding to an entire CD 123 binding protein or a portion thereof. A reference sequence may be an amino acid sequence that corresponds to a particular domain or domains (e.g., an alpha helix, a loop region) or a combination of domains (e.g., a combination of a loop and an alpha helix). Alignment for purposes of determining percent sequence identity (e.g., amino acid sequence identity) can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST (Basic Local Alignment Search Tool), BLAST-2, ALIGN, MEGALIGN (DNASTAR), CLUSTALW, CLUSTAL OMEGA, MUSCLE, or BioPython software. For a discussion of basic issues in searching sequence databases, see Altschul et al. (1994) NATURE GENETICS 6: 119-129, which is incorporated by reference herein. Those skilled inthe art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.

[0061] As used herein, the term “synthetic” refers to a molecule that is (i) not naturally occurring, (ii) not present in nature, (iii) does not comprise entirely natural components, or (iv) a combination of any one of (i), (ii) and (iii). For example, a synthetic peptide does not exist naturally, is produced, or otherwise modified by human intervention, such as techniques including recombinant or cell-free synthesis, and / or the peptide may comprise one or more non-naturally occurring amino acids.

[0062] As used herein, the phrase “pharmaceutically acceptable” refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0063] As used herein, the phrase “pharmaceutically acceptable carrier” as used herein refers to an agent (e.g., excipient, carrier, buffer, etc.) suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. Standard pharmaceutical carriers may include, for example a phosphate buffered saline solution, water, emulsions (e.g., such as an oil / water or water / oil emulsions), and various types of wetting agents. The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see e.g., Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rded. 2020).

[0064] As used herein, the term “epitope” refers to a region of a protein that is specifically recognized by a binding partner, such as an antibody or another binding protein. The epitope may generally span a portion of the protein. Often, proteins may have multiple such regions where binding partners can attach. Epitopes typically fall into two classes: continuous epitopes (also known as linear epitopes), which are epitopes defined by linear sequences of consecutive amino acids, and discontinuous epitopes (also known asconformational epitopes), which are epitopes defined by discontinuous amino acids that are brought together into spatial proximity when a protein is in its folded state.

[0065] As used herein, the term "paratope" refers to the specific region of a binding molecule (e.g., a binding protein, e.g., a CD 123 binding protein) that recognizes and binds an epitope of a target molecule (e.g., CD123). A paratope of a given binding molecule typically comprises 5-20 amino acids that are solvent accessible and in close proximity in three-dimensional space.

[0066] As used herein, the terms “subject” and “patient” refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably includes humans.

[0067] As used herein, “treat”, “treating”, and “treatment” refer to the treatment of a disease, disorder, or symptom or manifestation of such in a subject, e.g., in a human. This includes: (a) preventing a disease or disorder, (b) inhibiting the disease, disorder, etc., i.e., slowing or arresting its progress or development; and (b) relieving the disease, disorder, etc., i.e., causing regression of the disease state. As used herein, “prevent”, “preventing” and “prevention” refer to causing a disease, disorder, or symptom or manifestation of such not to occur for at least a period of time in at least some subjects.

[0068] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited processing steps. Similarly, throughout the description, where compositions are described as consisting essentially of specific components, or where processes and methods are described as consisting essentially of specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist of the recited components, and that there are processes and methods according to the present disclosure that consist of the recited processing steps.

[0069] Throughout the text, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or theelement or component can be selected from a group consisting of two or more of the recited elements or components.

[0070] Further, it should be understood that elements and / or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present disclosure, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present disclosure and / or in methods of the present disclosure, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated that embodiments may be variously combined or separated without parting from the present teachings and any invention provided herein. For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of any invention described and depicted herein.

[0071] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present disclosure and does not pose a limitation on the scope of any invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of any invention disclosed herein.

[0072] It should be understood that the expression “at least one of’ includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use.

[0073] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0074] It should be understood that the order of steps or order for performing certain actions is immaterial so long as disclosed invention(s) remain operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0075] As used herein, all numerical values or numerical ranges include whole integers within or encompassing such ranges and fractions of the values or the integers within or encompassing ranges unless the context clearly indicates otherwise. Thus, for example,reference to a range of 90-100%, includes 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, etc.II. CLUSTER OF DIFFERENTIATION 123 (CD123)

[0076] Provided herein are synthetic binding proteins (also referred to as miniproteins), that bind cluster of differentiation 123 (CD123) which is also known as interleukin-3 receptor alpha subunit (IL-3Ra), which binds interleukin-3 (IL-3). IL-3 is a cytokine that can impact survival, expansion, and differentiation of a variety of hematopoietic cells. See, e.g., Kan et al., (2023) CANCER DISCOV. 13(8): 1922-1947. IL-3 and other cytokines IL-5 and granulocyte-monocyte colony stimulating factor (GM-CSF) are part of the beta common family of cytokines, which all have receptors that share a common beta subunit (Pc also known as CD 131), but have unique alpha subunits to which the respective cytokines bind. See, e.g., Dougan et al. (2019) IMMUNITY 50(4): 796-811; Hercus et al. (2018) COLD SPRING HARB PERSPECT BIOL. 10(6): a028514. The a subunits of each of these receptors share a conserved extracellular cytokine receptor module domain with two repeats of a fibronectin type Ill-like (Fnlll) domain. See, e.g., Hercus et al. (2018), supra. The Pc is required for intracellular signaling. See, e.g., Dougan et al. (2019), supra; Hercus et al. (2018), supra.

[0077] IL-3 can bind to CD 123 monomers with low affinity, but has a higher affinity for the IL-3R receptor complex of CD 123 heterodimerized to the common Pc subunit (CD131), and binding of IL-3 to CD123 can also cause structural changes that promote heterodimerization. See, e.g., O’Shea et al. (2013) CLINICAL IMMUNOLOGY (FOURTH EDITION), 9 - Cytokines and Cytokine Receptors, pg. 108-135. As depicted schematically in FIG. 1A, IL-3 can bind to CD123 ( a subunit of IL-3R receptor complex), which heterodimerizes with CD131 (P subunit of IL-3R receptor complex). See, e.g., Dougan et al. (2019), supra. It is believed that heterodimerization activates intracellular activity of downstream pathways such as JAK-STAT, PI3K-AKT, and RAS-MAPK. See, e.g., Dougan et al. (2019), supra; Hercus et al. (2018), supra; Guthridge et al. (1998) STEM CELLS 16(5):301. Downstream gene activation through these signaling pathways promotes growth, proliferation, survival, and differentiation. While the IL-3R complex has been shown to exhibit a variety of stoichiometries that may influence signaling biases, without wishing to be bound by theory, the disclosure contemplates that antagonizing IL-3 binding to CD123 can disrupt heterodimerization with CD131, reducing or preventing downstreamsignaling, including survival and proliferation. See, e.g., Kan et al., (2023), supra; Testa et al. (2019) CANCERS 11(9): 1358.

[0078] IL-3R is expressed on early hemopoietic progenitor cells. See, e.g., Achi et al. (2020) C NCERS 12(11):3087; Guthridge et al. (1998), supra. Moreover, malignant cells of hematopoietic origin express CD123. See Achi et al., (2020), supra. IL3-R has also been found to be overexpressed by leukemic stem cells and on cells from patients with difficult to treat cancers including acute myeloid leukemia (AML), blastic plasmacytoid dendritic neoplasm (BPDCN), hairy cell leukemia (HCL), acute lymphoblastic leukemia, etc. See, e.g., Achi, (2020), supra; Bras et al. (2019) CYTOMETRY B CLIN CYTOM. 96(2): 134-142. In AML, it is thought that overexpression of CD123 may lead to aberrant IL-3 signaling and constitutive STAT5 activation which can increase resistance to apoptosis. See, e.g., Achi, (2020), supra. High levels of CD123 in AML patients are associated with decreased survival. Id. BPDCN is considered an extremely aggressive blood cancer and can be diagnosed, in part, through detection of CD 123. Id. Additional hematopoietic malignancies are marked by CD 123 overexpression including HCL, Hodgkin Lymphoma, and chronic myeloid leukemia (CML). Id.

[0079] Agonism of intracellular signaling by IL-3 binding to CD123 / CD131 heterodimers is thought to contribute to increase in proliferation of malignant cells. Thus, interest has increased in development of blood cancer therapeutics that target CD123 including, e.g., modified cytokines (e.g., recombinant IL-3 fused to diphtheria toxin), antibodies or antibody-drug conjugates, and certain CAR-T cells. See, e.g., Achi et al. (2020), supra; Testa et al. (2019), supra. Tagraxofusp, a recombinant IL-3 linked to a diphtheria-toxin payload, has been approved for BPDCN, and there are several clinical trials evaluating the efficacy of CD 123 -directed therapeutics for targeted cancer treatments. See, e.g., Achi et al. (2020), supra; Testa et al. (2019), supra.

[0080] Thus, among other things, the disclosure contemplates that a targeted therapeutic binding protein with highly-specific binding to CD 123, which, among other things, can also antagonize IL-3-mediated signaling is a promising strategy for targeted killing of CD123 expressing cancer cells (such as depicted in FIG. IB, where a CD123 binding protein stops IL-3 from binding, which can, in some embodiments, prevent heterodimerization with CD131 and subsequent downstream signaling).III. CD123 BINDING PROTEINS

[0081] The disclosure provides synthetic CD123 binding proteins, and methods of identifying, making, characterizing, formulating, and using such CD123 binding proteins. The synthetic CD 123 binding proteins have improved manufacturability, increased specificity, and affinity as compared to existing agents (e.g., antibodies, modified cytokines, CAR-T cells) that bind to CD123. For example, other CD123 antibodies and / or binding proteins may not specifically block IL-3 from binding to CD123. Without wishing to be bound by theory, the disclosure contemplates that blocking IL-3 from interacting with CD123 can decrease proliferation of malignant cells. Similarly, large molecules such as biologies (e.g., monoclonal antibodies that bind to CD 123 such as talacotuzumab) are expensive to produce, are challenging to produce in uniform batches of drug substance (including, especially, e.g., at commercially-scalable amounts), and can be challenging to formulate, transport, store, and administer to subjects.

[0082] The synthetic CD123 binding proteins disclosed herein avoid certain such disadvantages as they have high binding affinity to CD123, can be engineered to have desired pharmacodynamic and pharmacokinetic properties (e.g, a desirable circulating half-life in plasma), are chemically and thermally stable, and are resistant to protease degradation (e.g, via L-X-R-R sites, wherein X represents any amino acid residue), resistant to deamination, lack post-translational modification (e.g., glycosylation, e.g., N- linked glycosylation, e.g., glycosylation through N-X-S / T, wherein X represents any amino acid residue), and are stable in different redox environments.

[0083] A synthetic CD123 binding protein described herein can have mM-level solubility. In some embodiments, the CD123 binding protein can have a solubility greater than 0.01 mg / mL, 0.05 mg / mL, 0.1 mg / mL, 0.5 mg / mL, 1 mg / mL, 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, or 80 mg / mL in an aqueous solution.

[0084] The CD 123 binding proteins described herein are designed to specifically bind CD 123, and, in certain embodiments, block IL-3 from binding to CD 123. Depending upon the circumstances, the synthetic CD 123 binding proteins bind CD 123 and prevent downstream cellular signaling (such as in a hematopoietic cell, e.g., a cancer cell, see, e.g.,schematic in FIG. IB). Synthetic CD123 miniproteins as provided herein can, in some embodiments, bind with an affinity at least at a level of a reference CD 123 binding protein.

[0085] Synthetic CD123 binding proteins disclosed herein can be conjugated to or with conjugated (e.g., chemically conjugated or as fusion proteins) one or more effector molecules. For the example, the CD123 binding proteins described herein can be bound to one or more other synthetic binding proteins, which can be the same or different. The resulting proteins can be monovalent, bivalent, or multivalent. For example, a first CD 123 binding protein described herein can be conjugated to a second CD123 binding protein that can be the same as or different from the first CD 123 binding protein. The resulting molecule is bivalent. Alternatively, a first CD123 binding protein described herein can be conjugated to a second binding protein that binds a different target of interest such as, e.g., a cell-specific surface protein (e.g., on a second, different cell, e.g., a CAR-expressing cell, e.g., a CAR-T cell, e.g., an effector binding protein, e.g., on a therapeutic cell such as a chimeric antigen receptor expressing cell to engage a CD 123 -expressing cancer cell to a therapeutic cell), serum albumin (e.g., to extend serum half-life), etc., which may optionally be bound to a third binding protein that can be, in some embodiments, a second CD123 binding protein or the protein to the second target. The resulting molecule is bivalent or trivalent and can bind to cells expressing CD123 (e.g., cancer cells) and another cell expressing another target (e.g., an effector binding protein, e.g., a therapeutic cell such as a cell expressing a CAR, e.g., a CAR-T cell, etc.). In addition, it is contemplated that the resulting molecule can be multivalent and contain two, three, four, five, six or seven miniproteins, which can be the same or different.

[0086] The synthetic CD123 binding proteins disclosed herein can be to or with conjugated (e.g., chemically conjugated or as fusion proteins) one or more effector molecules. For the example, the CD 123 binding proteins described herein can be bound to one or more other synthetic binding proteins, which can be the same or different. The resulting proteins can be monovalent, bivalent, or multivalent. For example, a first CD 123 binding protein described herein can be attached to (e.g., by a linker) and / or produced as a conjugate with another agent such as a small molecule or chelator that is recognized by a second binding protein that itself is attached to a target cell (e.g., a modified chimeric antigen receptor associated with a cell). By way of non-limiting example, the effector can be or comprise a fhiorophore (e.g., fluorescein, fluorescein derivatives, indocyanines, indocyanine derivatives, cyanines, cyanine derivatives), or chelator (e.g., tetraxetan (DOTA)), or othersmall molecules. For example, the fluorophore may be fluorescein isothiocyanate (FITC), fluorescein 5-maleimide, fluorescein-5-carboxamide, fluorescein-6-carboxamide, or 6- FAM phosphoramidite.

[0087] An effector can also be a standalone molecule, such as, for example, a detectable label and / or a toxic molecule. In either case, a detectable label may be used in combination with a synthetic CD123 binding protein for example, to detect and / or monitor a cell type e.g., a cancer cell), such as in a subject or in an assay. In certain embodiments, a CD123 binding protein can comprise an effector with a cytotoxic molecule, such that when the CD123 binding protein binds to CD123, the toxic molecule is delivered to the cell.Exemplary cytotoxic molecules include diphtheria toxin or one or more fragments thereof, auristatins (e.g., monomethyl auristatin (MMAE)), taxanes e.g. docetaxel, paclitaxel, etc.), radionuclides, cytotoxic antibiotics, or fragments thereof e.g., doxorubicin, etc.).

[0088] Depending on circumstances, an effector may comprise an endogenous target (e.g., one that is present on a particular cell, such as a target cell. In certain embodiments, the target cell can be an engineered cell (e.g., such as a cell expressing a chimeric antigen receptor (CAR), e.g., a CAR-T cell) designed to express a protein that binds to a CD 123 binding protein).

[0089] When an effector that binds to a target on a second cell (e.g., a cell expressing a CAR, e.g., a CAR-T cell) is combined with a synthetic CD123 binding protein, the resulting molecule is a conjugate that can bind CD 123 on a first cell (e.g., a cancer cell) through the CD 123 binding protein as well as the second target molecule on a second cell (e.g., a CAR-T cell, etc.), through the effector.

[0090] In one aspect, the disclosure provides a synthetic CD123 binding protein that comprises:(a) an amino acid sequence from 35 amino acids to 100 amino acids in length;(b) a net negative charge in phosphate buffered saline (PBS);(c) a binding affinity for CD 123 stronger than 1 pM; and(d) a stability profile such that the protein (i) retains at least 90% binding affinity to CD123 upon cooling to room temperature after thermal denaturation at 95°C in PBS for at least about five minutes relative to the protein prior to thermal denaturation; (ii) retains at least 90% binding affinity to CD 123 after incubation for 16 hours at 37°C of incubation in PBS relative to the protein under the same conditions prior to incubating; and / or (iii) retains at least 90% bindingaffinity to CD 123 in PBS following chemical denaturation in 4 M urea for 1 hour at room temperature relative to the protein prior to chemical denaturation.

[0091] In another aspect, the disclosure provides a synthetic CD123 binding protein, the binding protein comprising:(a) an amino acid sequence from 30 amino acids to 95 amino acids in length;(b) a net negative charge in PBS;(c) a binding affinity for CD 123 stronger than 10 pM;(d) at least three alpha helices;(e) at least two amino acid loops, where a first loop having a first amino acid sequence connects a terminal amino acid (e.g., a C-terminal amino acid) of a first alpha helix to a terminal amino acid (e.g., a N-terminal amino acid) of a second alpha helix, and a second loop having a second amino acid sequence connects a second, terminal amino acid (e.g., a C-terminal amino acid) of the second alpha helix to a terminal amino acid (e.g., an N-terminal amino acid) of a third alpha helix; and(f) a hydrophobic core defined by at least two hydrophobic amino acids present in at least one, two or three of the three alpha helices.

[0092] Synthetic CD123 binding proteins of the present disclosure may include naturally- occurring or non-naturally occurring amino acids. It is understood that certain amino acids may have and / or take on different characteristics (e.g., hydrophobic, hydrophilic, neutral, etc.) depending upon the context (e.g., macro and / or micro-environment including, but not limited to surrounding amino acids, environmental conditions such as solvent type, pH, etc.). Various terms and phrases may be used herein to describe, identify, and / or characterize amino acids. In addition, a single amino acid, at any given time, may have more than one characteristic or identity. Depending upon the context, a hydrophobic amino acid may be selected from alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine, tryptophan, tyrosine, lysine, and arginine. A hydrophilic amino acid may be an amino acid selected from cysteine, aspartic acid, glutamic acid, histidine, lysine, asparagine, glutamine, arginine, serine, threonine, tryptophan, and tyrosine. A charged amino acid may be an amino acid selected from arginine, histidine, lysine, aspartic acid (aspartate), and glutamic acid (glutamate). A positively-charged amino acid may be an amino acid selected from arginine, histidine, and lysine. A negatively- charged amino acid may be an amino acid selected from aspartic acid (aspartate) andglutamic acid (glutamate). An uncharged or neutral amino acid may be selected from alanine, cysteine, phenylalanine, glycine, histidine, isoleucine, leucine, methionine, asparagine, proline, glutamine, serine, threonine, valine, tryptophan, and tyrosine. Synthetic CD123 binding proteins of present disclosure have a primary structure comprising certain key features. For example, synthetic CD123 binding proteins can have amino acid sequences that include various combinations of hydrophobic and solvent accessible amino acids organized into certain domains. Primary structures (i.e., amino acid sequences) of the synthetic CD 123 binding proteins will have a combination of one or more types (e.g., hydrophobic, e.g., solvent accessible) amino acids.

[0093] In certain embodiments, the synthetic CD123 binding protein comprises one or more of the following features (a) the first, second, and / or third alpha helix each contains at least one hydrophobic amino acid; (b) the first, second, and / or third alpha helix each contains at least two or three hydrophobic amino acids; (c) the first, second, and / or third alpha helix each contain at least one or two solvent accessible amino acids; (d) the first and / or second loop contains at least one hydrophobic amino acid, or (e) the binding protein comprises any combination of elements selected from (a), (b), (c), and (d).

[0094] Similarly, it is contemplated that the CD123 binding can comprise one or more of the following features: (a) the second and third alpha helix each contains at least two hydrophobic amino acids; (b) the first, second, and third alpha helix each contains at least one solvent accessible amino acid; (c) the first, second, and third alpha helix each contain at least two hydrophobic and one solvent accessible amino acids; (e) the second and third alpha helix each contains at least four solvent accessible amino acids; and / or (f) the first loop contains at least one hydrophobic amino acid.

[0095] In each of the foregoing aspects, the synthetic CD123 binding protein comprises one or more of the following features: (a) free of tryptophan amino acids; (b) free of methionine amino acids; (c) free of lysine amino acids; (d) does not comprise an unpaired cysteine amino acid when cysteine amino acids are present in the protein (e) free of N- linked glycosylation sites (e.g., N-X-S / T, wherein X represents any amino acid residue); (f) free of protease cleavage sites (e.g., L-X-R-R sequences, wherein X represents any amino acid residue); and (g) soluble up to at least 0.5 mM in PBS at 4°C for one month.

[0096] Preferably, the CD 123 binding sites are designed to be free of glycosylation sites (e.g., free of N-linked glycosylation sites), for example, free of peptide sequences that are substrates for glycosylation (e.g., N-X-S / T (wherein X represents any amino acid residue)),which can be a substrate for an oligosaccharyltransferase (OST) complex). Alternatively or in addition, the CD 123 binding sites are designed to be free of protease cleavage sites, for example, free of peptide sequences that are substrates for proteases (e.g., L-X-R-R (wherein X represents any amino acid residue), which can be a substrate for a Kexin / KEX2 protease). Similarly, the CD123 binding proteins may also be designed to be free of other protease cleavage sites for other proteolytic enzymes such as trypsin, chymotrypsin, elastase, subtilisin, etc. Some synthetic CD123 binding proteins may be designed to avoid cleavage by certain other enzymes, including depending upon linkers and fusion protein partners.

[0097] It is contemplated that the synthetic CD123 binding protein can comprise from 35 amino acids to 100 amino acids in length, from 35 amino acids to 70 amino acids in length, from 35 amino acids to 75 amino acids in length, from 35 amino acids to 65 amino acids in length, from 35 amino acids to 60 amino acids in length, from 35 amino acids to 55 amino acids in length, from 35 amino acids to 50 amino acids in length, from 35 amino acids to 45 amino acids in length, from 40 amino acids to 75 amino acids in length, from 40 amino acids to 80 amino acids in length, from 40 amino acids to 70 amino acids in length, from 40 amino acids to 65 amino acids in length, from 40 amino acids to 60 amino acids in length, from 40 amino acids to 55 amino acids in length, from 45 amino acids to 70 amino acids in length, from 45 amino acids to 65 amino acids in length, from 45 amino acids to 60 amino acids in length, from 50 amino acids in length to 60 amino acids in length, from 45 amino acids to 55 amino acids in length, from 50 amino acids to 70 amino acids in length, from 50 amino acids to 65 amino acids in length, or from 40 amino acids to 50 amino acids in length. In certain embodiments, the synthetic CD123 binding protein comprises 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids.

[0098] Depending upon the circumstances, the N-terminus of the first alpha helix is preceded by one or more N-terminal amino acids and / or the C-terminus of the third alpha helix is followed by one or more C-terminal amino acids. The at least one N-terminal amino acid can be 1, 2, 3, 4, or more amino acids. For example, a miniprotein with a consensus sequence comprising that of SEQ ID NO: 25 or SEQ ID NO: 29 can have a sequence of SGY, where the tyrosine can be attached, through a peptide bond, to the first (N-terminal) amino acid of DI. The at least one C-terminal amino acid can be 1, 2, 3, 4, or more amino acids. For example, a miniprotein with a consensus sequence comprising that of SEQ ID NO: 25 can comprise a sequence of RX45GS, wherein X45 can be Y, K, H, R,or E, or a miniprotein with a consensus sequence comprising that of SEQ ID NO: 29 can have a sequence of RX45GS, wherein X45 can be Y, K, or E, and, in each instance of a miniprotein comprising a consensus sequence of SEQ ID NO: 25 or SEQ ID NO: 29 where the N-terminal arginine of RX45GS can be attached, through a peptide bond, to the last (C- terminal) amino acid of D3.

[0099] In certain embodiments, the N-terminus of the first alpha helix in the synthetic CD123 binding protein is preceded by one or more N-terminal amino acids, and / or the C- terminus of the third alpha helix in the synthetic CD123 binding protein is followed by one or more C-terminal amino acids. Depending on the circumstances, the N-terminus of the first alpha helix comprises an N-terminal extension, e.g., comprising 5, 10, 15, 20, 25, 30, 35, 36, 37, 38, 39, 40 or more amino acids in length. For example, in some embodiments, an N-terminal extension comprises an amino acid sequence comprising that of SEQ ID NO: 34. In certain embodiments, the C-terminus of the third alpha helix is followed by one or more C-terminal amino acids. In some embodiments, the C-terminus of the third alpha helix comprises a C-terminal extension, e.g., comprising 5, 10, 15, 20, 25, 30, 35, 36, 37, 38, 39, 40 or more amino acids in length. For example, in some embodiments, the C- terminal extension comprises an amino acid sequence comprising that of SEQ ID NO: 37.

[0100] Among other things, the disclosure provides a means for extending the N- and / or C-terminus of a miniprotein (e.g., a Reference Miniprotein, e.g., comprising an amino acid sequence of SEQ ID NOs: 1-24). For example, an N-terminal extension and / or a C- terminal extension can be or comprise one or more tags (e.g., a myc tag, a hemagglutinin (HA) tag, etc.). Sequences of myc tags and HA tags are known to those of skill in the art. An exemplary HA tag sequence is set forth in SEQ ID NO: 38 (YPYDVPDYA). An exemplary myc tag sequence is set forth in SEQ ID NO: 39 (EQKLISEEDL).

[0101] An N-terminal extension and / or a C-terminal extension can be a linker or can comprise a linker. A linker can be an ordered or disordered linker. A linker can be a peptide linker, such as set forth in SEQ ID NOs: 36 (GSGSGGGSGGSSGGS) and / or SEQ ID NO: 40 (SSGGSSSSGSGSGSGGGGGGS). Combinations of tags and linkers (e.g., including those set forth in Table 4A and / or Table 9) can be used to make up N- and / or C- terminal extensions as disclosed herein.

[0102] For example, in certain embodiments, a miniprotein of the disclosure may further comprise an N-terminal extension comprising or consisting of a polypeptide having an amino acid sequence of SEQ ID NO: 34(KDNSSTIEGRYPYDVPDYALQAGSGSGGGSGGSSGGS) and / or a C-terminal extension comprising or consisting of a polypeptide having an amino acid sequence of SEQ ID NO: 37 (SSGGSSSSGSGSGSGGGGGGSEQKLISEEDL).

[0103] An N-terminal extension can comprise a polypeptide having an amino acid sequence of KDNSSTIEGRYPYDVPDYALQA (SEQ ID NO: 35) and / or GSGSGGGSGGSSGGS (SEQ ID NO: 36). In certain embodiments, polypeptides of SEQ ID NOs: 35 and 36 can be linked by a peptide bond between the C-terminal alanine of SEQ ID NO: 35 and the N-terminal glycine of SEQ ID NO: 36 to form an N-terminal extension as set forth in SEQ ID NO: 34.

[0104] A C-terminal extension can comprise a polypeptide having an amino acid sequence of SEQ ID NO: 39 and / or SSGGSSSSGSGSGSGGGGGGS (SEQ ID NO: 40). The polypeptides of SEQ ID NOs: 39 and 40 can be linked by a peptide bond between the C-terminal serine of SEQ ID NO: 40 and the N-terminal glutamic acid of SEQ ID NO: 39 to form an N-terminal extension as set forth in SEQ ID NO: 37.

[0105] CD123 binding proteins of the disclosure can have both an N- and C-terminal extension, such as set forth in SEQ ID NOs: 34 and 37 and still bind CD123. By way of non-limiting example, a CD 123 binding protein (e.g., Reference Miniprotein 1 of SEQ ID NO: 1) that is being tested in a cell-based assay such as a yeast display (see, e.g., are capable of binding CD123) can have an N- and C-terminal extension, such as set forth in SEQ ID NO: 34, or 41-64). Such CD123 binding proteins bind to CD123 with at least 20 nM affinity as measured by yeast-display assay.

[0106] A synthetic CD123 binding protein may have an amino acid sequence comprising, consisting essentially of, or consisting of an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more or 100% identity with or to a reference sequence or component thereof, wherein the reference sequence is selected from any of SEQ ID NOs: 1-24 or 41-64, and portions (e.g., domains) thereof. In some embodiments, a synthetic CD123 binding protein may have an amino acid sequence comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% or more or 100% identity with or to a reference sequence or component thereof selected from any of SEQ ID NOs: 1-24 or 41-64, and portions thereof.

[0107] By way of non-limiting example, a structural arrangement in a synthetic CD 123 binding protein may be depicted as N-Zn6-HniLn2Hn3Ln4Hn5-Zn7-C, where H is an alpha helix, L is a loop, Z optionally comprises between 0-4 additional amino acids on each of the N and / or C-terminal regions flanking the N- and C-terminal sides of the first and third alpha helix, each of ni-n? represents an integer indicating the number of amino acids in that structural domain, N and C represent N-terminal and C-terminal domains, respectively.The number of H amino acids does not have to be the same across helices, for example, m, ns, and ns may be, but do not have to be, the same numbers. Similarly, n2 and may be, but do not have to be, the same number. In addition, ne and n? can be 0, 1, 2, 3, or 4. For example, an exemplary formula of a synthetic CD 123 binding protein may comprise N- Z3H11L4H13L3H9-Z4-C as depicted pictorially below denoting the amino acids in a helix domain (H) or a loop domain (L):N- Z Z Z -HHHHHHHHHHHLLLLHHHHHHHHHHHHHLLLHHHHHHHHH- Z Z Z Z -C.

[0108] Any given H domain (e.g., Hni) in an alpha helix that is part of a synthetic CD123 binding protein may independently contain between about 4 amino acids and about 20 amino acids in length. An H domain may independently comprise 4, 5, 6, 7, 8, 9, 10, 11,12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids in length. Depending on circumstances, in certain embodiments, an H domain can comprise more than 20 amino acids in length.

[0109] Loops disposed between alpha helices may also be of the same or different lengths. Each loop may independently comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 amino acids in length. In some embodiments, a loop can be more than 25 amino acids.

[0110] Each alpha helix may comprise the same number of amino acids in each of its H domains or different numbers of amino acids in length in reference to the primary structure of each helical region. That is, in some synthetic CD123 binding proteins each H domain in the binding protein is the same length. In some synthetic CD123 binding proteins one or more H domains has a different length relative to other H domains in the binding protein. In some embodiments, an H domain has zero, one, two, three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, or more conserved amino acids (e.g. relative to other CD 123 binding proteins).

[0111] A person skilled in the art can determine which amino acids of a given sequence constitute a loop or a helix (see, e.g., Mirdita, et al. (2022) NAT. METHODS (19): 679-682).

[0112] In an exemplary CD123 binding protein disclosed herein, the binding protein comprises at least three alpha helices, and at least two loops (a first loop and a second loop), wherein the first loop has a first amino acid sequence that connects a terminal amino acid (e.g., a C-terminal amino acid) of a first alpha helix to a terminal amino acid (e.g., a N- terminal amino acid) of a second alpha helix, and a second loop having a second amino acid sequence connects a second, terminal amino acid (e.g., an C-terminal amino acid) of the second alpha helix to a terminal amino acid (e.g., an N-terminal amino acid) of a third alpha helix.

[0113] A synthetic CD 123 binding protein may have one of several consensus sequence structures. Consensus sequences will generally have certain “fixed” amino acid positions as well as those that can be varied, such as by changing to another amino acid. Sometimes changing amino acids at certain positions can alter the function of the synthetic binding protein by increasing or decreasing affinity for the target (i.e., CD123). However, all the binding proteins disclosed herein, although having different primary structures have a minimal “threshold” binding affinity to CD 123. In some embodiments, a threshold binding affinity may be greater than about 10 mM, about 1 mM, about 750 nM, about 250 nM, about 100 nM, about 75 nM, about 50 nM, about 40 nM, about 30 nM, about 20 nM, about 10 nM, or about 1 nM.

[0114] A synthetic CD123 binding protein disclosure may be represented according to a formula shown as one or more domains, wherein each domain optionally has one or more conserved amino acid residues and / or a particular structure (e.g., loop, e.g., helix). For example, an exemplary synthetic CD123 binding protein comprises an amino acid sequence arranged in a primary structure ofN-Z1-D1-L1-D2-L2-D3-Z2-C (Formula I) where DI, D2 and D3 represent alpha helices and LI and L2 represent loops connecting the alpha helices.

[0115] Depending upon the circumstances, the N-terminus of the first alpha helix is preceded by one or more N-terminal amino acids and / or the C-terminus of the third alpha helix is followed by one or more C-terminal amino acids. The at least one N-terminal amino acid can be 1, 2, 3, 4, or more amino acids (e.g, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 36, 37, 38, 39, or 40). For example, a miniprotein with a consensus sequence comprising that of SEQ ID NO: 25 or SEQ ID NO: 29 can have a Z1 domain comprising an amino acid sequence of SGY, where the tyrosine can be attached, through a peptide bond, to the first(N-terminal) amino acid of DI. The at least one C-terminal amino acid can be 1, 2, 3, 4, or more amino acids (e.g., 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 36, 37, 38, 39, or 40). For example, a miniprotein with a consensus sequence comprising that of SEQ ID NO: 25 can comprise a Z2 domain comprising a sequence of RX45GS, wherein X45 can be Y, K, H, R, or E, or a miniprotein with a consensus sequence comprising that of SEQ ID NO: 29 can have a Z2 domain comprising a sequence of RX45GS, wherein X45 can be Y, K, or E, and, in each instance of a miniprotein comprising a consensus sequence of SEQ ID NO: 25 or SEQ ID NO: 29 where the N-terminal arginine of RX45GS can be attached, through a peptide bond, to the last (C-terminal) amino acid of D3.

[0116] The amino acid sequences of the exemplary consensus sequences for various miniproteins developed in Examples 1, 2, and 6 are set forth in TABLE 1. Bold residues represent helices and correspond to DI, D2, and D3 in order along a given consensus sequence. Exemplary consensus sequences for DI, D2, and D3 for each miniprotein are set forth in TABLE 2A. Positions for each variable amino acid along the length of a consensus sequence as set forth in TABLE 1 are set forth in TABLE 2B.TABLE 1. Exemplary CD123 Synthetic Binding Protein Consensus Sequences*Bolded and italicized text indicates amino acids present in a first alpha helix (positions 4-14), a second alpha helix (positions 19-31), and a third alpha helix (positions 35-43).TABLE 2A. Exemplary CD123 Synthetic Binding Protein Domain Consensus SequencesTABLE 2B. Exemplary Miniprotein Variable SubstitutionsA. Paratope Identification

[0117] A synthetic CD 123 binding protein can bind to its target via a paratope. A paratope of the disclosure can be represented by paratope consensus sequence. Consensus sequences sometimes have certain “fixed” amino acid positions as well as those that can be varied, such as by changing an amino acid at a certain position to another amino acid. At the paratope, typically only certain amino acid changes can be made without materially decreasing CD 123 binding potency (e.g., to a binding strength of less than 20 nM as measured by on-cell yeast affinity). Approaches such as saturation site mutagenesis can be used to determine which paratope positions cannot tolerate substitutions (e.g., without thebinding potency of the CD 123 binding protein materially decreasing) or can tolerate one or more substitutions, as well as which amino acid substitutions are tolerable.

[0118] Sometimes changing amino acids at certain positions, such as the paratope, can alter the function of the synthetic binding protein by increasing or decreasing affinity for its target (z.e., CD123). Using SSM, for example, binding affinities can be evaluated by making changes to each identified paratope residue and determining a minimal “threshold” binding affinity to CD123 to determine if a paratope residue is tolerant or intolerant to (i) substitution and, if so, (ii) which amino acid(s) can be substituted into the position where a paratope residue is tolerant to substitution. In some embodiments, a threshold binding affinity may be stronger than about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, or 5 nM.

[0119] Loss of binding potency can be measured by metrics such as fold-change (e.g, relative to the binding potency prior to one or more substitutions at the paratope), and / or as binding strength (e.g., binding strength that decreases relative to prior to the substitution, as measured in molar values). Binding affinity can be measured using a variety of approaches, including, but not limited to SPR and cell-based assays. One such cell-based approach to measuring binding affinity is a yeast display assay. For example, to evaluate binding affinity in a CD123 binding protein, the CD123 binding protein can be displayed on the surface of yeast cells, and contacted with a soluble CD123 protein. The CD123 protein can be presented at varying concentrations, such as 10 pM to 1 M. An affinity curve can be generated using concentrations at upper and lower levels of the range, to determine binding affinity of the miniprotein being evaluated. In a given context, e.g., in a particular assay, a change in binding strength (reflected by a numerical increase in binding affinity, e.g., from about 1 nM to about 10 nM is a decrease in strength) beyond a certain threshold can also be considered material, provided however, that binding affinities are all about 20 nM or stronger. It is contemplated that, in some embodiments, a CD123 binding protein with a yeast on-cell affinity of weaker than about 20 nM is inefficient at blocking IL-3 mediated signaling. In embodiments where the CD123 binding protein comprises an effector, is inefficient at binding to, e.g., a second, different cell such as a therapeutic cell (e.g, a CAR-expressing cell, etc.) comprising an effector binding protein, thus, inhibiting the effector from serving its purpose. In certain embodiments, a range of acceptable concentrations and / or fold changes within which a substitution is deemed “tolerable” can be determined based on the affinity curves and measurements. That is, in someembodiments, a miniprotein can show binding saturation at 1 nM, but if an amino acid substitution decreases the binding affinity to weaker than about 20 nM, the substitution is not considered tolerated, as binding potency to CD123 will have decreased materially. In certain embodiments, a binding potency of weaker than about 20 nM (e.g, about 30 nM, about 35 nM, about 40 nM, about 50 nM, or higher, etc.) as measured using on-cell affinity in a yeast display assay, where a CD123 binding protein of SEQ ID NO: 4 (Reference Miniprotein 4) has an on-cell affinity of 75 pM in a yeast display assay, is considered a material loss of binding potency. Thus, depending upon a given context, a CD123 binding protein with an on-cell binding potency of about 20 nM or weaker (e.g., about 30 nM, about 35 nM, about 40 nM, about 50 nM, or higher, etc.) when the comparator has an on- cell binding potency of stronger than about 75 pM (e.g., about 75 pM, about 70 pM, about 65 pM, about 60 pM, about 55 pM, about 50 pM, about 45 pM, or less, etc.) can be considered a material loss in binding potency. Binding potency can also be measured using other approaches such as SPR. In such embodiments, a material loss in binding strength can be represented by a miniprotein with a binding affinity of about 20 nM or greater, as compared to a CD 123 binding protein with a binding affinity of less than about 20 nM. In other words, a material loss in binding potency as measured by SPR could occur if a CD123 binding protein (e.g., such as provided herein, for example, in Tables 1, 2A, 2B, 6, 7 A, and 9) having a binding affinity of about 20 nM or stronger (e.g., about 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, 0.01 nM or less ) is changed to contain one or more substitutions that results in a binding affinity weaker than about 20 nM (e.g., about 30 nM, about 40 nM, about 50 nM, or higher, etc.).

[0120] In certain embodiments, a decrease in binding strength after an amino acid substitution from about 1 nM to above about 20 nM can be considered a material decrease in binding strength. As a result, any amino acid change (e.g., substitution) at a position that results in a binding affinity of greater than about 20 nM or weaker would not be considered tolerable. In some embodiments, when a reference miniprotein (e.g., such as a CD123 binding protein as provided herein, e.g., Reference Miniprotein 4, etc.) has an on-cell binding affinity of about 75 pM, a test miniprotein (e.g, having one or more substitutions relative to the reference) with an on-cell binding affinity of about 20 nM or weaker (e.g, about 25 nM, about 30 nM, about 40 nM, about 50 nM, or higher, etc.) is considered to have a material loss of binding potency relative to the reference miniprotein.

[0121] Tolerated substitutions in CD123 binding proteins of the disclosure are those in which a CD123 binding protein with one or more substitutions relative to a reference miniprotein (e.g., as provided herein) has a binding affinity of about 20 nM or stronger (e.g., 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, 0.01 nM or less). Such numbers may also be expressed in fold-change based on the disclosure and parameters provided herein.

[0122] Paratope identity was determined using more than 700 miniproteins. A conformational paratope of CD123 binding proteins of the disclosure can be set forth according to the formula:X6 - X7 - X9 - X10 - XI 1 - X39 - X43, where position numbers 6, 7, 9, 10, 11, 39, and 43 correspond to linear, ordinal positions of amino acid residues relative to SEQ ID NO: 25 or 29, each of which is a 47-mer. Thus, paratope residues can be found at positions 6, 7, 9, 10, 11, 39, and 43 relative to SEQ ID NOs: 25 or 29. Certain paratope residues can be immutable in that they are completely intolerant to substitution without material loss of function (e.g., as a CD123 binding protein, e.g., having a binding potency beyond a threshold, such as provided herein).

[0123] In certain embodiments, the synthetic CD 123 binding protein binds to CD 123 through a paratope of the CD123 binding protein, which paratope is defined by amino acids X6 - X7 - X9 - X10 - XI 1 - X39 - X43, wherein X6 can be Y or F, X7 can be A, S, or G, X9 is E, X10 can be Y, F, or W, XI 1 can be L or I, X39 can be L, M, I, or V, and X43 can be H or Y, and wherein the amino acid numbering corresponds to the numbering of SEQ ID NOs: 25 or 29.

[0124] In one aspect, the disclosure provides a means for blocking binding of IL-3 to CD123. In another aspect, the disclosure provides a means for binding CD123. In some embodiments, the means for binding CD123 comprises a conformational paratope defined by amino acids X6 - X7 - X9 - X10 - XI 1 - X39 - X43, wherein X6 can be Y or F; X7 can be A, S, or G; X9 is E; X10 can be Y, F, or W; XI 1 can be L or I; X39 can be L, M, I, or V; and X43 can be H or Y; and wherein the amino acid numbering corresponds to the numbering of SEQ ID NOs: 25 or 29.

[0125] In some embodiments, the conformational paratope is as follows: X6 is Y, X7 is A, X9 is E, X10 is Y, XI 1 is L, X39 is L, and X43 is H. Without material loss of binding potency (e.g., as measured by binding at 250 nM or stronger), X6 in addition to Y, can also be F; X7 in addition to A can also be S or G; X10 in addition to Y, can also be F or W; XI 1in addition to L can also be I; X39 in addition to L, can also be M, I, or V; and X43, in addition to H, can also be Y. X9 cannot be substituted without material loss of binding potency. The paratope can be made up of any combination of provided amino acids as set forth as permissible substitutions in X6 - X7 - X9 - X10 - XI 1 - X39 - X43.

[0126] In one aspect, the disclosure provides a synthetic CD123 binding protein comprising:(i) an amino acid sequence arranged in a primary structure of Z1-D1-L1-D2-L2-D3-Z2 (Formula I), wherein DI, D2, and D3 are domains 1, 2, and 3, respectively; LI, and L2, are loops 1, and 2, respectively; and Z1 and Z2 are N- and C-terminal regions, respectively; and(ii) an amino acid of SEQ ID NO: 25, wherein DI, D2, and D3, independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 26, wherein X4 is A or V;X5 is Y, E, H, Q, F, or V; and X14 is E, L, or I;(b) D2 comprises an amino acid sequence of SEQ ID NO: 27, wherein X20 is E or T; X25 is L, Y, Q, S, G, or N; X26 is R, K, V, or I; X27 is H, V, or A; X30 is E, L, or D; and X31 is R, I, or Q; and(c) D3 comprises an amino acid sequence of SEQ ID NO: 28, wherein X35 is V, K, R, E, H, G, Q, or S; X37 is Q, or S; X41 is D, S, or E; and X43 is H or Y.

[0127] Furthermore, LI can comprise an amino acid sequence of GX16IS, wherein X16 is A or F; and / or L2 comprises an amino acid sequence of GDD. Depending on circumstances, Z1 can comprise an amino acid sequence of SGY and / or Z2 can comprise an amino acid sequence of RX45GS, wherein X45 is Y, K, H, R, or E.

[0128] In certain embodiments, the synthetic CD123 binding protein comprises an amino acid sequence of SEQ ID NO: 25, wherein X4 is A or V; X5 is Y, E, H, Q, F or V; X14 is E, L, or I; XI 6 is A or F; X20 is E or T; X25 is L, Y, Q, S, G, or N; X26 is R, K, V, or I; X27 is H, V, or A; X30 is E, L, or D; X31 is R, I, or Q; X35 is V, K, R, E, H, G, Q, or S; X37 is Q, or S; X41 is D, S, or E; X43 is H or Y; and X45 is Y, K, H, R, or E.

[0129] In certain embodiments, the CD123 binding protein comprises an amino acid sequence selected from any of SEQ ID NOs: 1-24 and 41-64.

[0130] In one aspect, the disclosure provides a synthetic CD123 binding protein comprising:(i) an amino acid sequence arranged in a primary structure of Z1-D1-L1-D2-L2-D3-Z2 (Formula I), wherein DI, D2, and D3 are domains 1, 2, and 3, respectively; LI, and L2, are loops 1, and 2, respectively; and Z1 and Z2 are N- and C-terminal regions, respectively; and(ii) an amino acid of SEQ ID NO: 29, wherein DI, D2, and D3, independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 30, wherein X5 is Y, Q, or F;(b) D2 comprises an amino acid sequence of SEQ ID NO: 31, wherein X25 is L, Q, S, G, or N; and X26 is R, K, or I; and(c) D3 comprises an amino acid sequence of SEQ ID NO: 32, wherein X35 is V, K, E, H, G, or S; and X43 is H or Y.

[0131] Furthermore, LI can comprise an amino acid sequence of SEQ ID NO: 33; and / or L2 comprises an amino acid sequence of GDD. Depending on circumstances, Z1 can comprise an amino acid sequence of SGY and / or Z2 can comprise an amino acid sequence of RX45GS, wherein X45 is Y, K, or E.

[0132] In certain embodiments, the synthetic CD123 binding protein comprises an amino acid sequence of SEQ ID NO: 29, wherein X5 is Y, Q, or F; X25 is L, Q, S, G, or N; X26 is R, K, or I; X35 is V, K, E, H, G, or S; X43 is H or Y; and X45 is Y, K, or E.

[0133] In certain embodiments, the CD123 binding protein comprises an amino acid sequence selected from any of SEQ ID NOs: 1-9 or 41-49.

[0134] The synthetic CD123 binding proteins disclosed herein can have a binding affinity from between about 1 pM to about 0.001 nM; about 1 pM to about 0.01 nM; about 1 pM to about 0.75 nM; about 1 pM to about 0.5 nM; about 1 pM to about 0.25 nM; about 1 pM to about 1 nM; about 0.5 pM to about 1 nM; about 0.25 pM to about 1 nM; about 0.10 pM to about 1 nM; about 75 nM to about 1 nM; about 50 nM to about 1 nM; about 25 nM to about 1 nM; about 10 nM to about 1 nM; and about 5 nM to about 1 nM. Furthermore, the synthetic CD 123 binding protein can have a binding affinity stronger than about 1 pM, about 0.75 pM, about 0.5 pM, about 0.25 pM, about 0.1 pM, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, about 0.1 nM, about 0.01 nM, and about 0.001 nM.

[0135] In some embodiments, a threshold binding affinity may be stronger than about 1 pM, about 100 nM, about 10 nM, about 1 nM, about 100 pM, about 10 pM, or about 1 pM.

[0136] It is contemplated that optimization of synthetic binding proteins may be achieved using optimized designs, such as, for example, modifications of one or more amino acids by substitution at one or more positions with a different amino acid. Optimization, such as by amino acid modifications, allows tunability of certain characteristics such as changes to (e.g., increases in) binding affinity and / or avidity.

[0137] Synthetic CD123 binding proteins can be optimized by affinity maturation techniques. For example, affinity maturation may be used on a sequence of a binding protein to create another synthetic CD 123 binding protein with at least the same or better selectivity and / or affinity for CD123 as compared to the starting sequence. Affinity maturation can be accomplished using techniques known to those of ordinary skill in the art, including, for example, generating libraries using error prone PCR, degenerate codons, synthetic oligonucleotide pools, or a combination thereof. These libraries can then be transformed into yeast and improved variants may be isolated by methods such as magnetic, flow cytometric, and / or FACS-based approaches. Computational design / redesign strategies may also be used when affinity maturing proteins and computer programs for implementing such approaches are known in the art. Prior to affinity maturation, synthetic binding proteins may be characterized to determine functional and structural features, such as binding affinity (e.g., for CD123) and conformation.

[0138] Synthetic CD123 binding proteins provided herein are engineered to have certain characteristics (e.g., binding affinity / avidity, binding specificity, e.g., for a target, e.g., for CD123). Various in silico, in vitro, and in vivo characterization assays may be used to evaluate these CD123 binding proteins. For example, binding assays can be used to determine competitive binding to a target, e.g., CD 123 as compared to binding of another molecule to the same target such as, e.g., IL-3 to CD123. Other assays can be used to determine binding affinity of a binding protein, e.g., a CD123 binding protein for its target, which can include for example, surface plasmon resonance (SPR), and flow cytometry.

[0139] The synthetic CD123 binding proteins of the present disclosure are designed to have certain stability characteristics. For example, a binding protein is stable in that it may retain at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 9%, 94%, 95%, 96%, 97%, 98%, 99% or substantially all of its binding affinity to CD123 upon cooling to room temperature after thermal denaturation at 95°C in a solution (e.g.,phosphate buffered saline (PBS)) for at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60 or more minutes relative to the synthetic CD 123 binding protein prior to thermal denaturation.

[0140] In some embodiments, a synthetic CD123 binding protein is stable in that it may retain at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 9%, 94%, 95%, 96%, 97%, 98%, 99% or substantially all of its binding affinity to CD123 after incubation at 37°C (e.g., for 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more hours) relative to the synthetic CD 123 binding protein prior to incubation.

[0141] Synthetic CD123 binding proteins of the present disclosure may also display stability in resistance to chemical denaturation and / or retention of stability after exposure to chemical denaturants. For example, a synthetic CD123 binding protein may be stable in that it may retain at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 9%, 94%, 95%, 96%, 97%, 98%, 99% or substantially all of its binding affinity to CD123 in PBS following exposure to a denaturing chemical (e.g., 4 M urea) at room temperature for about 15 minutes, 30 minutes, 45 minutes, 1, 2, 3, 4, 5, 6, 7, 8 or more hours relative to the binding affinity of the synthetic CD 123 binding protein prior to exposure to the chemical denaturant.

[0142] Synthetic CD123 binding proteins engineered, developed, and produced herein are selected and / or specific for human CD123.

[0143] In some embodiments, a synthetic CD 123 binding protein blocks binding of another binding protein (e.g., IL-3) to CD123. In some embodiments, a synthetic CD123 binding protein binds to another receptor, but binds to human CD 123 with a much greater affinity. For example, the binding affinity of a synthetic CD123 binding protein may be between 1 and 200-fold greater than that for any other binding partner (e.g., mouse CD123).

[0144] Affinity of a synthetic CD123 binding protein may be modified and may vary depending on modifications made to, for example, its primary sequence. A binding affinity may be at least 1-fold greater, at least 2-fold greater, at least 3-fold greater, at least 4-fold greater, at least 5-fold greater, at least 6-fold greater, at least 7-fold greater, at least 8-fold greater, at least 9-fold greater, 10-fold greater, at least 20-fold greater, at least 30-fold greater, at least 40-fold greater, at least 50-fold greater, at least 60-fold greater, at least 70- fold greater, at least 80-fold greater, at least 90-fold greater, at least 100-fold greater, at least 125-fold greater, at least 150-fold greater, at least 175-fold greater, at least 200-foldgreater than the affinity of the synthetic CD 123 binding protein for an unrelated (e.g., different) target (e.g., mouse CD123).IV. DESIGN OF CD123 SYNTHETIC BINDING PROTEINS

[0145] As provided herein, the synthetic CD 123 binding proteins bind strongly to and are highly specific for human CD123 (e.g., as compared to mouse CD123). It is contemplated that the specificity of the proteins developed and disclosed is due in part at least to the unique paratope residues interacting with human CD 123. A synthetic CD 123 binding protein in accordance with the present disclosure may have certain conserved or allowable residues in particular locations. For example, with respect to Reference Miniprotein 4 (SEQ ID NO: 4), it is contemplated that a synthetic CD123 binding protein with certain substitutions in accordance with TABLE 6 will maintain strong and specific binding to human CD123. It is further contemplated that certain residues are critical to specific binding, as set forth in TABLE 6 and described in Example 6.V. SYNTHESIS OF CD123 BINDING PROTEINS

[0146] The synthetic CD 123 binding proteins described herein may be produced by methods known to those of ordinary skill in the art. Methods may include, for example, biological approaches, such as recombinant approaches and / or chemical approaches, such as solid phase and / or liquid phase chemical synthesis, etc., or combinations thereof.

[0147] With regard to recombinant approaches, a variety of methodologies can be implemented to produce the binding proteins disclosed herein. For example, DNA molecules encoding the binding proteins can be synthesized chemically and / or cloned / produced using recombinant DNA methodologies. The resulting DNA molecules encoding binding proteins of interest can be ligated to other nucleotide sequences, including, for example, expression control sequences, to produce a gene expression construct (i.e., expression vector). Thereafter, the resulting expression vectors are introduced into host cells using conventional transfection or transformation techniques. Exemplary host cells include d, coli cells, Bacillus subtilis cells, Pichia Pastoris cells, Saccharomyces cerevisiae cells, Kluyveromyces lactis cells, Chinese hamster ovary (CHO) cells, human embryonic kidney 293 (HEK 293) cells, HeLa cells, baby hamster kidney (BHK) cells, monkey kidney cells (COS), and human hepatocellular carcinoma cells (e.g., Hep G2). The transformed host cells can be grown under conditions that permit the host cells to express the genes that encode the binding proteins.

[0148] Specific expression and purification conditions will vary depending upon the expression system employed. For example, if a gene is expressed in E. coli, it is first cloned into an expression vector by positioning the engineered gene downstream from a suitable bacterial promoter, e.g., T7, lac, Trp or Tac, and, in some contexts, a prokaryotic signal sequence or fusion to a protein such as, e.g., Trx, MBP, SUMO, or OsmY. The expressed protein may be secreted. The expressed protein can be harvested after disruption of the cells by French press or sonication (e.g., in the presence of 4-6 M urea).Alternatively, or in addition, the binding proteins can be harvested and purified or isolated from cell extracts using techniques known in the art, e.g., affinity tags such as glutathione- S-transf erase (GST) or histidine tags. Protease cleavage with SUMO Protease (Ulp), thrombin, enterokinase, TEV protease, 3C protease may be used to cleave affinity tags and fusion proteins from the miniprotein binder. The protein may be further purified with reverse phase HPLC using a C-18 column and eluted in a solvent gradient (e.g., gradient of acetonitrile). The protein may then be lyophilized to remove solvent and may be resuspended in phosphate buffered saline. Purification by reverse phase HPLC may be used to remove endotoxin from samples expressed in E. coli.

[0149] If the engineered gene is expressed in eukaryotic host cells, e.g., CHO cells, it is first inserted into an expression vector containing a suitable eukaryotic promoter, a secretion signal, a poly A sequence, and a stop codon. Optionally, the vector or gene construct may contain enhancers. The vector may also optionally contain fusion domains which can be used to facilitate expression and secretion. Vectors may also optionally contain enzyme (e.g., protease) cleavage sites. The gene construct can be introduced into eukaryotic host cells using conventional transfection (e.g., for mammalian) and transformation (e.g., for yeast).

[0150] In addition, the synthetic binding proteins may be produced in cell-free systems. For example, chemical synthesis such as organic chemical synthesis using liquid and / or solid phase chemical processes may be used. Such processes and tools for performing such processes, such as various automatic synthesizers, are well known to those of ordinary skill in the art and such tools are widely commercially available. More specifically, methods of chemically synthesizing polypeptides are well known in the art and include, but are not limited to, solid-phase peptide synthesis, liquid-phase peptide synthesis, and organic synthesis methods. In some synthetic approaches, an amino group of one amino acid (or amino acid derivative) is linked to a carboxyl group of another amino acid (or amino acidderivative) that has been activated by reacting it with a reagent such as dicyclohexylcarbodiimide (DCC). When the free amino group attacks the activated carboxyl group, a peptide bond is formed and dicyclohexylurea is released. In such methods, other potentially reactive groups (such as the a-amino group of the N-terminal amino acid or amino acid derivative and the carboxyl group of the C-terminal amino acid or amino acid derivative) may be blocked (“protected”) from participating in the chemical reaction. Thus, only particular active groups react such that the desired product is formed. Blocking groups useful for this purpose include, without limitation, tertbutoxycarbonyl groups (t-Boc) and benzoyloxycarbonyl groups to protect amine groups; and simple esters (such as methyl and ethyl groups) and benzyl esters to protect carboxyl groups. Blocking groups can typically be subsequently removed with a treatment that leaves peptide bonds intact (for example, treatment with dilute acid). This process of protecting reacting groups that should not react, coupling to form a peptide bond, and deprotecting reactive groups may be repeated. A peptide may be synthesized by sequentially adding amino acids to a growing peptide chain.

[0151] Both liquid-phase and solid phase peptide synthesis methods can be used to make the binding proteins described herein. In solid-phase peptide synthesis, the growing peptide chain is typically linked to an insoluble matrix (such as, for example, polystyrene beads) by linking the carboxyterminal amino acid to the matrix. At the end of synthesis, the peptide can be released from the matrix using a cleaving reagent that does not disrupt peptide bonds, such as hydrofluoric acid (HF). Protecting groups are also typically removed at this time. Automated, high throughput, and / or parallel peptide synthesis methods may also be used in accordance with the disclosure. For more information about peptide synthesis methods, see, e.g., Merrifield (1969) ADV. ENZYMOL. RELAT. AREAS MOL. BIOL., 32: 221-96; Fridkin et al. (1974) ANN. REV. BIOCHEM. 43(0): 419-43; Merrifield (1997) METH. ENZYMOL. 289: 3-13; Sabatino et al. (2009) CURR. OPIN. DRUG DISCOV. DEVEL., 11(6): 762-70.

[0152] Once synthesized, the binding proteins can be purified using standard approaches including, for example, chromatographic (e.g., reverse phase HPLC) and affinity binding approaches. The resulting binding proteins can then be characterized using a variety of chemical, biological and biophysical approaches.VI. CHARACTERIZATION OF CD123 BINDING PROTEINSA. Biophysical Characterization

[0153] The synthetic binding proteins described herein may be characterized using a variety of approaches to determine, e.g., secondary and tertiary conformation, binding affinity, binding selectivity, stability (e.g., thermostability, chemical stability, propensity to degrade, etc.), solubility, etc.

[0154] For example, protein conformation may be measured via circular dichroism spectroscopy, infrared spectroscopy, NMR, X-ray crystallography, cryo-electron microscopy and AlphaFold (alphafold.ebi.ac.uk / ). Binding affinity and / or selectivity may be determined using assays such as flow cytometric analyses using, e.g., yeast or mammalian cells, biolayer interferometry and / or surface plasmon resonance measurements, each of which will be able to determine different types and specificities of binding.

[0155] Binding affinity and / or avidity can be determined by measuring the equilibrium dissociation constant (KD) of a synthetic CD123 binding protein to a target. In some embodiments, the binding affinity (KD) of synthetic CD 123 binding proteins in the range of 10'5M or less, or ranging down to 10'10M or lower, (e.g., about 10'6, 10'7, 10'8, 10'9, 10'10M or less).

[0156] In some embodiments, the synthetic CD123 binding protein comprises a binding affinity characterized by a dissociation constant ranging from about 1 pM to 1 pM. In some embodiments, the binding affinity is between about 0.001 nM to about 1 pM; about 0.01 nM to about 1 pM; about 0.1 nM to about 1 pM; about 1 nM to about 1 pM; about 1 nM to about 0.5 pM; about 1 nM to about 0.25 pM; about 1 nM to about 0.10 pM; about 1 nM to about 75 nM; about 1 nM to about 50 nM; about 1 nM to about 25 nM; about 1 nM to about 10 nM; and about 1 nM to about 5 nM. In some embodiments, the binding affinity is stronger than about 1 pM, about 0.75 pM, about 0.5 pM, about 0.25 pM, about 0.1 pM, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, about 0.1 nM to about 0.01 nM, and about 0.01 nM to about 0.001 nM. One of ordinary skill in the art will readily know how to calculate equilibrium dissociation constants using measured Ka (1 / sec) and Kd (1 / secM) of the synthetic CD 123 binding proteins.

[0157] Other analytical techniques (some of which are also used for synthesis and purification) include, without limitation, HPLC, LCMS, quantitative thin layer chromatography and others known to those of skill in the art. Stability can be measured using assays that expose binding proteins to elevated temperatures (e.g., 37 °C, e.g., 95 °C, etc.) and / or chemical denaturants (e.g., urea, and guanidine hydrochloride) and then observe whether the protein refolds into its pre-exposure structure / conformation and / or regains binding activity to a given target molecule. Degradation can be evaluated using techniques such as reverse phase HPLC or gel electrophoresis to monitor resistance of a synthetic binding protein to degradation.B. Biochemical Characterization

[0158] The biological activity of the binding proteins can be determined via various assays, such as in vitro binding assays on chips and / or on primary cells (see, e.g., Examples 4, 5, and 6). Such assays can be used to determine whether a binding protein has antagonistic (or agonistic) properties. For example, suitable assays can be performed to determine whether a synthetic CD123 binding protein disclosed herein can, e.g., bind to a tumor cell in a way that will allow it to be engaged with another cell (e.g., a modified cell such as a cell expressing a CAR, etc.) when connected (e.g., bound) to another protein (e.g., an anti-effector molecule and / or entity (e.g., an anti-fluorescein binding protein, e.g., an anti-FITC binding protein, e.g., an anti -DOT A binding protein, e.g., as part of a chimeric antigen receptor). Depending on circumstances, a CD123 binding protein can be used to antagonize IL-3 binding. For example, a synthetic CD 123 binding protein can bind to CD 123 on a cell such as a cancer cell, preventing IL-3 from binding and promoting proliferation and survival of the cancer cell. Given context, in certain embodiments, such a CD123 binding protein can also include an effector comprising a cytotoxic payload that can kill the cancer cell or otherwise identify or target it for killing. An effector may also have a visualizable or otherwise detectable effector, so that cancer cells can be detected for diagnosis or monitoring. In certain embodiments, a CD123 binding protein can simultaneously act as an antagonist of IL-3 while also bringing a cancer cell into contact with a therapeutic cell as described herein.

[0159] As will be understood to those in the art, given context, various assays may be used to characterize phenotype and activity of synthetic CD123 binding proteins. For example, to evaluate efficacy of synthetic CD 123 binding proteins and determine their ability to engage CD 123 -mediated signaling and cytotoxicity cell markers and functionalassays may be used. To give but one example, cell surface markers associated with cancer cell phenotype and different stages of activation (e.g., CD123 expression) may be evaluated. Evaluation may include various assays and methods known to those of skill in the art including, but not limited to, immunocytochemistry including flow cytometric and flow activated cell sorting (FACS) analyses, western blotting, etc. In some embodiments, functional assays determining, for example, how many target cells (e.g., cancer cells) are killed by a synthetic CD123 binding protein further comprising an effector that binds to an antigen on a second, different cell (e.g., a CAR-expressing cell, e.g., a CAR-T cell, etc.).

[0160] Cellular phenotype or function, e.g., through a cell receptor such as CD 123, can be measured using any number of commercial assays used to characterize, e.g., cellular phenotype using surface markers, cell death of cancer cells (e.g, achieved via CAR- expressing-cell, e.g., CAR-T cell-induced cytotoxicity, etc.). For example, a FACS-based assay may be used to identify cell surface markers such as CD123, which is a known cancer cell marker. Expression of CD123 may be used to phenotype and “stage” a cancer cell, e.g., to which a synthetic CD123 binding protein of the disclosure has bound.

[0161] Assays conducted with cells, such as primary tumor cells from subjects with a disease (e.g, cancer), PBMCs from healthy human subjects, PMBCs from human subjects with a disease (e.g., cancer), etc. may be used to evaluate and characterize synthetic CD123 binding proteins. For example, the cells may have a visualizable reporter that is detectable upon CD 123 -mediated binding. In some embodiments, CD123 binding is measured by contacting a population of cells with a known labeled CD123 miniprotein and measuring level of binding to the cells (e.g., to cancer cells, etc.). Such a measurement may be compared to a measurement made after contacting a population of cells with a synthetic CD 123 binding protein as provided herein. The amount of signaling activity can be reduced or decreased contacting the population of cells (before, concomitant with, or after exposure to a ligand) with a synthetic CD123 binding protein (including, e.g., as compared to binding with a different CD 123 binding protein such as IL-3, which can induce downstream signaling).

[0162] Characterization assays may also be conducted in vivo. For example, synthetic CD 123 binding proteins may be tested for selective binding by comparing a control CD 123 binding protein (e.g., a scaffold with the same size and shape as synthetic CD123 binding proteins, but without any identity in paratope regions or with a molecule that is a naturalligand for CD123 such as, e.g., IL-3) to synthetic CD123 binding proteins as provided herein (e.g., as in Table 4A, Table 7A, and Table 9).VII. CD123-BINDING PROTEIN CONJUGATES

[0163] It is contemplated that the synthetic binding proteins may be engineered to modify certain desired properties (e.g., binding affinity, binding avidity, bi or multi-specificity, or pharmacokinetic or pharmacodynamic properties, etc.). That is, a CD123 binding protein may also include a CD123 binding protein conjugate. Production of such conjugates can be achieved by conjugating (e.g., chemical conjugation or via a fusion protein) a synthetic binding protein to an effector molecule. For example, the synthetic binding protein can be conjugated to an effector. As provided herein, an effector can be, for example, second binding molecule, e.g., a second synthetic binding molecule, which can be the same or different from the first synthetic binding protein, or an antibody or antibody fragment) or a molecule that directly (e.g., bovine serum albumin (BSA), murine serum albumin (MSA), or human serum albumin (HSA)) or indirectly (e.g., an engineered binding site for BSA, MSA, or HSA) enhances the PK or PD properties of the binding molecule. By way of nonlimiting example, half-life can be extended by a variety of approaches known to those of skill in the art including use of polyethylene glycol (PEG), fusion proteins (e.g., Fc fusions, albumin fusions), engineered Fc binding such as engineered binding to neonatal Fc receptor (FcRn), antibody conjugation (e.g., to an antibody or fragment thereof), e.g., other protein engineering approaches that change the stability and / or clearance of a protein from an organism. It is contemplated that certain half-life extension approaches such as PEGylation may reduce clearance and prolong circulation. Fusions, such as to Fc or albumin, and / or addition of binding domains such as engineered HSA domains may prolong half-life due to circulatory half-life properties of the molecules (e.g., longer circulatory half-life via binding to albumin in the serum). For instance, a serum albumin binding domain may facilitate longer half-life via binding to albumin in circulating blood. One mechanism that extension could occur may include via recycling of a synthetic CD 123 binding protein by binding to neonatal Fc receptor (FcRn), which could thereby extend the serum half-life of the synthetic CD 123 binding protein.

[0164] It is contemplated that a variety of effector molecules can be used to modify the properties of the synthetic CD123 binding protein disclosed herein. Furthermore, effector molecules may be, for example, binding proteins that bind to a different target on adifferent cell. Such effector molecules may “engage” the CD 123 -positive cell (e.g., hematopoietic cell, e.g., cancer cell) and bring it apposed to the different cell, such as a therapeutic cell (e.g., a chimeric antigen receptor cell), expressing an effector binding protein that facilitates binding the effector to the therapeutic cell. An effector molecule may be selected from a variety of entities. An effector may comprise a small molecule, a chelator, a biologic molecule (or fragment thereof), any of which can bind to a protein or fragment thereof on a surface of a cell (e.g., an effector binding protein, e.g., an anti- fluorescent tag binding protein, e.g., an anti-fluorescein or derivative thereof, e.g., anti- FITC binding protein, e.g., an anti-DOTA binding protein, etc.), such as a therapeutic engineered cell (e.g., CAR-expressing cell, e.g., CAR-T cell, etc.).

[0165] Furthermore, it is contemplated that the effector may be a cytotoxic molecule (e.g., toxin that will be endocytosed into a cell, e.g., to kill a target cell such as a cancer cell) or detectable label (e.g., radiolabel or fluorescent tag) which can be used in a detection assay, e.g., a diagnostic assay, or, e.g., in an assay intended to target CD123 expressing cells for delivery of a molecule, etc. in a condition where CD123 cells may themselves be targeted for destruction (e.g., cancer, e.g., AML, etc.). The cytotoxic molecule may be a small molecule, biologic or fragment thereof, radionuclide, etc. Exemplary effectors can include, e.g., diphtheria toxin, auristatins (e.g., monomethyl auristatin (MMAE)), taxanes (e.g. docetaxel, paclitaxel, etc.), radionuclides, cytotoxic antibiotics e.g., doxorubicin, etc.) or any fragments thereof. In certain embodiments, the cytotoxic molecule is a diphtheria toxin. In certain embodiments, the cytotoxic molecule is an auristatin. In some embodiments, the cytotoxic molecule is a taxane. In some embodiments, the cytotoxic molecule is a radionuclide. In some embodiments, the cytotoxic molecule is an antibiotic.

[0166] It is also contemplated that the effector molecule can be chemically conjugated to the synthetic binding protein or can be incorporated into the synthetic binding protein as a fusion protein. The chemical conjugation can be accomplished by including a conjugation site into the synthetic binding protein, e.g., via inclusion of a derivatizable amino acid (e.g., a lysine, cysteine, histidine, or non-natural amino acid). For example, a conjugation site can be a lysine residue, a cysteine residue, a histidine residue, or a non-natural amino acid residue. The conjugation site can then be used to link the effector molecule to the binding protein, either directly or indirectly (e.g., via a linker, such as homobifunctional or a heterobifunctional cross-linking agent).

[0167] A conjugation site, such as at a particular residue (e.g., a lysine, cysteine, histidine, or non-natural amino acid residue), can be occur or be placed (e.g., engineered, e.g., inserted by substitution, etc.) at different locations in a miniprotein. A conjugation site can also be at an N- and / or C-terminus of a synthetic CD123 binding protein. Location of a conjugation site can depend on a variety of factors and conditions (e.g., accessibility of the site and / or accessibility of the conjugated entity to its target (e.g., accessibility of the effector), impact on folding of the synthetic binding protein and / or any effectors, etc.).

[0168] A conjugation site can be at an N- and / or C-terminal region of a CD123 binding protein, and / or can be in an alpha helical region, a loop, or one or more of an N- and / or C- terminus, alpha helical region, or loop region.

[0169] Conjugation sites may also be at a certain distance from the N- or C-terminus of a synthetic binding protein. In certain embodiments, a CD 123 binding protein can have more than one conjugation site. In CD123 binding proteins with more than one conjugation site, the disclosure contemplates that one or more different entities (e.g., two different effectors) can be placed at each unique (e.g., at a different position relative to the length of an amino acid sequence of a CD123 binding protein) conjugation site. In some embodiments, the CD123 binding protein has 1, 2, 3, 4, or 5 conjugation sites. Depending on circumstances, in certain embodiments, a conjugation site may be located no more than 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 residues from the N- and / or C-terminus of a synthetic CD 123 binding protein.

[0170] Depending on circumstances, an amino acid for conjugation (e.g., a lysine, cysteine, histidine, non-natural amino acid, etc.) may be introduced at an amino acid in the second and / or third alpha helices. The disclosure provides CD123 binding proteins that can be modified (e.g., by inclusion of one or more binding sites for conjugation of an effector molecule). By way of non-limiting example, in Reference Miniproteins of the disclosure, any one of positions X26, X35, or X45 may be a lysine, cysteine, histidine, or non-natural amino acid, and can serve as a conjugation site such as a small molecule (e.g., FITC with or without a linker) or chelator (e.g., DOTA). Such a conjugate can be represented according to a formula of (i): CD123 binding protein - effector (XR), wherein the effector is directly conjugated to the binding protein; or (ii) CD 123 binding protein - linker (L) - effector (XR), where a linker is used to connect the effector to the binding protein. Depending on circumstances, the linker can, in some embodiments, include at least one lysine, cysteine, histidine, or non-natural amino acid (e.g., a lysine, cysteine,histidine, or non-natural amino acid located on the N- or C-terminal end of the linker). For example, in certain embodiments, a conjugate comprises a CD123 miniprotein, a linker connected to an amino acid of the miniprotein, a lysine, cysteine, histidine, or non-natural amino acid on the C-terminal end of the linker, and an effector, which can be attached to the lysine, cysteine, histidine, or non-natural amino acid of the linker using conjugation techniques known to those of skill in the art.

[0171] Conjugation sites can occur directly within a linear polypeptide (e.g, positions X26, X35, or X45 as set forth in SEQ ID NOs: 25 or 29). For example, Reference Miniproteins 22-27 each have a lysine at position 26, 35, or 45. Reference Miniproteins 22 (SEQ ID NO: 4) and 25 (SEQ ID NO: 22) each have a lysine at position 26; Reference Miniproteins 23 (SEQ ID NO: 23) and 26 (SEQ ID NO: 3) each have a lysine at position 35; and Reference Miniproteins 24 (SEQ ID NO: 24) and 27 (SEQ ID NO: 4) each have a lysine at position 45. The lysine residues can serve as conjugation sites for an effector, such as a small molecule (e.g, fluorescein, fluorescein isothiocyanate (FITC), e.g., an amine-reactive FITC (e.g., NHS-FITC), etc.) or chelator (e.g., tetraxetan (DOTA), e.g., an amine-reactive tetraxetan (e.g., NHS-DOTA)). Structures of certain exemplary effectors are shown in TABLE 3A.TABLE 3A. Exemplary effector structures

[0172] By way of non-limiting example, Reference Miniproteins 25, 26, and 27 each have a fluorescein isothiocyanate conjugated to the lysine. No linker is present in those exemplary Reference Miniproteins; however, the disclosure contemplates that, in some embodiments, and depending upon position of conjugation, a linker may be used to attach an effector to the synthetic CD123 binding protein. For example, a linker could be attached to an N- or C-terminus of a reference miniprotein, which may also optionally include a lysine residue just prior to the site of an effector.

[0173] By way of non-limiting example, linkers may be or include hydrazone, PEG, bifunctional 4-(4-acetylphenoxy) butanoic acid moiety, maleimidocaproyl, maleimidomethyl cyclohexane- 1 -carboxylate, maleimidocaproyl group with a tetrapeptide portion consisting of the amino acid sequence, glycine-glycine-phenylalanine-glycine (SEQ ID NO: 88), and / or maleimidocaproyl group with PEG. Exemplary homo- and heterobifunctional cross-linking agents can also include, for example, EDC (l-ethyl-3-(3- dimethylaminopropyl) carbodiimide hydrochloride), sulfo-NHS (N-hydroxysulfo succinimide), NHS (N-hydroxysuccinimide), dimethyl pimelimidate dihydrochloride, suberic acid bis(N-hydroxysuccinimide ester), DSG (disuccinimidyl glutarate), DSS (disuccinimidyl suberate), DSP (dithiobis(succinimidyl propionate)), sulfo-SMCC (sulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane- 1 -carboxylate), SPDP ((succinimidyl 3-(2-pyridyldithio)propionate), N-succinimdyl oxy carbonylethylmethanethio sulfonate, 6-Maleimidohexanoic acid N-hydroxysuccinimide ester, 6- Maleimidocaproic acid sulfo-N-succinimidyl ester, maleimidoacetic acid N- hydroxysuccinimide ester, N-succinimidyl iodoacetate, and 4-(4-maleimidophynyl) butyric acid N-hydroxysuccinimide ester.

[0174] Linkers may be used to link two or more synthetic CD 123 binding proteins to one another, and may also be used to link one or more synthetic CD123 binding proteins to one or more effectors. A linker may be a peptide linker or a chemical linker. Linkers may be covalently bound (e.g., to an amino acid) to a synthetic CD 123 binding protein and covalently bound to a second agent (e.g., an effector, e.g., a second binding protein). The composition and / or length of the linker may be designed with a particular functionality in mind, but preferably is non-immunogenic. A linker may contain one or more glycine amino acids and / or one or more serine amino acids. Exemplary linkers can comprise one or multiples of (Gly2Ser)n, (Gly3Ser)n, or (Gly4Ser)n, where n can be 1, 2, 3 etc. For example, linkers can include any of those set forth in TABLE 3, which provides exemplary interm olecular linkers that can be used, for example, to attach effectors to CD 123 binding proteins.TABLE 3B. Intermolecular Linkers

[0175] It is contemplated that the synthetic CD123 binding proteins disclosed herein can monovalent or multivalent. Multivalent proteins may include, but are not limited to bivalent and trivalent formats. A multivalent molecule may include two, three, four, or more monovalent synthetic CD 123 binding proteins or, for example, one synthetic CD 123 binding protein and another binding protein that binds another target (e.g., serum albumin). In any of these formats, at least one linker can connect a C-terminal amino acid of a first monovalent binding protein to an N-terminal amino acid of a second monovalent synthetic binding protein, such that the first and the second monovalent synthetic CD 123 binding proteins are linked together. For example, in some embodiments, a multivalent (e.g., bivalent) miniprotein may comprise two or three miniproteins, each independently between about 35 and 100 amino acids and associated (e.g., linked, conjugated) with one another. In some embodiments, multivalent molecules may be fused and / or combined to another molecule, such as an effector molecule, e.g., a half-life extender (e.g., a site or a protein that binds to serum albumin to extend serum half-life, etc.) or a multivalent protein may comprise a miniprotein (e.g., a synthetic CD123 binding protein), and a miniprotein engineered to bind to serum albumin (e.g., to extend serum half-life). In some such embodiments, a half-life extension molecule may be fused or conjugated to either a C- terminal amino acid or an N-terminal amino acid (or to both the N- and C-terminal amino acids) of a synthetic CD123 binding protein disclosed herein.

[0176] In certain embodiments, the disclosure provides a multivalent e.g., bivalent) protein comprising a multiple synthetic CD123 binding proteins disclosed herein. The multivalent protein can comprise a first synthetic CD123 binding protein and a second synthetic CD123 binding protein linked together through at least one linker. A linker (e.g., a glycine and serine containing linker (e.g., GGS or others, including, as set forth in Table 3B), can connect a C-terminal amino acid of the first synthetic CD 123 binding protein to an N-terminal amino acid of the second synthetic CD 123 binding protein. Depending upon the circumstances, the multivalent binding protein can have a binding affinity stronger than the binding affinity of each synthetic CD 123 binding protein alone.

[0177] In certain embodiments, the multivalent protein comprises a synthetic CD123 binding protein having an amino acid sequence, wherein the amino acid sequence comprises an amino acid sequence set forth in TABLE 9.VIII. PHARMACEUTICAL COMPOSITIONS

[0178] Once produced, a synthetic CD123 binding protein disclosed herein can be formulated into a pharmaceutical composition.

[0179] For therapeutic use, a synthetic CD123 binding protein disclosed herein is combined with a pharmaceutically acceptable carrier. Various carriers (e.g., diluents, excipients, etc.) used in formulating and preparing pharmaceutical compositions are known and / or readily accessible to those of skill in the art. Depending upon the circumstances, a carrier can include a liquid (e.g., a sterile liquid) or a solid. A carrier may be selected from or comprise water, aqueous solvents, non-aqueous solvents, dispersion media, surfactants, antioxidants, buffers, adjuvants, tonicity agents, stabilizers, bulking agents, lyoprotectants, metal ions, chelating agents, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art. Typically a carrier is approved by United States Food and Drug Administration and meets the standards of the United States Pharmacopoeia (USP), the European Pharmacopoeia (EP), the British Pharmacopoeia, and / or other International Pharmacopoeia. Suitable formulations for use in the present disclosure are found in see e.g., Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rded. 2020). For a brief review of methods for drug delivery, see, e.g., Langer (1990) SCIENCE 249: 1527-1533. The resulting pharmaceuticalcompositions are suitable for administration to a subject e.g., an animal, e.g., a mammal, e.g., a human).

[0180] A pharmaceutical composition may contain formulation materials for modifying, maintaining, or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption, or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogensulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt-forming counterions (such as sodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, polyethylene glycol (PEG), sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients and / or pharmaceutical adjuvants (see e.g, Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rded. 2020)).

[0181] In certain embodiments, a pharmaceutical composition may contain a sustained- or controlled-delivery formulation. Techniques for formulating sustained- or controlled- delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. Sustained-release preparations may include, e.g, porous polymeric microparticles or semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gammaethyl-L-glutamate, poly (2-hydroxyethyl-inethacrylate), ethylene vinyl acetate, or poly- D(-)-3 -hydroxybutyric acid. Sustained release compositions may also include liposomes that can be prepared by any of several methods known in the art.

[0182] Depending upon the circumstances, a pharmaceutical composition may contain nanoparticles, or lipid droplets, e.g., polymeric nanoparticles, liposomes, or micelles (see Anselmo et al. (2016) BIOENG. TRANSL. MED. 1 : 10-29).

[0183] Pharmaceutical compositions containing a synthetic CD 123 binding protein can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intravenous (IV), intraperitoneal, intradermal, inhalation, transdermal, topical, transmucosal, intrathecal and rectal administration. In certain embodiments, the synthetic peptide is administered by subcutaneous administration.

[0184] Useful formulations can be prepared by methods known in the pharmaceutical art. For example, see e.g., Adeboye Adejare, REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY (23rded. 2020). Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.

[0185] For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, poly ethoxylated castor oil or phosphate buffered saline (PBS). The carrier should be stable under the conditions of manufacture and storage, and should be preserved against microorganisms. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures thereof.

[0186] Pharmaceutical formulations preferably are sterile. Formulations can be sterilized, for example, by methods appropriate to retain activity and stability of the synthetic CD 123 binding protein included therein. Sterilization can be accomplished by any suitable method, e.g., filtration through sterile filtration membranes. Where the composition islyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.

[0187] Depending upon the drug substance and formulation, the resulting dosage forms can be stable for extended periods of time, such as 1 month, 3 months, 6 months, 1 year, 2 years, 3 years, or more, when the dosage form is a liquid or solid. The formulations can be stable at room temperature or higher. It is contemplated that the dosage form is stable at ambient conditions in PBS. Alternatively the dosage form is frozen (e.g., a liquid or a lyophilizate) and stable under appropriate temperatures such as, e.g., -20°C, -80°C).

[0188] Depending upon the circumstances, the dosage forms can be formulated as a unit dose, which can include, for example, about 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 1.25 mg, 1.5 mg, 1.75 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 25 mg, 50 mg, 100 mg, 250 mg, 500 mg, 1 g, 1.5 g, 2.5 g, 5 g, or 10 g of the drug substance.

[0189] The compositions described herein may be administered locally or systemically. It is contemplated that the compositions described herein are generally administered by parenteral administration. Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. In certain embodiments, the pharmaceutical composition is administered subcutaneously or may be administered intravenously, e.g., via intravenous infusion. In certain embodiments, it is contemplated that the synthetic constructs disclosed herein can be administered by systemic administration.

[0190] Generally, a therapeutically effective amount of active component, for example, a synthetic CD123 binding protein disclosed herein, is in the range of 0.01 pg / kg to 250 mg / kg, e.g., 0.1 pg / kg to 25 mg / kg, 1 pg / kg to 15 mg / kg, 10 pg / kg to 10 mg / kg, 0.1 mg / kg to 100 mg / kg, 2.5 mg / kg to 175 mg / kg, 5 mg / kg to 250 mg / kg. In certain embodiments, the effective amount is 0.01 pg / mg. In certain embodiments, the effective amount is 0.1 mg / kg. In certain embodiments, the effective amount is 1 mg / kg. In certain embodiments, the effective amount is 15 mg / kg. In certain embodiments, the effective amount is 30 mg / kg. In certain embodiments, the effective amount is 50 mg / kg. In certain embodiments, the effective amount is 100 mg / kg. A dose may also be a flat dose, for example, about 0.25 mg to 25 mg, and depending on context (e.g., administration route such as intravenous vs. subcutaneous vs. oral), dose can change. In certain embodiments (e.g., intravenous administration) a dose may be about 0.075 mg to about 100 mg, 0.1 mg to 100 mg, 1 mg to 100 mg, 0.1 mg to 50 mg or 1 mg to 50 mg. In certain embodiments(e.g., subcutaneous administration) a dose may be about 0.25 mg to about 2.5 mg (e.g., about 0.25 mg, 0.5 mg, 0.75 mg, 1 mg, 1.25 mg, 1.5 mg, 2 mg, or 2.5 mg. In other embodiments, (e.g., oral administration) a dose may be about 5 mg to about 25 mg (e.g., about 5 mg, 7.5 mg, 10 mg, 12.5 mg, 15 mg, 20 mg, or 25 mg). The amount administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health of the patient, the in vivo potency of the active component, the pharmaceutical formulation, and the route of administration. The initial dosage can be increased beyond the upper level in order to rapidly achieve the desired blood-level or tissue-level. Alternatively, the initial dosage can be smaller than the optimum, and the daily dosage may be progressively increased during the course of treatment. Human dosage can be optimized, e.g., in a conventional Phase I dose escalation study. Dosing frequency can vary, depending on factors such as route of administration, dosage amount, serum half-life of the synthetic peptide, and the disease, disorder, or condition being treated. Exemplary dosing frequencies are once per day, once per week and once every two weeks.IX. METHODS OF USE AND TREATMENT

[0191] The CD 123 binding proteins provided herein can be used in a variety of different approaches or contexts, including, for example, as a therapeutic and / or diagnostic agent. For example, the synthetic CD123 binding proteins of the disclosure may also be used to compete with binding of IL-3 to native CD 123, thereby preventing or reducing intracellular signaling in cancer cells, and slowing proliferation and / or survival of the cancerous cells. Alternatively, the synthetic CD123 binding proteins of the disclosure can be coupled with one or more detectable labels (e.g., a fluorescent label), to determine if a binding protein specifically binds to CD 123. In addition, such labeled CD 123 miniproteins can be used to determine if binding occurs to monomeric or dimerized (or otherwise bound to one or more additional subunits) CD 123. Such methods may be used, for example, as a diagnostic or screening agent to identify proteins that specifically bind to CD 123.

[0192] The synthetic CD123 binding proteins of the present disclosure can be used in a method of targeting a cell expressing CD123 to another component or cell (e.g., a therapeutic cell), such as a method which comprises contacting a cell that expresses CD123 on its cell surface with a composition comprising a synthetic CD123 binding protein disclosed herein, wherein the CD123 binding protein further comprises an effector, and theeffector binds to a cell-surface antigen on another cell, thereby engaging the other cell to target the CD 123 -expressing cell for death.

[0193] For example, the binding proteins can be used in a method of targeting CD 123. The method comprises contacting a cell that expresses CD123 on its cell surface with a composition comprising the synthetic CD123 binding protein disclosed herein. In addition, the CD 123 binding proteins described herein can be used to modulate CD 123 -positive cell activity (e.g., intracellular signaling activity, e.g., JAK-STAT, etc., e.g., proliferation, survival, etc.). The method comprises contacting a cell that expresses CD 123 on its cell surface with a composition comprising the synthetic CD123 binding protein disclosed herein. In each method, the CD 123 binding protein or the pharmaceutical composition comprising a CD123 binding protein further comprises an effector molecule. In each of the foregoing methods, the synthetic CD 123 binding protein or the pharmaceutical composition can leverage CD 123 expression to (1) bind to a cancer cell and (2) engage the bound cell to come into proximity with another cell (e.g., therapeutic cell, e.g., a chimeric antigen receptor cell, e.g., a chimeric antigen receptor cell expressing an effector binding component (e.g., antibody to an effector, etc.) on its surface) via an effector.

[0194] It is further contemplated that CD123 binding proteins provided herein can be used to target CD 123 -expressing cells (e.g., cancer cells) for delivery of a molecule, etc. in a condition where CD 123 -expressing cells may themselves be targeted for destruction (e.g., cancer, etc.).

[0195] The CD 123 binding proteins provided herein may be used in treatment of a disease, disorder, or condition mediated by CD 123 -positive cell activity. CD123 is expressed on healthy and malignant hematopoietic cells. The cancer cells can be targeted by (i) antagonism of IL-3 signaling using synthetic CD123 binding proteins disclosed herein, thus, preventing intracellular signaling and disrupting or decreasing survival and / or proliferation of the cancer cell; and / or (ii) by another component binding to an effector on the synthetic CD123 binding protein and bringing a therapeutic cell (e.g., a chimeric antigen receptor, e.g., comprising a surface-exposed effector binding component) into proximity with the CD 123 -expressing cancer cell.

[0196] In certain embodiments, the disclosure provides a method of targeting CD123- by contacting a cell that expresses CD123 on its cell surface with a composition comprising a synthetic CD 123 binding protein as provided herein.

[0197] In one aspect, the disclosure provides a method of targeting CD123, the method comprising contacting a cell that expresses CD123 on its cell surface with a composition comprising a synthetic CD123 binding protein or pharmaceutical composition as provided herein.

[0198] In one aspect, the disclosure provides a method of antagonizing IL-3R signaling, the method comprising contacting a cell that expresses CD123 on its cell surface with a composition comprising a synthetic CD123 binding protein or pharmaceutical composition as provided herein, thereby preventing, inhibiting, and / or decreasing binding of IL-3 to CD 123. Such a change in binding of IL-3 can then decrease IL-3 mediated signaling responses such as proliferation and survival and, given context, reduce proliferation and survival of certain cell types, such as cancer cells.

[0199] In one aspect, the present disclosure provides a method of modulating cancer cell activity, the method comprising contacting cancer cell that expresses CD123 on its cell surface with a composition comprising a synthetic CD123 binding protein or a pharmaceutical composition as provided herein, and under conditions that permit the CD 123 binding protein to bind to the CD 123 on the cancer cell. Depending upon the circumstances, the synthetic CD123 binding protein optionally further comprises an effector, wherein the effector comprises a cytotoxic agent (e.g., small molecule, radionuclide, cytotoxic protein, etc.) capable of killing a cancerous cell.

[0200] In one aspect, the present disclosure provides a method of modulating cancer cell activity, the method comprising contacting cancer cell that expresses CD123 on its cell surface with a composition comprising a synthetic CD123 binding protein or a pharmaceutical composition as provided herein, and under conditions that permit the CD 123 binding protein to bind to the CD 123 on the cancer cell. Depending upon the circumstances, the synthetic CD123 binding protein optionally further comprises an effector, wherein the effector binds to a protein on another different cell (e.g., a therapeutic cell) and the activity of the cancer cell is modulated in that it does not signal through the CD 123 signaling pathway and the second, different cell effects a cytotoxic response against the cancer cell.

[0201] In certain embodiments, a CD123 expressing cell is a hematopoietic cell, for example, a cancer cell. In some embodiments, a CD123 expressing cell is a cancer cell that is not of hematopoietic origin. In certain embodiments, a second different cell or a therapeutic cell is an engineered cell, such as an engineered cell comprising a chimericantigen receptor (CAR), including, for example, a CAR-T cell. The engineered cell can be designed to attack the cancer cell after being brought into contact with the cancer cell via a complex comprising a CD123 binding protein that binds the cancer cell and an effector molecule that binds to the therapeutic cell (e.g., through an effector binding protein, such as, for example, an anti-fluorescein or anti-fluorescein derivative protein or anti-DOTA protein, etc.).

[0202] Without being bound by theory, it is contemplated that a synthetic CD123 binding protein as provided by the present disclosure reduces or decreases IL-3-mediated activity in a target cell (e.g., a cancer cell) relative to cell-mediated activity in the target cell in the absence of the CD123 binding protein.

[0203] In one aspect, the disclosure provides a method of treating one or more cancers or populations of cancer cells (e.g., tumor) in a subject in need thereof, the method comprising administering to the subject an effective amount of a synthetic CD123 binding protein or pharmaceutical composition as provided herein.

[0204] The disclosure provides a method of treating cancer by administering to a subject in need thereof a fusion protein comprising a synthetic CD123 binding protein or a pharmaceutical composition as provided herein, wherein the effector comprises a cytotoxic molecule or wherein the effector targets a therapeutic cell. Upon administration, the cytotoxic molecule or the therapeutic cell is brought into proximity with the CD123 expressing cancer cell, thereby promoting or increasing CD 123 -mediated cytotoxicity in the cancer cell.

[0205] Subjects that can be treated include those suspected as having, having, or at risk of having a disease, disorder, or condition that would benefit from targeted cell toxicity by CD123 expressing cells. The methods described herein may include a step of selecting a treatment for a subject in need thereof. The method includes (a) identifying (e.g., diagnosing) the subject with such a disease, disorder, or condition, and (b) selecting a synthetic CD 123 binding protein as described herein, to treat the subject.

[0206] When administered to a subject, and depending on context (including type of effector), the synthetic CD 123 binding proteins result in cytotoxicity or therapeutic-cell mediated death of one or more CD 123 -expressing cancer cells.

[0207] The disclosure also provides methods of treating a subject in need thereof by administering an effective amount of the synthetic CD123 binding protein to the subject. The methods and compositions described herein can be used alone or in combination withother therapeutic agents and / or modalities. The phrase administered “in combination,” as used herein, is understood to mean that two (or more) different treatments are delivered to the subject during the course of the subject’s affliction with the disorder, such that the effects of the treatments on the patient overlap at a point in time. In certain embodiments, the delivery of one treatment is still occurring when the delivery of the second begins, so that there is overlap in terms of administration. This is sometimes referred to herein as “simultaneous” or “concurrent delivery.” In other embodiments, the delivery of one treatment ends before the delivery of the other treatment begins. In certain embodiments of either case, the treatment is more effective because of combined administration. For example, the second treatment is more effective, e.g., an equivalent effect is seen with less of the second treatment, or the second treatment reduces symptoms to a greater extent, than would be seen if the second treatment were administered in the absence of the first treatment, or the analogous situation is seen with the first treatment. In certain embodiments, delivery is such that the reduction in a symptom, or other parameter related to the disorder is greater than what would be observed with one treatment delivered in the absence of the other. The effect of the two treatments can be partially additive, wholly additive, or greater than additive. The delivery can be such that an effect of the first treatment delivered is still detectable when the second is delivered.

[0208] It is contemplated that therapy can be accomplished using a synthetic CD123 binding as part of a combination therapy wherein the CD 123 binding protein further optionally comprises an effector and, optionally, one or more additional agents, which agent(s) may be administered prior, concomitant with, and / or after treatment with a synthetic CD123 binding protein comprising an effector.

[0209] The combination therapy comprising the CD123 binding protein and effector may include one or more additional agents or therapeutic approaches known to those of skill in the art and may have been previously used, be already ongoing, or added to a treatment for a subject in need thereof.

[0210] In methods of the present disclosure, where administration occurs, administration can be before, during, or after administration or use of one or more other treatments. In some embodiments, one or more other treatments may be a biological agent (e.g., biologies, gene therapy, peptides), a small molecule (e.g., chemotherapy, corticosteroids, antivirals, antibiotics, anti-inflammatory agents, etc.), one or more cells (e.g., immunotherapy), and / or one or more mechanical interventions (e.g., surgery, cryotherapy,radiation). Exemplary small molecules can include, paclitaxel, cyclophosphamide. Exemplary biologies can include, for example, immune checkpoint modulator targeting antibodies such as anti-PD-1 or anti-CTLA-4 antibodies. Exemplary other treatments include, but are not limited to, cell therapy such as ex vivo expanded and differentiated chimeric antigen receptor cells (e.g., CAR-T cells). In certain embodiments, the additional therapy may include a combination of therapeutics of different classes.

[0211] Exemplary diseases, disorders, or conditions that may be treated with the CD123 binding proteins disclosed herein include those such as cancer or population of cancerous cells such as, for example, solid tumors (including, e.g., lymphomas that have developed into solid tumors in lymph), gastric cancer, brain cancer (e.g., glioma), mesothelioma, ovarian cancer, pancreatic cancer, prostate cancer, myeloma (e.g., multiple myeloma), leukemia (e.g., acute myeloid leukemia (AML), acute lymphoblastic leukemia), lymphoma (e.g., mantle cell lymphoma, follicular lymphoma, non-Hodgkin lymphoma, Hodgkin lymphoma), blastic plasmacytoid dendritic neoplasm (BPDCN), hairy cell leukemia (HCL), etc.

[0212] Compositions of the present disclosure may be used to treat a subject diagnosed as having or at risk of having a one or more such conditions. In some embodiments, the subject has been diagnosed as having cancer and / or a cancer or population of cancerous cells.

[0213] The disclosure also provides a method of targeting a population of CD123- expressing cancer cells, the method comprising contacting the population with a composition comprising a synthetic CD123 binding protein, wherein the synthetic binding protein optionally further comprising an effector that includes a toxic molecule. In certain embodiments, the effector binds to a binding protein on a therapeutic cell (e.g., a CAR cell, e.g., a CAR-T cell, etc.). After contact with the composition, a greater portion of the population of CD123 expressing cells is killed relative to treatment without the effector and / or without the synthetic CD123 binding protein.

[0214] It is contemplated that a subject may be evaluated, e.g., by a healthcare provider, before, during, and / or after treatment with a composition provided herein. Depending on the outcome of the evaluation, a treatment may be continued or ceased, treatment frequency or dosage may change, or the patient may be treated with a different synthetic CD 123 binding protein and / or effector. Subjects may be administered a composition comprising the synthetic CD123 binding protein for a discrete period of time according to dosageparadigms described herein, including, optionally, until the disease, disorder, or condition is treated.X. KITS

[0215] Synthetic CD123 binding proteins of the present disclosure may be included as part of a kit. A kit may comprise a container comprising or consisting essentially of a unit of a pharmaceutical composition comprising a synthetic CD123 binding protein, instructions for use, and optionally, one or more agents (e.g., a buffer or diluent, if appropriate, to dissolve the binding protein or dilute a solution containing the binding protein), and a dispenser. A kit may include a label indicating the intended use of the contents of the kit. The contents of the kit may be used for treating, monitoring and / or diagnosing a subject in need thereof.

[0216] The present disclosure is further illustrated by the following examples which should not be construed as further limiting.EXAMPLESEXAMPLE 1: INITIAL SCREENING

[0217] This Example describes an initial in vitro screen of a synthetic protein library using yeast display to identify synthetic proteins capable of binding human CD123. The library was designed in silico and was screened for members having certain characteristics, such as binding to the target (CD123) at particular concentrations of target (e.g., 1 pM). Once identified, certain synthetic binding proteins were synthesized for use in downstream screening and discovery processes.

[0218] A synthetic binding protein library having greater than 1 billion members was screened for CD123 binding by yeast display. Each binding protein contained an N- terminal extension of SEQ ID NO: 34 and a C-terminal extension of SEQ ID NO: 37 to facilitate expression and detection in yeast display. Each protein was about 115 amino acids in length. Synthetic proteins that bound to CD123 were isolated from non-binding proteins through iterative rounds of magnetic and fluorescent selection, using standard labeling and selection techniques (see, e.g., Chao et al., (2006) NAT. PROTOC. 1(2):755- 68). Briefly, magnetic selection was performed using streptavidin-coated beads saturated with biotinylated CD123. The beads were used to isolate yeast displaying miniproteins that bound to CD123. Isolated cells were grown so that additional rounds of selection could becompleted. For fluorescence-mediated selection, yeast cells were labeled with biotinylated CD123 at 1 pM for a first selection round. Cells were then labeled with streptavidin-APC and cells displaying miniproteins that bound to CD123 were sorted on APC fluorescence (Sony SH800 cell sorter). All cells that showed a greater fluorescent signal than cells not labeled with CD123 were isolated. These cells were then grown so that additional selection rounds under more stringent conditions including lower concentrations of soluble CD 123 could be completed.

[0219] After multiple selection rounds the amino acid sequences of synthetic proteins that bound to CD 123 expressed on yeast were determined using next-generation sequencing methods. Briefly, plasmid DNA encoding the miniprotein sequences was isolated from the selected yeast cells using standard DNA isolation techniques for yeast cells. PCR was then used to amplify specific DNA fragments that encoded miniprotein sequences and append necessary DNA elements to enable sequencing on an Illumina Mi-Seq instrument. Unique miniprotein sequences were identified that bind CD 123 at a strength of equal to or less than of 1 pM. Representative exemplary sequences were selected for further validation.Miniproteins having these sequences were expressed in and purified from E. coli. Binding of each miniprotein to CD123 was measured by surface plasmon resonance (SPR).

[0220] As part of the miniprotein preparation, the CD 123 binding miniproteins were exposed to 4M urea at temperatures of at least 50°C. These proteins retained their properly folded three-dimensional structure as confirmed by circular dichroism spectroscopy.Separately, each of these miniproteins also retained their properly folded three-dimensional structure after heating to about 95°C as confirmed by circular dichroism spectroscopy (but not including urea exposure).

[0221] A miniprotein showing binding to CD123 was chosen as the basis to create a new library with greater than 1 billion variants of that miniprotein. The library was screened for binding to human CD123 using standard yeast surface display techniques. Miniprotein variants that bound to CD123 were isolated from non-binding miniproteins through iterative rounds of magnetic and fluorescent selection, using labeling and selection techniques as described earlier in this Example. Thousands of unique miniprotein amino acid sequences were identified, and one was selected for further optimization.EXAMPLE 2: OPTIMIZATION OF CD123 MINIPROTEINS

[0222] This Example describes the optimization and affinity maturation of a CD123 miniprotein from Example 1. The amino acid sequence of the selected miniprotein was redesigned to increase thermal stability, and then was affinity matured, which resulted in 18 new proteins with CD 123 binding affinity stronger than 750 nM, referred to as Reference Miniproteins 1, 5-10, and 11-21 (SEQ ID NOs: 1, 5-10, and 11-21) as shown in TABLE 4A. The sequences of each protein with its N- and C-terminal extension used in Example 1 are also shown (Reference Miniproteins 41, 45-50, and 54-64 of SEQ ID NOs: 41, 45-50, and 54-64, respectively, each having an amino acid sequence 115 amino acids in length). Each of the Reference Miniproteins herein had the same secondary structural arrangement of loop amino acids and alpha helix amino acids the parent. Miniproteins encoded by each of these sequences were expressed in and purified from E. coli. Parameters of each protein (molecular weight, isoelectric point, and charge at pH 7.4) are set forth in TABLE 4B. Binding characteristics of each miniprotein to human CD123 were measured as shown in TABLE 4C, and binding activities (Ka, Kd, and KD values) of each miniprotein to human CD 123 were determined using SPR.

[0223] The resulting 18 Series 1 proteins were similar to one another. The amino acid sequences of Reference Miniproteins 1, 5-10, and 11-21 share about 90% identity with one another and Reference Miniproteins 1-9 (Subseries 1.1), share about 91% identity with one another.TABLE 4A. Exemplary Series 1 and Subseries 1.1 Miniprotein Reference SequencesTABLE 4B. Protein Parameters of Exemplary Reference Miniproteins* protein parameters are calculations based on each 47 amino acid sequence.N.D. = not measured in this Example, but can be calculated given the amino acid sequenceTABLE 4C. Binding Characteristics of Exemplary Reference Miniproteinsinteraction half-life is time after initial binding at which half of the starting pool of a plurality of CD 123 binding proteins remain bound to CD 123.EXAMPLE 3: CD123 MINIPROTEIN STABILITY

[0224] This Example describes in vitro chemical stability of various Reference Miniproteins 1-24 (SEQ ID NOS. 1-24) using biophysical characterization after exposure to exemplary chemical denaturants.

[0225] Each of these CD 123 miniproteins developed and characterized in Example 2 were exposed to 4M urea and showed stability (data not shown). That is, after exposure tourea and subsequent dilution into PBS, the Reference Miniproteins each folded into their expected, respective, molecular structures (as prior to urea exposure) at 25°C as measured by CD spectroscopy (data not shown).

[0226] In addition, miniproteins were thermally stable during heating and showed proper refolding during cooling. Measurements of whether these proteins had properly folded three-dimensional structure after heating to 95°C and then returning to 25°C as measured by circular dichroism (CD) spectroscopy were determined. An example is shown in FIG. 2A, demonstrating that exemplary Reference Miniprotein 4 (SEQ ID NO: 4) was thermally-stable at about 25°C, through to being heated up to about 91 °C, and refolded into the same configuration about cooling to about 25°C. Furthermore, FIG. 2B shows that Reference Miniprotein 4 was stable during heating in 10°C increments from 25°C - 95°C.EXAMPLE 4: COMPETITION FOR CD123 BINDING & ANTAGONISM OF IL-3

[0227] This Example describes binding location of CD123 miniproteins before and after exposure to interleukin 3 (IL-3), a cytokine that is a natural ligand for CD 123 (also known as IL-3Ra). This study assessed whether synthetic CD123 miniproteins of the present disclosure would be able to saturate CD123 monomers and / or if addition of IL-3 would displace synthetic CD123 miniproteins from binding to CD123. A chip containing immobilized CD 123 monomers was used and, at point A on FIG. 3, Reference Miniproteins 2, 3, and 4, each at 100 nM, or running buffer alone were added. As seen in FIG. 3, binding signal increased with each of the synthetic CD 123 binding proteins, as equilibrium was reached. Then, as indicated by the arrow and “IL-3” on the graph of FIG. 3, IL-3 (at 100 nM) was added to each sample (each miniprotein and buffer). As shown by the binding curves, samples that were already saturated by the Reference Miniproteins binding to the CD 123 monomers did not show any signal increase when IL-3 was added. In contrast, in the control buffer channel, increase in signal, indicating binding to CD 123, was observed upon injection of IL-3. Lack of increase in signal in the samples with synthetic CD 123 binding proteins indicates that the CD 123 miniproteins are competitive binders, as, if the binding was non-competitive with IL-3, an increase in signal upon injection of IL-3 into the Reference Miniprotein samples would have been expected. This is also supported by modeling as shown in FIGs. 4A and 4B. FIG. 4A shows the binding site of IL-3 on CD 123 and FIG. 4B shows the expected binding site of exemplary syntheticCD 123 binding proteins, indicating that the CD 123 miniproteins bind at the same site as IL-3 on CD 123.EXAMPLE 5: SUBSTITUTION TOLERANCE

[0228] This Example describes use of single-site saturation mutagenesis (SSM) for identification and interrogation binding tolerance at each of 47 amino acid positions in an exemplary CD 123 Miniprotein.

[0229] Reference Miniprotein 4 (SEQ ID NO: 4) was selected according to its strong binding affinity for CD 123. To better understand the sequence requirements for binding, single amino acid substitutions were made at each of the 47 linear amino acid positions, from N-terminus to C-terminus, of SEQ ID NO: 4.

[0230] Allowable residues in linear positions from N-to-C terminus in Formula I are set forth in TABLE 5, with reference to SEQ ID NO: 4 (Reference Miniprotein 4). The “allowable residues” represent those residues which may be changed from the reference residue at a given position in Reference Miniprotein 4 (SEQ ID NO: 4). For example, position 6 in TABLE 5 is listed as having “F” as an allowable residue, but position 6 in SEQ ID NO: 4 is Y, so the allowable residues are in addition to those set forth in SEQ ID NO: 4. To give but another example, position 11 in TABLE 5 is listed as L or I as allowable residues, but SEQ ID NO: 4 has an L at position 11, thus, allowable residues should be considered to be those in addition to the residues present in SEQ ID NO: 4.For example, at position 1, serine (S) was substituted with alanine, asparagine, aspartic acid, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, arginine, threonine, tryptophan, tyrosine, or valine. The binding affinity (KD) for CD123 was assessed using yeast surface display. This process was continued for each of the remaining 46 amino acids in SEQ ID NO: 4, and a map of substitutions tolerances was developed as shown in TABLE 5. To be considered an allowable residue, binding of the mutated miniproteins had to occur at an affinity 20 nM or stronger. As shown in TABLE 5, there were allowable residues at positions other than 9, which were immutable in that they could not be varied (to another amino acid residue) and still maintain binding at an affinity of 20 nM or stronger.TABLE 5. Allowable Residues in Synthetic Miniproteins*Relative to linear positions and amino acids in SEQ ID NO: 4. Allowable residues are in addition to those already present in SEQ ID NO: 4.EXAMPLE 6: PARATOPE IDENTIFICATION

[0231] This Example describes the identification and interrogation of the paratope of CD123 miniproteins using substitution tolerance data from Example 5.

[0232] Affinity measurements (KD) were determined using titration of soluble CD 123 used to label yeast cells expressing individual substitutions of Reference Miniprotein 4 (SEQ ID NO: 4) generated as described in Example 5. The binding signal at 0 pM, 244 pM, 488 pM, 976 pM, 1.95 nM, 3.91 nM, 7.81 nM, 15.6 nM, 31.3 nM, 62.5 nM, 125 nM, 250 nM, 500 nM, and 1 pM of CD123 was measured. The Hill equation (Formula II),Formula IIis the Hill coefficient, Ko.s is the half-saturation constant, Y is the output response, I is the input concentration. The structure of Reference Miniprotein 4 (SEQ ID NO: 4) was predicted with AlphaFold2 and used to identify solvent accessible surface residues. These residues were then analyzed within the context of single-site-mutational data to assess mutational tolerance in terms of binding affinity to CD 123. The paratope was identified manually with the SSM data derived from the Reference Miniprotein 4 (SEQ ID NO: 4) as described in Example 5. Paratope residues were identified as occurring at positions 6, 7, 9, 10, 11, 39, and 43 within the 47-mer of Reference Protein 4. Each position was substituted with each of the other 18 other naturally occurring amino acids (excluding cysteine), and binding was assessed using yeast display assays.

[0233] Briefly, each miniprotein being assessed was displayed on the surface of yeast at approximately 50,000 copies per yeast cell, and soluble CD123 was titrated against the yeast cells displaying the miniprotein at varying concentrations of CD123 from 10 pM to 1 pM. Each binding protein was engineered to contain an N-terminal extension of SEQ ID NO: 34 and a C-terminal extension of SEQ ID NO: 37 to facilitate expression and detection in yeast display, resulting in miniproteins as set forth in SEQ ID NOs: 41-64. These data were used to determine binding affinities of the miniproteins for CD 123.

[0234] Paratope residues were identified at positions corresponding to amino acid residues relative to SEQ ID NO: 4, which is a 47-mer, namely at positions 6, 7, 9, 10, 11, 39, and 43. One paratope residue was found to be immutable in that it was completely intolerant to substitution without loss of function (see, e.g., TABLE 6).TABLE 6. Paratope Residues and Tolerances

[0235] The amino acid corresponding to position 6 of SEQ ID NO: 25 or SEQ ID NO: 29 (with residues numbered linearly starting with 1 at the N-terminal amino acid, through 47 as the C-terminal amino acid) was 100% conserved over 21 sequences as provided herein in SEQ ID NOs: 1-24. Position 9 was 100% conserved across all Miniproteins (SEQ ID NOs: 1-24) and using SSM analysis of SEQ ID NO: 4 was found to be immutable in that it could not be changed from its identity as a glutamic acid (E) without materially decreasing CD123 binding potency. In this example, a decrease in binding potency was considered a material decrease in binding potency when a CD 123 binding protein had a binding affinity of weaker than 250 nM.

[0236] The amino acid corresponding to position 6 of SEQ ID NO: 25 or SEQ ID NO: 29 (with residues numbered linearly starting with 1 at the N-terminal amino acid, through 47 as the C-terminal amino acid) was 100% conserved in SEQ ID NOs: 1-24 and using SSM analysis of SEQ ID NO: 4, could only tolerate substitution from tyrosine (Y) to phenylalanine (F) without materially decreasing CD 123 binding potency to greater than 250 nM as measured by yeast on-cell affinity.

[0237] The amino acid corresponding to position 7 of SEQ ID NO: 25 or SEQ ID NO: 29 (with residues numbered linearly starting with 1 at the N-terminal amino acid, through 471Amino acid identities at positions in SEQ ID NO: 25 or 29. Paratope was determined using SSM data with Reference Miniprotein 4 (SEQ ID NO: 4, which is a Series 1.1 protein of consensus sequence SEQ ID NO: 4) as a starting sequence; position refers to linear, ordinal position with amino acid position 1 being the N-terminal amino acid in SEQ ID NO: 4 and amino acid 47 being the C-terminal amino acid in SEQ ID NO: 4.as the C-terminal amino acid) was 100% conserved in SEQ ID NOs: 1-24 and using SSM analysis of SEQ ID NO: 4, could only tolerate substitution from alanine (A) to serine (S) or glycine (G) without materially decreasing CD123 binding potency to greater than 250 nM as measured by yeast on-cell affinity.

[0238] The amino acid corresponding to position 10 of SEQ ID NO: 25 or SEQ ID NO: 29 (with residues numbered linearly starting with 1 at the N-terminal amino acid, through 47 as the C-terminal amino acid) was 100% conserved in SEQ ID NOs: 1-24 and using SSM analysis of SEQ ID NO: 4, could only tolerate substitution from tyrosine (Y) to phenylalanine (F) or tryptophan (W) without materially decreasing CD 123 binding potency to greater than 250 nM as measured by yeast on-cell affinity.

[0239] The amino acid corresponding to position 11 of SEQ ID NO: 25 or SEQ ID NO: 29 (with residues numbered linearly starting with 1 at the N-terminal amino acid, through 47 as the C-terminal amino acid) was 100% conserved in SEQ ID NOs: 1-24 and using SSM analysis of SEQ ID NO: 4, could only tolerate substitution from leucine (L) to isoleucine (I) without materially decreasing CD123 binding potency to greater than 250 nM as measured by yeast on-cell affinity.

[0240] The amino acid corresponding to position 39 of SEQ ID NO: 25 or SEQ ID NO: 29 (with residues numbered linearly starting with 1 at the N-terminal amino acid, through 47 as the C-terminal amino acid) was 100% conserved in SEQ ID NOs: 1-24 and using SSM analysis of SEQ ID NO: 4, could only tolerate substitution from leucine (L) to methionine (M), isoleucine (I), or valine (V) without materially decreasing CD123 binding potency to greater than 250 nM as measured by yeast on-cell affinity.

[0241] The amino acid corresponding to position 43 of SEQ ID NO: 25 or SEQ ID NO: 29 (with residues numbered linearly starting with 1 at the N-terminal amino acid, through 47 as the C-terminal amino acid) was either tyrosine (Y) or histidine (H) in SEQ ID NOs: 1-24 and using SSM analysis of SEQ ID NO: 4, could not tolerate other substitutions without materially decreasing CD123 binding potency to greater than 250 nM as measured by yeast on-cell affinity.

[0242] A conformational paratope of a CD123 binding protein of the present disclosure can be represented as X6 - X7 - X9 - X10 - XI 1 - X39 - X43 (SEQ ID NO: 87), where X6 is Y, X7 is A, X9 is E, X10 is Y, XI 1 is L, X39 is L, and X43 is H. Without losing binding potency as measured by binding at 20 nM or stronger, X6 could also be F; X7 could also be S or G; X10 could also be F or W; XI 1 could also be I; X39 could also be M,I, or V; and X43 could also be Y. X9 could not be substituted without loss of potency beyond the threshold.EXAMPLE 7: ENGINEERED CONJUGATION SITES

[0243] This Example describes the optimization of two CD123 miniproteins from Example 2 for placement of conjugation sites. Two Reference Miniproteins, 1, and 10, were selected for further engineering. Amino acid sequences of all billion proteins from the library in Example 1 were reviewed, and three sites were selected in which to rationally insert lysine residues as conjugation sites: position 26, position 35, and position 45, relative to SEQ ID NOs: 25 or 29. Selection criteria included exclusion of sites that may interfere with paratope binding. Thus, Reference Miniproteins 2-4 and 22-24 (SEQ ID NOs: 2-4 and 22-24) were developed, as shown in TABLE 7A. Reference Miniproteins 22, 23, and 24 belong to series 1 (according to the consensus sequence of SEQ ID NO: 25), and Reference Miniproteins 2, 3, and 4, belong to Series 1 and Subseries 1.1 (according to consensus sequences of SEQ ID NO: 25 and 29, respectively). Parameters of each protein (molecular weight, isoelectric point, and charge at pH 7.4) are set forth in TABLE 7B. Binding characteristics of each miniprotein to human CD123 were measured as shown in TABLE 7B, and binding activities (Ka, Kd, and KD values) of each miniprotein to human CD 123 were determined using SPR.TABLE 7A. Exemplary CD123 binding proteins with engineered lysine conjugation sitesTABLE 7B. Protein Parameters of Exemplary Reference Miniproteins* protein parameters are calculations based on each 47 amino acid sequence.TABLE 7C. Binding Characteristics of Exemplary Reference Miniproteinsinteraction half-life is time after initial binding at which half of the starting pool of a plurality of CD 123 binding proteins remain bound to CD 123.

[0244] Reference Miniprotein 2 had a lysine conjugation site placed at position 26(relative to SEQ ID NO: 25), Reference Miniprotein 3 and 23 each had a lysine conjugation site placed at position 35 (relative to SEQ ID NO: 25), and Reference Miniprotein 4 and 24each had a lysine conjugation site placed at position 45 (relative to SEQ ID NO: 25). Reference Miniproteins 2, 3, 4, were used to conjugate exemplary effectors as described in Example 8.EXAMPLE 8: CONJUGATION OF SELECTED CD123 MINIPROTEINS TO EXEMPLARY EFFECTORS & IN VITRO BINDING CHARACTERIZATION

[0245] This example describes the preparation of synthetic CD123 binding proteins comprising an exemplary fluorophore. Each of Reference Miniproteins 2, 3, and 4 (SEQ ID NOs: 2, 3, and 4, respectively) were used to conjugate an exemplary effector.Reference Miniprotein 2 had a lysine conjugation site placed at position 26 (relative to SEQ ID NO: 25)), Reference Miniprotein 3 had a lysine conjugation site placed at position 35 (relative to SEQ ID NO: 25) and is, and Reference Miniprotein 4 had a lysine conjugation site placed at position 45 (relative to SEQ ID NO: 25) and is referred to as Reference Miniprotein 27 (SEQ ID NO: 4 with 5-FL at position 45). Reference Miniprotein 28 was generated by chemically synthesizing a CD 123 binding protein comprising SEQ ID NO: 4, with a non-canonical lysine-fluorescein incorporated into position 45.

[0246] Reference Miniproteins 2, 3, and 4 (were conjugated to an NHS-functionalized fluorescein effector with no linker (NHS-5-FL) as shown in TABLE 8A and according to procedures known to those of skill in the art, but modified to use higher temperatures and DMSO at concentrations that are not tolerated by native proteins, but are tolerated by the synthetic CD123 miniproteins. The conjugated miniprotein sequences are referred to as Reference Miniprotein 25 (SEQ ID NO: 2 with 5-FL at position 26), Reference Miniprotein 26 (SEQ ID NO: 3 with 5-FL at position 35), and Reference Miniprotein 27 (SEQ ID NO: 4 with 5-FL at position 45) and their respective structures are set forth in TABLE 8B, indicating effector and position.TABLE 8A. Exemplary effector structure

[0247] Yield, purity, and DAR of the conjugates were assessed via HPLC at 280 nm and 493 nm (data not shown).

[0248] Conjugates were purified by size exclusion chromatography (SEC) using Sephadex G15 or G25 columns to remove unbound activator. Anion exchange chromatography (AEX) was then performed using a Pierce Strong Anion Exchange spin column to isolate the DAR = 1 species. The purified conjugates were then analyzed by liquid chromatography-mass spectroscopy (LC-MS) to verify the molecular weight, purity, and conjugation location of each sample.

[0249] Binding characteristics of the exemplary CD123 binding proteins in TABLE 8B are provided in TABLE 8C.

[0250] Synthetic CD123 binding proteins provided herein were successfully conjugated to a fluorescent effector with and without linker, and can be used in applications that involve bringing a therapeutic cell into close proximity for cancer cell death (e.g., a CAR expressing an anti-FITC binding protein).TABLE 8B. Exemplary CD123 binding proteins with a FITC-5-FL effector conjugated to a lysine at position 26, 35, or 45TABLE 8C. Binding Characteristics of Exemplary Reference Miniproteins of 8Binteraction half-life is time after initial binding at which half of the starting pool of a plurality of CD 123 binding proteins remain bound to CD 123.INCORPORATION BY REFERENCE

[0251] All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc. , whether supra or infra, are hereby incorporated by reference in their entirety for all purposes. To the extent the material incorporated by reference contradicts or is inconsistent with this specification, the specification will supersede any such material.EQUIVALENTS

[0252] The invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The foregoing embodiments are therefore to be considered in all respects illustrative rather than limiting on the invention described herein. Scope of the invention is thus indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.TABLE 9. Table of Sequences

Claims

CLAIMSWhat is Claimed is:

1. A synthetic CD123 binding protein, the binding protein comprising:(a) an amino acid sequence from 35 amino acids to 100 amino acids in length;(b) a net negative charge in phosphate buffered saline (PBS);(c) a binding affinity for CD 123 stronger than 1 pM; and(d) a stability profile such that the protein (i) retains at least 90% binding affinity to CD123 upon cooling to room temperature after thermal denaturation at 95°C in PBS for at least about five minutes relative to the protein prior to thermal denaturation; (ii) retains at least 90% binding affinity to CD 123 after incubation for 16 hours at 37°C of incubation in PBS relative to the protein under the same conditions prior to incubating; and / or (iii) retains at least 90% binding affinity to CD 123 in PBS following chemical denaturation in 4 M urea for 1 hour at room temperature relative to the protein prior to chemical denaturation.

2. A synthetic CD123 binding protein, the binding protein comprising:(a) an amino acid sequence from 30 amino acids to 95 amino acids in length;(b) a net negative charge in PBS;(c) a binding affinity for CD 123 stronger than 10 pM;(d) at least three alpha helices;(e) at least two amino acid loops, where a first loop having a first amino acid sequence connects a terminal amino acid (e.g., a C-terminal amino acid) of a first alpha helix to a terminal amino acid (e.g., a N-terminal amino acid) of a second alpha helix, and a second loop having a second amino acid sequence connects a second, terminal amino acid (e.g., a C- terminal amino acid) of the second alpha helix to a terminal amino acid (e.g., an N-terminal amino acid) of a third alpha helix; and(f) a hydrophobic core defined by at least two hydrophobic amino acids present in at least one, two or three of the three alpha helices.

3. The synthetic CD 123 binding protein of claim 1 or 2, wherein binding to CD 123 occurs through a paratope of the CD123 binding protein, which paratope is defined by amino acids X6 - X7 - X9 - XI 0 - XI 1 - X39 - X43, wherein X6 is selected from Y or F, X7 is selected from A, S, or G, X9 is E, XI 0 is selected from Y, F, or W, XI 1 is selected from L or I, X39 is selected from L, M, I, or V, and X43 is selected from H or Y, and wherein the amino acid numbering corresponds to the numbering of SEQ ID NOs: 25 or 29.

4. A synthetic CD123 binding protein comprising a conformational paratope defined by amino acids X6 - X7 - X9 - X10 - XI 1 - X39 - X43, wherein X6 is selected from Y or F, X7 is selected from A, S, or G, X9 is E, XI 0 is selected from Y, F, or W, XI 1 is selected from L or I, X39 is selected from L, M, I, or V, and X43 is selected from H or Y, and wherein the amino acid numbering corresponds to the numbering of SEQ ID NOs: 25 or 29.

5. The synthetic CD123 binding protein of claim 3 or 4, wherein X6 is Y, X7 is A, X9 is E, X10 is Y, XI 1 is L, X39 is L, and X43 is H.

6. The synthetic CD123 binding protein of any one of claims 1-5, wherein the binding protein comprises one or more of the following features:(a) free of tryptophan amino acids;(b) free of methionine amino acids;(c) free of lysine amino acids;(d) does not comprise an unpaired cysteine amino acid when cysteine amino acids are present in the protein;(e) free of glycosylation sites;(f) free of protease cleavage sites; and(g) soluble up to at least 1 mM in PBS at 4°C for one month.

7. The synthetic CD123 binding protein of any one of claims 1-6, wherein the binding affinity is between about 10 pM to about 0.1 nM; about 7.5 pM to about 0.75 nM; about 5 pM to about 0.5 nM; about 2.5 pM to about 0.25 nM; about 1 pM to about 1 nM; about 0.75 pM to about 1 nM, about 0.5 pM to about 1 nM; about 0.25 pM to about 1 nM; about 0.10 pM to about 1 nM; about 75 nM to about 1 nM; about 50 nM to about 1 nM; about 25 nM to about 1 nM; about 10 nM to about 1 nM; and about 5 nM to about 1 nM.

8. The synthetic CD 123 binding protein of any one of claims 1-7, wherein the binding affinity is stronger than about 1 pM, about 0.75 pM, about 0.5 pM, about 0.25 pM, about 0.1 pM, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, about 0.1 nM, about 0.01 nM, and about 0.001 nM.

9. The synthetic CD123 binding protein of any one of claims 2-8, wherein the N-terminus of the first alpha helix is preceded by one or more N-terminal amino acids.

10. The synthetic CD123 binding protein of any one of claims 2-9, wherein the C-terminus of the third alpha helix is followed by one or more C-terminal amino acids.

11. The synthetic CD123 binding protein of any one of claims 2 and 6-10, wherein(a) the first, second, and / or third alpha helix each contains at least one hydrophobic amino acid, wherein, optionally, one or more of the at least one hydrophobic amino acids is not solvent accessible;(b) the first, second, and / or third alpha helix each contains at least two or three hydrophobic amino acids, wherein, optionally, one or more of the at least two or three hydrophobic amino acids is not solvent accessible;(c) the first, second, and / or third alpha helix each contain at least one or two solvent accessible amino acids;(d) the first and / or second loop contains at least one hydrophobic amino acid, or(e) the binding protein comprises any combination of elements selected from (a), (b),(c), (d), and (e).

12. The synthetic CD123 binding protein of claim 11, wherein:(a) the second and third alpha helix each contains at least two hydrophobic amino acids;(b) the first, second, and third alpha helix each contains at least one solvent accessible amino acid;(c) the first, second, and third alpha helix each contain at least two hydrophobic and one solvent accessible amino acids;(d) the second and third alpha helix each contains at least four solvent accessible amino acids; and / or(e) the first loop contains at least one hydrophobic amino acid.

13. The synthetic CD 123 binding protein of any one of the preceding claims, wherein the N-terminus of the first alpha helix is preceded by one or more N-terminal amino acids.

14. The synthetic CD123 binding protein of claim 13, wherein the N-terminus of the first alpha helix comprises an N-terminal extension.

15. The synthetic CD123 binding protein of claim 14, wherein the N-terminal extension has an amino acid sequence comprising that of SEQ ID NO: 34.

16. The synthetic CD123 binding protein of any one of the preceding claims, wherein the C-terminus of the third alpha helix is followed by one or more C-terminal amino acids.

17. The synthetic CD123 binding protein of claim 16, wherein the C-terminus of the third alpha helix comprises a C-terminal extension.

18. The synthetic CD 123 binding protein of claim 17, wherein the C-terminal extension has an amino acid sequence comprising that of SEQ ID NO: 37.

19. The synthetic CD123 binding protein of any one of the preceding claims, wherein the protein comprises from 35 amino acids to 70 amino acids in length, from 35 amino acids to 75 amino acids in length, from 35 amino acids to 65 amino acids in length, from 35 amino acids to 60 amino acids in length, from 35 amino acids to 55 amino acids in length, from 35 amino acids to 50 amino acids in length, from 35 amino acids to 45 amino acids in length, from 40 amino acids to 75 amino acids in length, from 40 amino acids to 80 amino acids in length, from 40 amino acids to 70 amino acids in length, from 40 amino acids to 65 amino acids in length, from 40 amino acids to 60 amino acids in length, from 40 amino acids to 55 amino acids in length, from 45 amino acids to 70 amino acids in length, from 45 amino acids to 65 amino acids in length, from 45 amino acids to 60 amino acids in length, from 50 amino acids in length to 60 amino acids in length, from 45 amino acids to 55 amino acids in length, from 50 amino acids to 70 amino acids in length, from 50 amino acids to 65 amino acids in length, or from 40 amino acids to 50 amino acids in length.

20. The synthetic CD123 binding protein of claim 19, wherein the protein comprises 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 amino acids.

21. A synthetic CD123 binding protein comprising:(i) an amino acid sequence arranged in a primary structure of Z1-D1-L1-D2-L2-D3-Z2 (Formula I), wherein DI, D2, and D3 are domains 1, 2, and 3, respectively; LI, and L2, are loops 1, and 2, respectively; and Z1 and Z2 are N- and C-terminal regions, respectively; and(ii) an amino acid of SEQ ID NO: 25, wherein DI, D2, and D3, independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 26, wherein X4 is A or V;X5 is Y, E, H, Q, F, or V; and X14 is E, L, or I;(b) D2 comprises an amino acid sequence of SEQ ID NO: 27, wherein X20 is E or T; X25 is L, Y, Q, S, G, or N; X26 is R, K, V, or I; X27 is H, V, or A; X30 is E, L, or D; and X31 is R, I, or Q; and(c) D3 comprises an amino acid sequence of SEQ ID NO: 28, wherein X35 is V, K, R, E, H, G, Q, or S; X37 is Q, or S; X41 is D, S, or E; and X43 is H or Y.

22. The synthetic binding protein of claim 21, wherein LI comprises an amino acid sequence of GX16IS, wherein X16 is A or F; and / or L2 comprises an amino acid sequence of GDD.

23. The synthetic binding protein of claim 21 or 22, wherein Z1 comprises an amino acid sequence of SGY and / or Z2 comprises an amino acid sequence of RX45GS, wherein X45 is Y, K, H, R, or E.

24. The synthetic CD123 binding protein of any one of claims 21-23, comprising an amino acid sequence of SEQ ID NO: 25, wherein X4 is A or V; X5 is Y, E, H, Q, F or V; X14 is E, L, or I; XI 6 is A or F; X20 is E or T; X25 is L, Y, Q, S, G, or N; X26 is R, K, V, or I; X27 is H, V, or A; X30 is E, L, or D; X31 is R, I, or Q; X35 is V, K, R, E, H, G, Q, or S; X37 is Q, or S; X41 is D, S, or E; X43 is H or Y; and X45 is Y, K, H, R, or E.

25. The synthetic CD123 binding protein of any one of claims 21-24, comprising an amino acid sequence selected from any of SEQ ID NOs: 1-24 or 41-64.

26. A synthetic CD123 binding protein comprising:(i) an amino acid sequence arranged in a primary structure of Z1-D1-L1-D2-L2-D3-Z2 (Formula I), wherein DI, D2, and D3 are domains 1, 2, and 3, respectively; LI, and L2, are loops 1, and 2, respectively; and Z1 and Z2 are N- and C-terminal regions, respectively; and(ii) an amino acid of SEQ ID NO: 29, wherein DI, D2, and D3, independently comprise any of the following combinations:(a) DI comprises an amino acid sequence of SEQ ID NO: 30, wherein X5 is Y, Q, or F;(b) D2 comprises an amino acid sequence of SEQ ID NO: 31, wherein X25 is L, Q,S, G, or N; and X26 is R, K, or I; and(c) D3 comprises an amino acid sequence of SEQ ID NO: 32, wherein X35 is V, K, E, H, G, or S; and X43 is H or Y.

27. The synthetic CD123 binding protein of claim 26, wherein LI comprises an amino acid sequence of SEQ ID NO: 33; and / or L2 comprises an amino acid sequence of GDD.

28. The synthetic CD123 binding protein of claim 26 or 27, wherein, Z1 comprises an amino acid sequence of SGY and / or Z2 comprises an amino acid sequence of RX45GS, wherein X45 is Y, K, or E.

29. The synthetic CD123 binding protein of any one of claims 26-28. comprising an amino acid sequence of SEQ ID NO: 29, wherein X5 is Y, Q, or F; X25 is L, Q, S, G, or N; X26 is R, K, or I; X35 is V, K, E, H, G, or S; X43 is H or Y; and X45 is Y, K, or E.

30. The synthetic CD123 binding protein of any one of claims 21-29, comprising an amino acid sequence selected from any of SEQ ID NOs: 1-9 or 41-49.

31. The synthetic CD 123 binding protein of any one of claims 21-24 or 26-29, wherein X26 is K.

32. The synthetic CD123 binding protein of any one of claims 21-24 or 26-29, wherein X35 is K.

33. The synthetic CD 123 binding protein of any one of claims 23, 24, or 26-29, wherein X45 is K.

34. The synthetic CD123 binding protein of any one of claims 31-33, wherein an effector is conjugated to the lysine at position X26, X35, or X45.

35. The synthetic CD123 binding protein of claim 34, wherein the conjugation is directly to the lysine (e.g., via NHS-based chemistry).

36. The synthetic CD123 binding protein of claim 34, wherein the effector comprises a cytotoxic molecule.

37. The synthetic CD123 binding protein of claim 34, wherein the cytotoxic molecule comprises a diphtheria toxin or portion thereof.

38. The synthetic CD123 binding protein of claim 34, wherein the effector comprises a small molecule or chelator.

39. The synthetic CD123 binding protein of any one of claims 21-38, wherein the binding protein has a binding affinity for CD 123 stronger than 1 pM.

40. The synthetic CD123 binding protein of any one of claims 21-39, wherein the binding affinity is between about 1 pM to about 0.001 nM; about 1 pM to about 0.01 nM; about 1 pM to about 0.75 nM; about 1 pM to about 0.5 nM; about 1 pM to about 0.25 nM; about 1 pM to about 0.01 nM; about 1 pM to about 1 nM; about 0.5 pM to about 1 nM; about 0.25 pM to about 1 nM; about 0.10 pM to about 1 nM; about 0.10 pM to about 0.01 nM; about 75 nM to about 1 nM; about 50 nM to about 1 nM; about 25 nM to about 1 nM; about 10 nM to about 1 nM; and about 5 nM to about 1 nM.

41. The synthetic CD123 binding protein of any one of claims 21-40, wherein the binding affinity is stronger than about 1 pM, about 0.75 pM, about 0.5 pM, about 0.25 pM, about 0.1 pM, about 75 nM, about 50 nM, about 25 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 0.75 nM, about 0.5 nM, about 0.25 nM, about 0.1 nM, about 0.01 nM, and about 0.001 nM.

42. The synthetic CD123 binding protein of any one of claims 21-41, wherein the amino acid sequence of the binding protein has at least 85 (e.g., 75, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 90.5, 91, 91.5, 92, 92.5, 93, 93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, 99.5, 99.6, 99.7, 99.8, 99.9) percent identity to the amino acid sequence of any of SEQ ID NOs: 1-24 or 41-64.

43. The synthetic CD 123 binding protein of any one of claims 1-42, wherein the protein has an amino acid sequence comprising or according to one or more sequences set forth in Table 9.

44. The synthetic CD123 binding protein of any one of claims 1-43, wherein the protein has an amino acid sequence comprising or according to one or more sequences as set forth in Table 1, with one or more amino acid substitutions as set forth in Table 2B.

45. The synthetic binding protein of any one of claims 1, 2, or 6-44, wherein the synthetic binding protein comprises a paratope defined by a paratope represented by X28 - X29 - X32 - X39 - X41 - X42, where X28 is V or L, X29 is D, X32 is D, X39 is T or V, X41 is I or L, and X42 is R or Q, wherein the amino acid positions correspond to those of SEQ ID NOs: 25 or 29 from N-terminus to C-terminus.

46. The synthetic binding protein of any one of claims 1, 2, or 6-44, comprising a paratope defined by any combination of positions as set forth in Table 6.

47. A synthetic CD123 binding protein comprising an amino acid sequence with reference to any of SEQ ID NOs: 1-24, but having one or more changes to one or more amino acid residues as set forth in Table 6.

48. A pharmaceutical composition comprising the synthetic CD123 binding protein of any of claims 1-47; and a pharmaceutically acceptable carrier.

49. The pharmaceutical composition of claim 48, wherein the synthetic CD123 binding protein further comprises an effector molecule.

50. The pharmaceutical composition of claim 49, wherein the effector is attached to the synthetic CD 123 binding protein at a lysine residue.

51. The pharmaceutical composition of claim 50, wherein the lysine residue is at position 26, 35, or 45 relative to SEQ ID NO: 25 or SEQ ID NO: 29.

52. The pharmaceutical composition of claim 49, wherein the effector is attached to the synthetic CD 123 binding protein via a linker.

53. The pharmaceutical composition of claim 52, wherein the linker comprises an amino acid sequence selected from Table 3B.

54. The pharmaceutical composition of claim 52 or 53, wherein the linker comprises a lysine.

55. The pharmaceutical composition of claim 54, wherein the linker comprises a lysine on its C-terminal end.

56. A method of targeting CD123, the method comprising contacting a cell that expresses CD123 on its cell surface with a composition comprising the synthetic CD123 binding protein of any one of claims 1-47 or the pharmaceutical composition of claim 40 or 41.

57. The method of claim 56, wherein the synthetic CD123 binding protein decreases IL-3- mediated activity (e.g., proliferation, survival) in a cancer cell expressing CD123 relative to cell-mediated activity in the cancer cell in the absence of the CD 123 binding protein.

58. A method of antagonizing IL-3 activity in a cancer cell, the method comprising contacting a cancer cell with a synthetic CD 123 binding protein of any one of claims 1- 47.

59. A method of treating cancer by administering to a subject in need thereof a conjugate comprising a synthetic CD123 binding protein of any one of claims 1-47 and an effector, or the pharmaceutical composition of any one of claims 48-55, wherein the effector comprises a cytotoxic molecule, wherein the administration localizes the cytotoxic molecule to the CD 123 -expressing cancer cell, promoting or increasing cytotoxicity by the effector.

60. A method of treating one or more cancer-related conditions in a subject in need thereof, the method comprising administering to the subject an effective amount of the synthetic CD 123 binding protein of claims 1-47 or the pharmaceutical composition of any one of claims 48-55.

61. The method of claim 59, wherein the administration is before, during, or after administration or use of one or more other treatments.

62. The method of claim 61, wherein the one or more other treatments is or comprises a biological agent (e.g., biologies, gene therapy, peptides), a small molecule (e.g., chemotherapy, corticosteroids, antivirals, antibiotics, anti-inflammatory agents, etc.), one or more cells (e.g., immunotherapy), and / or one or more mechanical interventions (e.g., surgery, cryotherapy).

63. The method of any one of claims 59-62, wherein the subject is diagnosed as having or at risk of having a cancer or population of cancerous cells (e.g., myeloma, lymphoma, leukemia, a solid tumor).

64. The method of claim 63, wherein the subject has been diagnosed as having cancer and / or a population of cancerous cells.

65. The method of claim 56, wherein the contacting step comprises contacting a population of cells expressing CD123 with a composition comprising a synthetic CD123 binding protein, wherein the synthetic binding protein further comprises an effector that binds to an antigen on a second, different cell, which second different cell promotes cytotoxicity of the population, wherein after the contacting, a greater portion of the population is dead as compared to contacting without the effector and / or without the synthetic CD123 binding protein.

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