Flt3-ligand bifunctional molecule for thrombocytopenia and acute radiation syndrome

Abstract

Provided herein are polypeptides, compositions, and methods for treating cancer in individuals using polypeptides that contain thrombopoietin domains and Flt3 ligand domains.Also provided herein are nucleic acids encoding such polypeptides, expression vectors and cells that contain such nucleic acids, and methods for producing polypeptides that contain thrombopoietin domains and Flt3 ligand domains.Administering a fusion polypeptide that contains thrombopoietin domains and Flt3 ligand domains to a subject can treat and alleviate the symptoms of hematopoietic insufficiency, including thrombocytopenia and / or acute radiation syndrome.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 373,436, filed August 24, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Among the adverse effects of exposure to high doses of radiation are hematopoietic failure, thrombocytopenia, and acute radiation syndrome. In some cases, the toxicity of radiation treatment may limit the radiation dose for cancer patients. In some cases, the subject may have a disease that results in hematopoietic failure. Fms-like tyrosine kinase 3 (Flt3) ligand (FL) and thrombopoietin have been envisioned as possible treatments to counter the adverse effects of radiation exposure and / or increase hematopoietic activity. However, the effectiveness of FL and thrombopoietin may be limited by their short half-lives in the circulation. Summary of the Invention

[0003] Provided herein is a polypeptide, composition and method for supporting cancer treatment in individuals using a polypeptide that comprises thrombopoietin domain and Flt3 ligand domain.Also described herein is the nucleic acid that encodes such polypeptide, the expression vector and cell that comprise such nucleic acid, and the method for producing the polypeptide that comprises thrombopoietin domain and Flt3 ligand domain.

[0004] Described herein are polypeptides comprising a thrombopoietin domain and an Flt3 ligand domain. In some embodiments, the thrombopoietin domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO:9. In some embodiments, the Flt3 ligand domain comprises an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO:3 or 5. Described herein are polypeptides comprising an amino acid sequence at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO:1. In some embodiments, the polypeptide further comprises an immunoglobulin Fc polypeptide or a fragment thereof. In some embodiments, the immunoglobulin is immunoglobulin G1 (IgG1). In some embodiments, the polypeptide further comprises an EPO leader sequence and / or a TEV cleavage domain. In some embodiments, the polypeptide further comprises a linker. In some embodiments, the linker connects the amino acid sequence at least 80% identical to SEQ ID NO:9 to the immunoglobulin Fc polypeptide or a fragment thereof. In some embodiments, a linker connects a thrombopoietin domain to an immunoglobulin Fc polypeptide or a fragment thereof. In some embodiments, a linker connects a thrombopoietin domain to a second thrombopoietin domain. In some embodiments, a linker connects a TEV domain to a thrombopoietin domain or a second thrombopoietin domain. In some embodiments, a linker connects an amino acid sequence at least 80% identical to SEQ ID NO: 3 or 5 to an immunoglobulin Fc polypeptide or a fragment thereof. In some embodiments, a linker connects a Flt3 ligand domain to an immunoglobulin Fc polypeptide or a fragment thereof. In some embodiments, the Flt3 ligand domain is Flt3 ligand isoform 1. In some embodiments, the Flt3 ligand domain is human Flt3 ligand isoform 1. In some embodiments, the amino acid sequence is identical to SEQ ID NO: 1.In some embodiments, the polypeptide is an immunoglobulin Fc polypeptide or fragment thereof and comprises one or more modifications relative to the wild-type IgG Gc region identified in SEQ ID NO: 7. In some embodiments, the one or more modifications affect the immunological properties of the immunoglobulin Fc polypeptide or fragment thereof. In some embodiments, the one or more modifications comprise L234A, L235A, N297A, N297Q, P329Q, or a combination thereof according to EU numbering. In some embodiments, the one or more modifications comprise L234A and L235A according to EU numbering. In some embodiments, the one or more modifications comprise N297A according to EU numbering. In some embodiments, the one or more modifications comprise P329Q according to EU numbering. In some embodiments, the immunological properties comprise antigen-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated phagocytosis (ADCP), or a combination thereof. In some embodiments, administering the polypeptide to a subject increases the platelet count in the subject. In some embodiments, administering the polypeptide to a subject increases the number of dendritic cells in the subject's blood. In some embodiments, the polypeptide improves survival of hematopoiesis-related cells in treated cells compared to untreated cells. In some embodiments, the polypeptide stimulates proliferation of hematopoiesis-related cells in treated cells compared to untreated cells. In some embodiments, the polypeptide activates the MAPK pathway. In some embodiments, the polypeptide increases ERK phosphorylation in treated cells compared to untreated cells. In some embodiments, the polypeptide increases Erk1 / 2 phosphorylation in treated cells compared to untreated cells. In some embodiments, the polypeptide activates the PI3K / Akt pathway. In some embodiments, the polypeptide increases Akt phosphorylation in treated cells compared to untreated cells. In some embodiments, the polypeptide increases ERK / Akt phosphorylation in treated cells compared to untreated cells.In some embodiments, the polypeptide is configured to bind to fms-like tyrosine kinase 3 (FLT3). Provided herein is a composition comprising the polypeptide provided herein and a pharmaceutically acceptable excipient. In some embodiments, the composition is formulated to be administered intravenously, subcutaneously, or intratumorally.

[0005] Provided herein are methods for assisting cancer treatment in an individual, comprising administering to the individual any one of the polypeptides provided herein. Provided herein are methods for assisting cancer treatment in an individual, comprising administering to the individual any one of the compositions provided herein. Provided herein are methods for regulating an immune response in an individual, comprising administering to the individual any one of the polypeptides provided herein. Provided herein are methods for stimulating dendritic cell proliferation and activation, comprising administering to the individual any one of the polypeptides provided herein. In some embodiments, the method improves the survival of hematopoiesis-related cells in treated cells compared to untreated cells. In some embodiments, the method stimulates the proliferation of hematopoiesis-related cells in treated cells compared to untreated cells. In some embodiments, the method activates the MAPK pathway. In some embodiments, the method increases ERK phosphorylation in treated cells compared to untreated cells. In some embodiments, the method increases Erk1 / 2 phosphorylation in treated cells compared to untreated cells. In some embodiments, the method activates the PI3K / Akt pathway. In some embodiments, the method increases Akt phosphorylation in treated cells relative to untreated cells. In some embodiments, the method increases ERK / Akt phosphorylation in treated cells relative to untreated cells.

[0006] Provided herein is a nucleic acid encoding any one of the polypeptides provided herein.Described herein is an expression vector comprising any one of the nucleic acids provided herein.Described herein is a cell comprising a nucleic acid encoding any one of the polypeptides provided herein.Described herein is a method for producing any one of the polypeptides provided herein, the method comprising culturing cells under conditions sufficient to express the polypeptide.Described herein is any one of the polypeptides provided herein for use in a method for assisting in the treatment of cancer in an individual. [Brief explanation of the drawings]

[0007] The novel features described herein are set forth with particularity in the appended claims. A better understanding of the features and advantages of the features described herein will be obtained by reference to the following detailed description that sets forth illustrative examples in which the principles of the features described herein are utilized and the accompanying drawings. [Figure 1] FIG. 1 shows a schematic diagram of an exemplary fusion polypeptide comprising a FL domain, a human IgG1 domain, and a thrombopoietin domain. [Figure 2] Figure 2 shows a 4-12% Bis-Tris gel showing the expression of a fusion polypeptide containing an FL domain and a thrombopoietin domain, indicated by an arrow. [Figure 3] Figure 3 shows purified FLT3L-1-romiplostim protein on a 4-12% Bis-Tris precast SDS-PAGE gel. The SDS-PAGE gel shows protein bands of approximately 100 kDa under non-reducing conditions and approximately 70 kDa under reducing conditions (indicated by arrows), corresponding to the predicted molecular weight of the FL-romiplostim fusion polypeptide under each condition. The first lane shows the unstained protein ladder. [Figure 4] FIG. 4 shows flow cytometry results demonstrating FL-romiplostim fusion polypeptide rescue from apoptosis in OCI-AML5 cells deprived of FBS and growth factors. [Figure 5] FIG. 5 shows the percentage of apoptotic cells from the results of flow cytometry. [Figure 6A] 6A and 6B show the percentage of cells positive for phosphorylated Erk1 / 2 protein and the mean fluorescence intensity from the flow cytometry results. [Figure 6B] 6A and 6B show the percentage of positive cells and the mean fluorescence intensity for phosphorylated Erk1 / 2 protein from the results of flow cytometry. [Figure 7A] Figure 7A and Figure 7B show the percentage of cells positive for total Erk1 / 2 expression and mean fluorescence intensity using p44 / 42 MAPK (Erk1 / 2) antibody from flow cytometry results, providing a baseline for Erk1 / 2 expression of approximately 70-75% percent positive cells. [Figure 7B] Figure 7A and Figure 7B show the percentage of cells positive for total Erk1 / 2 expression and mean fluorescence intensity using p44 / 42 MAPK (Erk1 / 2) antibody from flow cytometry results, providing a baseline for Erk1 / 2 expression of approximately 70-75% percent positive cells. [Figure 8A] 8A and 8B show the percentage of positive cells and mean fluorescence intensity for PI3K-Akt phosphorylation from the flow cytometry results. [Figure 8B] 8A and 8B show the percentage of positive cells and mean fluorescence intensity for PI3K-Akt phosphorylation from the flow cytometry results. [Figure 9A] Figure 9A and Figure 9B show the percentage of positive cells and mean fluorescence intensity for PI3K-Akt expression using Akt(pan)(C67E7) antibody from flow cytometry results, providing a baseline for Akt expression of approximately 80-90% positive cells. [Figure 9B]Figure 9A and Figure 9B show the percentage of positive cells and mean fluorescence intensity for PI3K-Akt expression using Akt(pan)(C67E7) antibody from flow cytometry results, providing a baseline for Akt expression of approximately 80-90% positive cells. [Figure 10A] 10A and 10B show the percentage of positive cells and mean fluorescence intensity for Erk1 / 2 phosphorylation after treatment with thrombopoietin mimetic (TPOm) peptide. [Figure 10B] 10A and 10B show the percentage of positive cells and mean fluorescence intensity for Erk1 / 2 phosphorylation after treatment with thrombopoietin mimetic (TPOm) peptide. [Figure 11A] 11A and 11B show the percentage of positive cells and mean fluorescence intensity for Erk1 / 2 phosphorylation after treatment with romiplostim. [Figure 11B] 11A and 11B show the percentage of positive cells and mean fluorescence intensity for Erk1 / 2 phosphorylation after treatment with romiplostim. [Figure 12A] 12A and 12B show the percentage of positive cells and mean fluorescence intensity for Erk1 / 2 phosphorylation from the flow cytometry results. [Figure 12B] 12A and 12B show the percentage of positive cells and mean fluorescence intensity for Erk1 / 2 phosphorylation from the flow cytometry results. [Figure 13] FIG. 13 shows the results of an XTT assay of the M-07e cell line cultured with FL-romiplostim fusion polypeptide and assessed for its effect on cell proliferation. [Figure 14] FIG. 14 shows examples of the results of binding of FLT3L-romiplostim fusion polypeptides to human cMLP or mouse cMLP-expressing cells. [Figure 15] FIG. 15 shows an example of a 4-12% Bis-Tris gel showing the expression of FLT3L-romiplostim fusion polypeptide Fc mutants. [Figure 16]FIG. 16 shows an example of increased plasma concentrations following subcutaneous injection of FLT3L-romiplostim Fc mutants compared to wild-type Fc or control. [Figure 17] FIG. 17 shows an example of increased plasma concentrations following intravenous injection of FLT3L-romiplostim Fc mutants compared to controls. [Figure 18] FIG. 18 shows an example of an increase in platelet counts with injection of FLT3L-romiplostim versus controls. [Figure 19A] Figures 19A and 19B show examples of increases in platelets, spleen and blood DCs, respectively, from injection of FLT3L-romiplostim versus controls. [Figure 19B] Figures 19A and 19B show examples of increases in platelets, spleen and blood DCs, respectively, from injection of FLT3L-romiplostim versus controls. Detailed Description of the Invention

[0008] Individuals with hematopoietic deficiency, also known as bone marrow failure (BMF), may have a decreased production of one or more cell types in the hematopoietic lineage. Hematopoietic deficiency typically results in a decreased number of hematopoietic precursors and cytopenia in an individual's bone marrow. In some cases, hematopoietic deficiency can be hereditary or acquired. In some cases, exposure to high doses of radiation can result in acquired hematopoietic deficiency, including, but not limited to, thrombocytopenia and acute radiation syndrome. In some cases, cancer patients undergoing radiation treatment may experience severe side effects, including radiation syndrome, thrombocytopenia, and hematopoietic deficiency. In some cases, the toxicity of radiation treatment can limit the amount of radiation treatment a patient can receive, making it difficult to complete the treatment regimen and achieve tumor reduction. Reducing the effects of radiation syndrome, thrombocytopenia, and hematopoietic deficiency may allow cancer patients to receive more radiation treatments. In some cases, individuals may experience acute high-dose radiation exposure, which can result in adverse health outcomes such as radiation toxicity and acute radiation syndrome.

[0009] Thrombocytopenia is a disease that occurs when the number of platelets is too low. Because platelets play an important role in helping blood clot, thrombocytopenia can be accompanied by abnormal bleeding. In some cases, thrombocytopenia can result from decreased platelet production in the bone marrow, increased platelet breakdown in the bloodstream, and / or increased platelet breakdown in the spleen or liver.

[0010] Fms-like tyrosine kinase 3 (Flt3) ligand (FL) and thrombopoietin have been proposed as potential treatments for countering the adverse effects of radiation exposure and / or increasing hematopoietic activity and / or recovery. In some cases, FL injection has shown protective effects against high doses of radiation, including, but not limited to, increased bone marrow hematopoietic activity and hematopoietic recovery. Often, thrombopoietin can be used to increase platelet counts and reduce the risk of bleeding in individuals with thrombocytopenia. However, the effectiveness of FL and thrombopoietin may be limited by their short half-lives in the circulation. FL typically has a half-life of less than 5 hours after intraperitoneal injection in mouse models.

[0011] Often, the half-life can be extended when FL is presented as a fusion polypeptide, also referred to herein as a fusion protein. In some cases, a human FL fragment crystallizable (Fc) fusion polypeptide has a half-life of approximately 24 hours in a mouse model. Romiplostim, an Fc-peptide fusion protein (peptibody) analog of thrombopoietin (TPO), typically has a half-life ranging from 1 to 34 days, with a median of approximately 3.5 days. Sometimes, the presence of an Fc domain increases the half-life of the polypeptide. In some cases, the increased half-life may be due to the interaction of the Fc domain with the neonatal Fc receptor, which aids in the recycling of Fc fusion polypeptides encapsulated in the plasma membrane. In some cases, the presence of an Fc domain allows for cost-effective, single-step purification of the fusion polypeptide. In some cases, the presence of an Fc domain improves the solubility and stability of the partner domain in the fusion polypeptide. In some cases, the Fc domain contains one or more modifications compared to the wild-type IgG Fc region. In some cases, one or more modifications affect the immunological properties of the Fc domain, including, but not limited to, antigen-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated phagocytosis (ADCP).

[0012] Provided herein are polypeptides, compositions, and methods for treating cancer in individuals using polypeptides comprising thrombopoietin domains and FL domains. Also provided herein are nucleic acids encoding such polypeptides, expression vectors and cells comprising such nucleic acids, and methods for producing polypeptides comprising thrombopoietin domains and FL domains. Administration of a fusion polypeptide comprising a thrombopoietin domain and an FL domain to a subject can treat and reduce symptoms of hematopoietic insufficiency, including thrombocytopenia and / or acute radiation syndrome.

[0013] Polypeptides Provided herein are polypeptides comprising a thrombopoietin domain and an Flt3 ligand domain.

[0014] Fms-related tyrosine kinase 3 (FLT3) ligand (FL), encoded by the FLT3LG gene in humans, is a hematopoietic cytokine that regulates the proliferation of hematopoietic progenitor cells. FL binds to the Fms-like tyrosine kinase receptor Flt3 / Flk2. FL is a homodimer of four-helix bundle protomers and is structurally homologous to stem cell factor (SCF) and colony-stimulating factor 1 (CSF-1). FL does not stimulate proliferation of early hematopoietic cells by itself, but it can act as a major growth factor in synergy with other CSFs and interleukins to induce the proliferation and differentiation of various blood cell precursors and stimulate the proliferation of dendritic cells. Multiple isoforms of FL have been identified, including transmembrane and membrane-bound isoforms. The predominant biologically active form (209 a.a.) is anchored to the cell surface by the extracellular domain of the transmembrane protein. The membrane-bound isoform can be proteolytically cleaved to generate a biologically active soluble isoform. In some cases, the active form of FL refers to the physiologically active form that is membrane-anchored. In some cases, the active form of FL refers to the therapeutically active form that lacks a transmembrane segment.

[0015] Thrombopoietin (TPO), also known as megakaryocyte growth and development factor (MGDF), is a protein encoded by the THPO gene in humans. TPO is produced by the liver and kidney and regulates platelet production by stimulating megakaryocyte production and differentiation. TPO is a ligand for MLP / C MPL, a product of the myeloproliferative leukemia viral oncogene. In some cases, plasma TPO levels may be inversely correlated with the mass of megakaryocytes and platelets, which degrade TPO after binding to specific membrane receptors. In some cases, the function of TPO or a TPO domain may be assessed by a TPO performance assay, such as an ELISA or a bead-based multiplex assay. In some embodiments, the thrombopoietin domain herein refers to thrombopoietin or a functional fragment thereof, or romiplostim or a functional fragment thereof. Romiplostim is an Fc-fusion protein functional analog of thrombopoietin that increases platelet production through activation of the thrombopoietin receptor. Romiplostim is a dimeric Fc-peptide fusion protein (peptibody) with two identical single-chain subunits, each consisting of 269 amino acid (aa) residues. Each subunit consists of a human IgG1 Fc carrier domain covalently linked to a polypeptide sequence containing two binding domains that interact with the thrombopoietin receptor c-Mpl. Each binding domain consists of 14 aa. The amino acid sequence of romiplostim is not similar to that of endogenous thrombopoietin. When romiplostim binds to the TPO receptor, it can promote the proliferation of bone marrow megakaryocyte colony-forming cells, which leads to increased platelet production via the JAK2 and STAT5 kinase pathways. Romiplostim can be used to treat low blood platelet counts (thrombocytopenia) and help prevent bleeding in patients with idiopathic thrombocytopenia (ITP). In some cases, romiplostim can be used to reduce the effects of acute radiation syndrome (ARS). In some cases, romiplostim acts through a pathway similar to thrombopoietin. In some embodiments, romiplostim comprises a thrombopoietin domain with an Fc domain.

[0016] In some embodiments, the thrombopoietin domain comprises a functional analog or mimetic of thrombopoietin having substantially the same function. In some embodiments, the thrombopoietin domain comprises a functional analog or mimetic of romiplostim having substantially the same function. In some embodiments, when the thrombopoietin domain binds to a TPO receptor, the thrombopoietin domain can promote the growth of bone marrow megakaryocyte colony-forming cells. In some embodiments, when the thrombopoietin domain binds to a TPO receptor, the thrombopoietin domain can result in increased platelet production. In some embodiments, platelet production can be increased via the JAK2 and STAT5 kinase pathways. In some embodiments, administration of a composition comprising a thrombopoietin domain can treat thrombocytopenia and help prevent bleeding in patients with ITP. In some embodiments, administration of a composition, the thrombopoietin domain. In some embodiments, the function of the thrombopoietin domain can be assessed by ELISA or a TPO performance assay (e.g., a bead-based multiplex assay).

[0017] Provided herein are polypeptides, compositions, and methods for supporting cancer treatment in individuals using polypeptides comprising a thrombopoietin domain and an Flt3 ligand domain. Also provided herein are nucleic acids encoding such polypeptides, expression vectors and cells comprising such nucleic acids, and methods for producing polypeptides comprising a thrombopoietin domain and an Flt3 ligand domain. Administration of a fusion polypeptide comprising a thrombopoietin domain and an Flt3 ligand domain to a subject can treat and reduce symptoms of hematopoietic deficiency, including thrombocytopenia and / or acute radiation syndrome. Figure 1 shows a schematic diagram of an exemplary fusion polypeptide comprising an FL domain, a human IgG1 domain, and a thrombopoietin domain.

[0018] Provided herein are polypeptides comprising a human Flt3 ligand domain and a thrombopoietin domain. In some embodiments, the polypeptide is configured to bind to fms-like tyrosine kinase 3 (Flt3). In some embodiments, the polypeptide comprises a thrombopoietin domain and an Flt3 ligand domain. In some embodiments, the thrombopoietin domain comprises an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 9. In some embodiments, the thrombopoietin domain comprises the amino acid sequence of SEQ ID NO: 9. In some embodiments, the thrombopoietin domain comprises an amino acid sequence at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to romiplostim. In some embodiments, the Flt3 ligand domain comprises human Flt3 ligand isoform 1. In some embodiments, Flt3 ligand isoform 1 is membrane-bound. In some embodiments, the Flt3 ligand comprises a soluble Flt3 ligand. In some embodiments, the Flt3 ligand domain comprises an amino acid sequence at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO:3. In some embodiments, the Flt3 ligand domain comprises an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 5. In some embodiments, the Flt3 ligand domain comprises an amino acid sequence that is SEQ ID NO: 3. In some embodiments, the Flt3 ligand domain comprises an amino acid sequence that is SEQ ID NO: 5. In some embodiments, the Flt3 ligand domain is Flt3 ligand isoform 1. In some embodiments, the Flt3 ligand domain is human Flt3 ligand isoform 1. In some embodiments, the amino acid sequence is identical to SEQ ID NO: 1.

[0019] In some embodiments, the polypeptides provided herein comprise an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 1. In some embodiments, the polypeptide comprises an amino acid sequence that is SEQ ID NO: 1.

[0020] In some embodiments, the polypeptide further comprises an immunoglobulin Fc polypeptide or a fragment thereof. In some embodiments, the immunoglobulin is immunoglobulin G (IgG). In some embodiments, the immunoglobulin is immunoglobulin G1 (IgG1). In some embodiments, the immunoglobulin comprises a human immunoglobulin isotype. In some embodiments, the immunoglobulin comprises one or more of IgG1, IgG2, IgG3, IgG4, IgA, IgE, or IgM. In some embodiments, the polypeptide comprises one or more modifications to an immunoglobulin Fc polypeptide or a fragment thereof relative to the wild-type IgG Fc region identified in SEQ ID NO: 7. In some embodiments, the one or more modifications comprise L234A, L235A, N297A, N297Q, P329Q, or a combination thereof according to EU numbering. In some embodiments, the one or more modifications comprise L234A and L235A (LALA) according to EU numbering. In some embodiments, the one or more modifications comprise N297A according to EU numbering. In some embodiments, the one or more modifications include N297Q according to EU numbering. In some embodiments, the one or more modifications include P329Q according to EU numbering. In some embodiments, the immunoglobulin comprises an effector function mutation. In some embodiments, the effector function mutation includes L234A and L235A (LALA), N297A, N297Q, or P329Q, or a combination thereof. In some embodiments, the immunoglobulin Fc polypeptide or fragment thereof comprises an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO:7.

[0021] In some embodiments, the polypeptide further comprises one or more signal sequences. In some embodiments, the polypeptide further comprises an EPO leader sequence and / or a tobacco etch virus (TEV) cleavage domain. In some embodiments, the signal sequence comprises an EPO leader sequence. In some embodiments, the signal sequence comprises a TEV cleavage domain. In some embodiments, the EPO leader sequence comprises an amino acid sequence at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO:2. In some embodiments, the TEV cleavage domain comprises an amino acid sequence at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO:10. In some embodiments, the signal sequence comprises one or more of human OSM, VSV-G, mouse Igκ, mouse Ig heavy chain, BM40, secretome, human IgKVII, CD33, tPA, human chymotrypsinogen, human trypsinogen-2, Gaussia luc, albumin, influenza hemagglutinin, human insulin, or silkworm fibroin.

[0022] In some embodiments, the polypeptide further comprises a linker. In some embodiments, the linker attaches an amino acid to the immunoglobulin Fc polypeptide or a fragment thereof. In some embodiments, the linker attaches an amino acid at least 80% identical to SEQ ID NO: 3 or 5 to the immunoglobulin Fc polypeptide or a fragment thereof. In some embodiments, the linker attaches a thrombopoietin domain to the immunoglobulin Fc polypeptide or a fragment thereof. In some embodiments, the linker attaching the amino acid to the immunoglobulin Fc polypeptide or a fragment thereof is at least 80% identical to SEQ ID NO: 6. In some embodiments, the linker attaches a Flt3 ligand domain to the immunoglobulin Fc polypeptide or a fragment thereof. In some embodiments, the linker attaching the Flt3 ligand domain to the second ligand domain is at least 80% identical to SEQ ID NO: 4. In some embodiments, the linker attaches the Flt3 ligand domain to the second ligand domain. In some embodiments, the linker attaches a thrombopoietin domain to the second thrombopoietin domain. In some embodiments, the linker connects the TEV domain to the thrombopoietin domain or the second thrombopoietin domain. In some embodiments, the linker connects an amino acid sequence at least 80% identical to SEQ ID NO: 3 or 5 to the immunoglobulin Fc polypeptide or a fragment thereof. In some embodiments, the linker connects the Flt3 ligand domain to the immunoglobulin Fc polypeptide or a fragment thereof. In some embodiments, the linker comprises an amino acid sequence at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 4. In some embodiments, the linker comprises an amino acid sequence at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 6.In some embodiments, the linker comprises an amino acid sequence that is at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO:8.

[0023] In some embodiments, the polypeptide further comprises an affinity / epitope tag. In some embodiments, the affinity / epitope tag comprises a polyhistidine tag, also referred to herein as a His tag. In some embodiments, the His tag comprises a string of histidine residues. In some embodiments, the affinity / epitope tag is removed after production and purification of the polypeptide.

[0024] In some embodiments, the polypeptide improves survival of hematopoiesis-related cells in the treated cells relative to untreated cells, hi some embodiments, the polypeptide stimulates proliferation of hematopoiesis-related cells in the treated cells relative to untreated cells.

[0025] In some embodiments, the polypeptide activates the MAPK pathway. In some embodiments, the polypeptide increases ERK phosphorylation in treated cells compared to untreated cells. In some embodiments, the polypeptide increases Erk1 / 2 phosphorylation in treated cells compared to untreated cells.

[0026] In some embodiments, the polypeptide activates the PI3K / Akt pathway. In some embodiments, the polypeptide increases Akt phosphorylation in treated cells compared to untreated cells. In some embodiments, the polypeptide increases Akt phosphorylation in treated cells compared to untreated cells.

[0027] In some embodiments, one or more modifications of the Fc domain relative to a wild-type IgG Fc region affect the immunological properties of the Fc domain. In some embodiments, the immunological properties include antigen-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated phagocytosis (ADCP), or a combination thereof. In some embodiments, the immunological property includes ADCC. In some embodiments, the immunological property includes CDC. In some embodiments, the immunological property includes ADCP.

[0028] Treatment method Provided herein are methods for assisting cancer treatment in an individual, comprising administering a polypeptide provided herein to the individual. Provided herein are methods for treating thrombocytopenia, leukopenia, neutropenia, anemia, and other blood-related side effects associated with cancer treatment in an individual, comprising administering a composition provided herein to the individual. Such cancer treatments can affect the bone marrow, resulting in a decrease in the production of one or more blood-related cells, including, but not limited to, red blood cells, white blood cells, platelets, and neutrophils. In some cases, cancer treatments can result in a decrease in the production of one or more cells of the myeloid, lymphoid, or hematopoietic system. Provided herein are methods for increasing the production of one or more red blood cells, white blood cells, platelets, or neutrophils in an individual undergoing cancer treatment, comprising administering a composition provided herein to the individual. Provided herein are methods for increasing the production of one or more cells of the myeloid, lymphoid, or hematopoietic system in an individual undergoing cancer treatment, comprising administering a composition provided herein to the individual. Such cancer treatments may include, but are not limited to, chemotherapy, radiation therapy, immunotherapy, radiofrequency ablation, cryoablation, bone marrow transplantation, targeted drug therapy, and cell-based therapy.

[0029] Provided herein are methods for modulating an immune response in an individual, comprising administering a polypeptide provided herein to the individual. In some embodiments, modulating the immune response in an individual includes, but is not limited to, promoting the activation and infiltration of T cells, B cells, NK cells, dendritic cells, and other innate immune cells in tumors or infections. Provided herein are methods for modulating an immune response in an individual, comprising administering a composition provided herein to the individual. In some embodiments, the individual may be undergoing treatment, including, but not limited to, chemotherapy, radiation therapy, immunotherapy, radiofrequency ablation, cryoablation, bone marrow transplantation, and targeted drug therapy. In some embodiments, the treatment includes irreversible electroporation (IRE), microwave, low-intensity focused ultrasound (LOFU), high-intensity focused ultrasound (HIFU), radiofrequency energy, or cryotherapy, or a combination thereof. In some embodiments, the polypeptide is administered in combination with a treatment. In some embodiments, the polypeptide is administered before the treatment. In some embodiments, the polypeptide is administered after the treatment. In some embodiments, the polypeptide is administered within 1 hour, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks of treatment. In some embodiments, the polypeptide is administered 1 hour, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks before treatment. In some embodiments, the polypeptide is administered 1 hour, 6 hours, 12 hours, 1 day, 2 days, 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, or 4 weeks after treatment.

[0030] In some embodiments, the polypeptide stimulates an immune response against the tumor synergistically with the treatment. In some embodiments, administration of the polypeptide comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 doses. In some embodiments, the doses are spaced apart by at least 6 hours, several days, or even weeks.

[0031] Provided herein are methods for activating dendritic cells, the methods comprising administering to an individual a polypeptide provided herein. Provided herein are methods for increasing the proliferation and activation of dendritic cell precursors and mature cells, the methods comprising administering to an individual a polypeptide provided herein.

[0032] In some embodiments, administering a polypeptide to a subject stimulates platelet production in the subject. In some embodiments, administering a polypeptide to a subject stimulates platelet production in the subject compared to before administration. In some embodiments, administering a polypeptide to a subject stimulates platelet production in the subject compared to administration of a polypeptide comprising an FLT3L domain and an Fc domain that does not comprise a thrombopoietin domain. In some embodiments, administering a polypeptide to a subject stimulates platelet production in the subject in a manner similar to administration of a polypeptide comprising a thrombopoietin domain. In some embodiments, administering a polypeptide to a subject increases the platelet count in the subject. In some embodiments, administering a polypeptide to a subject increases the platelet count in the subject compared to before administration. In some embodiments, administering a polypeptide to a subject increases the platelet count in the subject compared to administration of a polypeptide comprising an FLT3L domain and an Fc domain that does not comprise a thrombopoietin domain. In some embodiments, administering a polypeptide to a subject increases the platelet count in the subject in a manner similar to administration of a polypeptide comprising a thrombopoietin domain.

[0033] In some embodiments, administering the polypeptide to a subject increases the number of dendritic cells in the subject's spleen. In some embodiments, administering the polypeptide to a subject increases the number of dendritic cells in the subject's blood. In some embodiments, administering the polypeptide increases dendritic cell maturation. In some embodiments, administering the polypeptide to a subject increases dendritic cell activation in the subject. In some embodiments, administering the polypeptide to a subject increases dendritic cell maturation in the subject. In some embodiments, administering the polypeptide to a subject increases dendritic cell proliferation in the subject.

[0034] In some embodiments, one or more modifications of the Fc domain relative to a wild-type IgG Fc region affect one or more pharmacokinetic and / or pharmacodynamic properties of the fusion polypeptide. In some embodiments, one or more modifications of the Fc domain relative to a wild-type IgG Fc region increase one or more pharmacokinetic and / or pharmacodynamic properties of the fusion polypeptide. In some embodiments, one or more modifications of the Fc domain relative to a wild-type IgG Fc region decrease one or more pharmacokinetic and / or pharmacodynamic properties of the fusion polypeptide. In some embodiments, one or more modifications of the Fc domain relative to a wild-type IgG Fc region maintain one or more pharmacokinetic and / or pharmacodynamic properties of the fusion polypeptide. In some embodiments, the pharmacokinetic property comprises bioavailability. In some embodiments, the pharmacodynamic property comprises platelet production. In some embodiments, the pharmacodynamic property comprises dendritic cell maturation, activation, generation, and / or proliferation.

[0035] In some embodiments, bioavailability is measured by plasma concentrations at various time points. In some embodiments, the various time points include one or more of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the various time points include one or more of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or 14 days. In some embodiments, the various time points include one or more of 1, 2, 3, or 4 weeks. In some embodiments, the various time points include 4 and 7 days. In some embodiments, the various time points include 14 days. In some embodiments, the various time points are immediately after administration. In some embodiments, the various time points include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24 hours after administration. In some embodiments, one or more modifications of the Fc domain fusion polypeptide result in increased bioavailability via intravenous injection compared to subcutaneous injection. In some embodiments, one or more modifications of the Fc domain fusion polypeptide result in increased bioavailability compared to thrombopoietin. In some embodiments, one or more modifications of the Fc domain fusion polypeptide result in bioavailability comparable to that of the FLT3L-Fc fusion polypeptide. In some embodiments, the change in bioavailability is measured 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after administration.

[0036] In some embodiments, the FLT3L plasma concentration is measured to determine one or more pharmacokinetic and / or pharmacodynamic properties. In some embodiments, the FLT3L plasma concentration is at least 100 pg / ml, 200 pg / ml, 300 pg / ml, 400 pg / ml, 500 pg / ml, 600 pg / ml, 700 pg / ml, 800 pg / ml, 900 pg / ml, 1000 pg / ml, 1500 pg / ml, 2000 pg / ml, 3000 pg / ml, 4000 pg / ml, 5000 pg / ml, or 6000 pg / ml. In some embodiments, the FLT3L plasma concentration is at most 100 pg / ml, 200 pg / ml, 300 pg / ml, 400 pg / ml, 500 pg / ml, 600 pg / ml, 700 pg / ml, 800 pg / ml, 900 pg / ml, 1000 pg / ml, 1500 pg / ml, 2000 pg / ml, 3000 pg / ml, 4000 pg / ml, 5000 pg / ml, or 6000 pg / ml. In some embodiments, the FLT3L plasma concentration is about 100 pg / ml, 200 pg / ml, 300 pg / ml, 400 pg / ml, 500 pg / ml, 600 pg / ml, 700 pg / ml, 800 pg / ml, 900 pg / ml, 1000 pg / ml, 1500 pg / ml, 2000 pg / ml, 3000 pg / ml, 4000 pg / ml, 5000 pg / ml, or 6000 pg / ml. In some embodiments, the FLT3L plasma concentration is measured on one or more of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 after administration. In some embodiments, the FLT3L plasma concentration on day 4 is at least 400 pg / ml. In some embodiments, the plasma concentration on day 4 is at least 2000 pg / ml. In some embodiments, the plasma concentration on day 4 is at least 6000 pg / ml. In some embodiments, the plasma concentration on day 4 is at most 400 pg / ml. In some embodiments, the plasma concentration on day 4 is at most 2000 pg / ml. In some embodiments, the plasma concentration on day 4 is at most 6000 pg / ml. In some embodiments, the plasma concentration on day 4 is about 400 pg / ml.In some embodiments, the plasma concentration on day 4 is about 2000 pg / ml. In some embodiments, the plasma concentration on day 4 is about 6000 pg / ml.

[0037] In some embodiments, when the thrombopoietin domain binds to a TPO receptor, the thrombopoietin domain can result in increased platelet production. In some embodiments, a fusion polypeptide comprising a thrombopoietin domain described herein results in an increase in platelet production or platelet count in a subject after administration. In some embodiments, platelet production is measured by platelet count. In some embodiments, the platelet count is collected by sampling the subject's blood at various time points. In some embodiments, the various time points include 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 days after administration, or a combination thereof. In some embodiments, the various time points are 4 and 6 days. In some embodiments, the various time points are 4 and 7 days. In some embodiments, one or more modifications of the Fc domain fusion polypeptide result in an increase in platelet count over a control. In some embodiments, one or more modifications of the Fc domain fusion polypeptide result in an increase in platelet count over FLT3L-Fc. In some embodiments, one or more modifications of the Fc domain fusion polypeptide result in an increase in platelet count comparable to romiplostim after 4 days.

[0038] In some embodiments, the platelet count is increased by at least 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 times more than the control. In some embodiments, the platelet count is obtained on one or more of days 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or 21 after administration. In some embodiments, the platelet count is increased by at least 1.5 times more than the control after 4 days. In some embodiments, the platelet count is increased by at least 2 times more than the control after 4 days. In some embodiments, the platelet count is increased by at least 150,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, or 2,000,000 per microliter more than the control. In some embodiments, the platelet count increases to at least 1.5 million per microliter after 4 days. In some embodiments, the platelet count increases to at least 2 million per microliter after 4 days. In some embodiments, the platelet count increases by at least 10%, 20%, 30%, 40%, 50%, 60%, 60A, 80%, 90%, or 100% over the control. In some embodiments, the platelet count increases by at least 50% over the control after 4 days. In some embodiments, the platelet count increases by at least 100% over the control after 4 days.

[0039] In some embodiments, the platelet count is increased by up to 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 times over the control. In some embodiments, the platelet count is increased by up to 1.5 times over the control after 4 days. In some embodiments, the platelet count is increased by up to 2 times over the control after 4 days. In some embodiments, the platelet count is increased by up to 150,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, or 2,000,000 per microliter over the control. In some embodiments, the platelet count is increased by up to 1,500,000 per microliter after 4 days. In some embodiments, the platelet count is increased by up to 2,000,000 per microliter after 4 days. In some embodiments, platelet counts are increased by up to 10%, 20%, 30%, 40%, 50%, 60%, 60A, 80%, 90%, or 100% over controls. In some embodiments, platelet counts are increased by up to 50% over controls after 4 days. In some embodiments, platelet counts are increased by up to 100% over controls after 4 days.

[0040] In some embodiments, the platelet count is increased by about 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, or 5 times over the control. In some embodiments, the platelet count is increased by about 1.5 times over the control after 4 days. In some embodiments, the platelet count is increased by about 2 times over the control after 4 days. In some embodiments, the platelet count is increased by about 150,000, 200,000, 300,000, 400,000, 500,000, 600,000, 700,000, 800,000, 900,000, 1,000,000, or 2,000,000 per microliter over the control. In some embodiments, the platelet count is increased to about 1,500,000 per microliter after 4 days. In some embodiments, the platelet count is increased to about 2,000,000 per microliter after 4 days. In some embodiments, the platelet count is increased by about 10%, 20%, 30%, 40%, 50%, 60%, 60A, 80%, 90%, or 100% over the control. In some embodiments, the platelet count is increased by about 50% over the control after 4 days. In some embodiments, the platelet count is increased by about 100% over the control after 4 days.

[0041] In some embodiments, dendritic cell production is measured by the percentage of dendritic cells (e.g., CD11c high and MHCII high) that are CD45+ cells. In some embodiments, the percentage of dendritic cells that are CD45+ cells is collected by sampling the patient's blood. In some embodiments, splenocytes are isolated from the sampling of the patient's blood. In some embodiments, uncoagulated blood is isolated from the sampling of the patient's blood. In some embodiments, splenic dendritic cells are measured from the isolated splenocytes. In some embodiments, blood dendritic cells are measured from the isolated uncoagulated blood. In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased over a control. In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased over thrombopoietin. In some embodiments, the percentage of blood dendritic cells that are CD45+ cells is increased over a control. In some embodiments, the percentage of blood dendritic cells that are CD45+ cells is increased over thrombopoietin. In some embodiments, the percentage of blood dendritic cells that are CD45+ cells is comparable to FLT3L-Fc.

[0042] CD45 is a lymphocyte common antigen, a receptor-binding protein tyrosine phosphatase expressed on leukocytes. In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold over a control. In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased by at least 8-fold over a control. In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased by at least 4-fold over a control. In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased by at least 2-fold over a control. In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 percent. In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased by at least 4 percent. In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased by at least 4 percent. In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased by at least 100% over a control. In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased by at least 300% over a control. In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased by at least 500% over a control. In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased by at least 700% over a control.

[0043] In some embodiments, the percentage of splenic dendritic cells of CD45+ cells is increased by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold over a control. In some embodiments, the percentage of splenic dendritic cells of CD45+ cells is increased by up to 8-fold over a control. In some embodiments, the percentage of splenic dendritic cells of CD45+ cells is increased by up to 4-fold over a control. In some embodiments, the percentage of splenic dendritic cells of CD45+ cells is increased by up to 2-fold over a control. In some embodiments, the percentage of splenic dendritic cells of CD45+ cells is increased by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 percent. In some embodiments, the percentage of splenic dendritic cells of CD45+ cells is increased by up to 8 percent. In some embodiments, the percentage of splenic dendritic cells of CD45+ cells is increased by up to 4 percent. In some embodiments, the percentage of splenic dendritic cells of CD45+ cells is increased by up to 100% over a control. In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased by up to 300% over a control. In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased by up to 500% over a control. In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased by up to 700% over a control.

[0044] In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10-fold over a control. In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased by about 8-fold over a control. In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased by about 4-fold over a control. In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased by about 2-fold over a control. In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased by about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 percent. In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased by about 8 percent. In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased by about 4 percent. In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased by about 100% over a control. In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased by about 300% over a control. In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased by about 500% over a control. In some embodiments, the percentage of splenic dendritic cells that are CD45+ cells is increased by about 700% over a control.

[0045] In some embodiments, the percentage of blood dendritic cells that are CD45+ cells is increased by at least 10-fold over a control. In some embodiments, the percentage of blood dendritic cells that are CD45+ cells is increased by at least 5-fold over a control. In some embodiments, the percentage of blood dendritic cells that are CD45+ cells is increased by at least 10 percent. In some embodiments, the percentage of blood dendritic cells that are CD45+ cells is increased by at least 5 percent. In some embodiments, the percentage of blood dendritic cells that are CD45+ cells is increased by at least 100% over a control. In some embodiments, the percentage of blood dendritic cells that are CD45+ cells is increased by at least 400% over a control. In some embodiments, the percentage of blood dendritic cells that are CD45+ cells is increased by at least 900% over a control.

[0046] In some embodiments, the percentage of blood dendritic cells that are CD45+ cells is increased by up to 10-fold over controls. In some embodiments, the percentage of blood dendritic cells that are CD45+ cells is increased by up to 5-fold over controls. In some embodiments, the percentage of blood dendritic cells that are CD45+ cells is increased by up to 10 percent. In some embodiments, the percentage of blood dendritic cells that are CD45+ cells is increased by up to 5%. In some embodiments, the percentage of blood dendritic cells that are CD45+ cells is increased by up to 100% over controls. In some embodiments, the percentage of blood dendritic cells that are CD45+ cells is increased by up to 400% over controls. In some embodiments, the percentage of blood dendritic cells that are CD45+ cells is increased by up to 900% over controls.

[0047] In some embodiments, the percentage of blood dendritic cells that are CD45+ cells is increased by about 10-fold over controls. In some embodiments, the percentage of blood dendritic cells that are CD45+ cells is increased by about 5-fold over controls. In some embodiments, the percentage of blood dendritic cells that are CD45+ cells is increased by about 10 percent. In some embodiments, the percentage of blood dendritic cells that are CD45+ cells is increased by about 5 percent. In some embodiments, the percentage of blood dendritic cells that are CD45+ cells is increased by about 100% over controls. In some embodiments, the percentage of blood dendritic cells that are CD45+ cells is increased by about 400% over controls. In some embodiments, the percentage of blood dendritic cells that are CD45+ cells is increased by about 900% over controls.

[0048] Provided herein are methods for increasing the viability, proliferation, and / or activation of hematopoiesis-related cells in an individual undergoing cancer treatment, comprising administering a composition provided herein to the individual. In some embodiments, the method improves hematopoietic cell survival in treated cells compared to untreated cells. In some embodiments, the method stimulates hematopoietic cell proliferation in treated cells compared to untreated cells. The cancer treatments described herein may result in a decrease in the viability and / or proliferation of hematopoietic-related cells. In some embodiments, the hematopoietic-related cells include hematopoietic stem cells. In some embodiments, the hematopoietic stem cells are CD34+. In some embodiments, the hematopoietic-related cells are CD34+. In some embodiments, the hematopoietic-related cells are capable of differentiating into one or more of erythrocytes, leukocytes, platelets, or neutrophils. In some embodiments, the hematopoietic-related cells are capable of differentiating into one or more of multipotent progenitor cells, common myeloid progenitor cells (CMPs), common lymphoid progenitor cells (CLPs), or mature hematopoietic cells.

[0049] In some embodiments, the method activates the MAPK pathway. In some embodiments, the method increases ERK phosphorylation in treated cells compared to untreated cells. In some embodiments, the method increases Erk1 / 2 phosphorylation in treated cells compared to untreated cells. In some embodiments, the method activates the PI3K / Akt pathway. In some embodiments, the method increases Akt phosphorylation in treated cells compared to untreated cells. In some embodiments, the method increases ERK / Akt phosphorylation in treated cells compared to untreated cells.

[0050] Provided herein are polypeptides comprising a thrombopoietin domain and an Flt3 ligand domain for use in methods of assisting in the treatment of cancer in an individual.

[0051] In some embodiments, polypeptides useful for the treatment of cancer or tumors are disclosed herein. Treatment refers to a method that seeks to improve or ameliorate the disease being treated. With respect to cancer, treatment includes, but is not limited to, reducing tumor volume, reducing tumor volume growth, increasing progression-free survival, or overall life expectancy. In some embodiments, treatment affects the remission of the cancer being treated. In some embodiments, treatment encompasses use as a prophylactic or maintenance dose intended to prevent the recurrence or progression of a previously treated cancer or tumor. Those skilled in the art will understand that not all individuals will respond equally or at all to an administered treatment, but that these individuals are nonetheless considered to be treated.

[0052] In some embodiments, the cancer or tumor is a solid cancer or tumor. In some embodiments, the cancer or tumor is a blood cancer or tumor. In some embodiments, the cancer or tumor includes tumors of the breast, heart, lung, small intestine, colon, spleen, kidney, bladder, head, neck, ovary, prostate, brain, pancreas, skin, bone, bone marrow, blood, thymus, uterus, testes, peritoneum, and liver. In some embodiments, tumors that can be treated with the polypeptides of the present disclosure include adenoma, adenocarcinoma, angiosarcoma, astrocytoma, epithelial carcinoma, germinoma, glioblastoma, glioma, hemangioendothelioma, angiosarcoma, hematoma, hepatocellular carcinoma, leukemia, lymphoma, medulloblastoma, melanoma, neuroblastoma, osteosarcoma, retinoblastoma, rhabdomyosarcoma, sarcoma, and / or teratoma. In certain embodiments, the tumor / cancer is selected from the group consisting of acral lentiginous melanoma, actinic keratosis, adenocarcinoma, adenoid cystic carcinoma, adenoma, adenosarcoma, adenosquamous carcinoma, astrocytic tumor, Bartholin's gland carcinoma, basal cell carcinoma, bronchial carcinoma, capillary carcinoma, carcinoma, carcinosarcoma, bile duct carcinoma, chondrosarcoma, cystadenoma, endodermal sinus tumor, endometrial hyperplasia, endometrial stromal sarcoma, endometrial adenocarcinoma, ependymal sarcoma, Ewing's sarcoma, focal nodular hyperplasia, gastric tumor, germ line tumor, glioblastoma, glucagonoma, hemangioblastoma, hemangioendothelioma, hemangioma, liver adenoma, liver adenomatosis, hepatocellular carcinoma, insulinoma, intraepithelial neoplasia, intraepithelial squamous neoplasia, invasive squamous cell carcinoma, large cell carcinoma, adipose tissue tumor, and leukocyte myeloma. The cancer is selected from the group consisting of sarcoma, lung cancer, lymphoblastic leukemia, lymphocytic leukemia, leiomyosarcoma, melanoma, malignant melanoma, malignant mesothelial tumor, nerve sheath tumor, medulloblastoma, medulloepithelioma, mesothelioma, mucoepidermoid carcinoma, myeloid leukemia, neuroblastoma, neuroepithelial adenocarcinoma, nodular melanoma, osteosarcoma, ovarian cancer, papillary serous adenocarcinoma, pituitary tumor, plasmacytoma, pseudosarcoma, prostate cancer, pulmonary blastoma, renal cell carcinoma, retinoblastoma, rhabdomyosarcoma, sarcoma, serous carcinoma, squamous cell carcinoma, small cell carcinoma, soft tissue carcinoma, somatostatin-secreting tumor, squamous cell carcinoma, squamous cell carcinoma, undifferentiated carcinoma, uveal melanoma, leukoplakia carcinoma, vaginal / vulvar carcinoma, VIP tumor, and Wilms' tumor.In some embodiments, tumors / cancers treated with one or more polypeptides of the present disclosure include brain cancer, head and neck cancer, colorectal cancer, acute myeloid leukemia, pre-B-cell acute lymphoblastic leukemia, bladder cancer, astrocytoma, preferably grade II, III, or IV astrocytoma, glioblastoma, glioblastoma multiforme, small cell carcinoma, and non-small cell carcinoma, preferably non-small cell lung cancer, lung adenocarcinoma, metastatic melanoma, androgen-independent metastatic prostate cancer, androgen-dependent metastatic prostate cancer, prostate cancer, and breast cancer, preferably ductal carcinoma, and / or breast cancer. In some embodiments, cancers treated with one or more polypeptides of the present disclosure include glioblastoma. In some embodiments, cancers treated with one or more polypeptides of the present disclosure include pancreatic cancer. In some embodiments, cancers treated with one or more polypeptides of the present disclosure include ovarian cancer. In some embodiments, cancers treated with one or more polypeptides of the present disclosure include lung cancer. In some embodiments, the cancer to be treated with one or more polypeptides of the present disclosure comprises prostate cancer. In some embodiments, the cancer to be treated with one or more polypeptides of the present disclosure comprises colon cancer. In some embodiments, the cancer to be treated comprises glioblastoma, pancreatic cancer, ovarian cancer, colon cancer, prostate cancer, or lung cancer. In some embodiments, the cancer is refractory to other treatments. In some embodiments, the cancer to be treated is recurrent.

[0053] In certain embodiments, the polypeptide can be administered to a subject in need thereof by any route suitable for administering a polypeptide-containing pharmaceutical composition, such as subcutaneous, intraperitoneal, intravenous, intramuscular, intratumoral, intracerebral, intraarterial, intrathecal, intravesical, intraocular, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, or intraarticular. In certain embodiments, the polypeptide is administered intravenously. In certain embodiments, the polypeptide is administered subcutaneously. In certain embodiments, the polypeptide is administered intratumorally. In certain embodiments, the polypeptide is administered according to a suitable dosing schedule, such as weekly, twice weekly, monthly, twice monthly, once every two weeks, once every three weeks, or once monthly. In certain embodiments, the polypeptide is administered once every three weeks. The polypeptide can be administered in any therapeutically effective amount. In certain embodiments, a therapeutically acceptable amount is from about 0.1 mg / kg to about 50 mg / kg. In certain embodiments, a therapeutically acceptable amount is from about 1 mg / kg to about 40 mg / kg. In some embodiments, a therapeutically acceptable amount is about 1 mg / kg to about 20 mg / kg. In some embodiments, a therapeutically acceptable amount is about 1 mg / kg to about 10 mg / kg. In some embodiments, a therapeutically acceptable amount is about 5 mg / kg to about 30 mg / kg. In some embodiments, a therapeutically acceptable amount is about 5 mg / kg to about 20 mg / kg. A therapeutically effective amount includes an amount sufficient to ameliorate one or more symptoms associated with the disease or affliction being treated.

[0054] composition Provided herein is a composition comprising the polypeptide provided herein and a pharmaceutically acceptable excipient.In some embodiments, the composition is formulated to be administered intravenously.In some embodiments, the composition is formulated to be administered intravenously, subcutaneously, or intratumorally.

[0055] In some embodiments, the polypeptide of the present disclosure is contained in a pharmaceutical composition comprising one or more pharmaceutically acceptable excipients, carriers, and diluents. Pharmaceutically acceptable excipients, carriers, and diluents can be included to increase the shelf life, stability, or administrability of the polypeptide. Such compounds include salts, pH buffers, surfactants, anticoagulants, and preservatives. In some embodiments, the polypeptide of the present disclosure is administered suspended in a sterile solution. In some embodiments, the solution contains about 0.9% NaCl. In some embodiments, the solution contains about 5.0% dextrose. In certain embodiments, the solution further comprises one or more of a buffering agent, e.g., acetate, citrate, histidine, succinate, phosphate, bicarbonate, and hydroxymethylaminomethane (Tris); a surfactant, e.g., polysorbate 80 (Tween 80), polysorbate 20 (Tween 20), and poloxamer 188; a polyol / disaccharide / polysaccharide, e.g., glucose, dextrose, mannose, mannitol, sorbitol, sucrose, trehalose, and dextran 40; an amino acid, e.g., glycine or arginine; an antioxidant, e.g., ascorbic acid, methionine; or a chelating agent, e.g., EDTA or EGTA.

[0056] In certain embodiments, polypeptides of the present disclosure can be shipped / stored, lyophilized, and reconstituted prior to administration. In certain embodiments, lyophilized polypeptide formulations include bulking agents such as mannitol, sorbitol, sucrose, trehalose, dextran 40, or combinations thereof. The lyophilized formulations can be contained in vials made of glass or other suitable non-reactive materials. When formulated, polypeptides, whether reconstituted or not, can be buffered at a specific pH, generally below 7.0. In certain embodiments, the pH can be 4.5-7.0, 4.5-6.5, 4.5-6.0, 4.5-5.5, 4.5-5.0, or 5.0-6.0.

[0057] Also described herein are kits comprising one or more of the polypeptides described herein in a suitable container and one or more additional components selected from instructions for use, diluents, excipients, carriers, and devices for administration.

[0058] In certain embodiments, described herein are methods for preparing a treatment for adverse effects of exposure to radiation exposure, such as hematopoietic failure, thrombocytopenia, and / or radiation syndrome, comprising mixing one or more pharmaceutically acceptable excipients, carriers, or diluents with a polypeptide of the present disclosure. In certain embodiments, described herein are methods for preparing a cancer treatment for storage or transportation, comprising lyophilizing one or more polypeptides of the present disclosure.

[0059] Polypeptide Expression and Production Provided herein is a nucleic acid encoding a polypeptide provided herein, comprising a thrombopoietin domain and an Flt3 ligand domain. Provided herein is a nucleic acid encoding a polypeptide provided herein, comprising a romiplostim domain and an Flt3 ligand domain.

[0060] Provided herein is an expression vector comprising a nucleic acid encoding a polypeptide comprising a thrombopoietin domain and an Flt3 ligand domain.Provided herein is a cell comprising a nucleic acid encoding a polypeptide comprising a thrombopoietin domain and an Flt3 ligand domain.Provided herein is a cell comprising a nucleic acid encoding a polypeptide comprising a romiplostim domain and an Flt3 ligand domain.

[0061] Provided herein are methods for producing a polypeptide comprising a thrombopoietin domain and an Flt3 ligand domain, comprising culturing cells under conditions sufficient to express the polypeptide.

[0062] In some embodiments, various polypeptide expression systems can be transfected with the expression vectors provided herein. In some embodiments, the expression system comprises a bacterial cell expression system, a yeast cell expression system, an insect cell expression system, or a mammalian cell expression system, or a combination thereof. In some embodiments, the mammalian cell expression system comprises HEK293 or Chinese hamster ovary (CHO) cells, or a combination thereof. In some embodiments, the insect cell expression system comprises SF9 or SF21, or a combination thereof. In some embodiments, the yeast cell expression system comprises Saccharomyces cerevisiae. In some embodiments, the bacterial cell expression system comprises Escherichia coli. In some embodiments, the cells of the expression system are cultured in a bioreactor.

[0063] In some embodiments, the polypeptide further comprises an affinity / epitope tag. In some embodiments, the affinity / epitope tag comprises a polyhistidine tag, also referred to herein as a His tag. In some embodiments, the His tag comprises a string of histidine residues. In some embodiments, the affinity / epitope tag is removed after production and purification of the polypeptide. In some embodiments, the polypeptide is secreted into the culture medium and purified from the culture medium. In some embodiments, the cells of the expression system are lysed to access the polypeptide, and the lysate is processed to isolate the polypeptide. In some embodiments, the processing of the lysate includes, but is not limited to, washing, solubilization, and affinity chromatography.

[0064] definition In the following description, certain specific details are set forth to provide a thorough understanding of various embodiments. However, those skilled in the art will understand that the provided embodiments may be practiced without these details. Unless the context requires otherwise, throughout the specification and the following claims, the word "comprise" and variations thereof, such as "comprises" and "comprising," should be construed in the inclusive sense, i.e., "including, but not limited to." As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. It should also be noted that the term "or" is generally used in its sense including "and / or" unless the content clearly dictates otherwise. Additionally, the headings provided herein are for convenience only and do not interpret the scope or meaning of the claimed embodiments.

[0065] As used herein, the term "about" refers to an amount closer to the stated amount by no more than 10%.

[0066] As used herein, the terms "individual," "patient," or "subject" refer to an individual diagnosed with, suspected of suffering from, or at risk of developing at least one disease that the described compositions and methods are useful for treating. In some embodiments, the individual is a mammal. In some embodiments, the mammal is a mouse, rat, rabbit, dog, cat, horse, cow, sheep, pig, goat, llama, alpaca, or yak. ​​In some embodiments, the individual is a human.

[0067] Fms-related tyrosine kinase 3 ligand is also referred to as Flt3 ligand, Flt3L, Flt3-ligand, Flt3-L, Flt3 ligand, Flt3L, Flt3-L, or FL. FL is a hematopoietic cytokine encoded by the FLT3LG gene in humans and can bind to the fms-like tyrosine kinase receptor FLT3 / Flk2. FL has a four-helix bundle and is structurally homologous to stem cell factor (SCF) and colony-stimulating factor 1 (CSF-1). Multiple isoforms of FL exist, including but not limited to transmembrane and membrane-bound isoforms. The transmembrane isoform is approximately 209 amino acids (aa). Mature human Flt3 ligand has a 158-aa extracellular domain (ECD) with a cytokine-like domain and juxtamembrane tether region, a 21-aa transmembrane segment, and a 30-aa cytoplasmic tail. Membrane-bound isoforms can be proteolytically cleaved to generate biologically active soluble isoforms. In some embodiments, the FL domain of a fusion polypeptide provided herein comprises an isolated, synthetic, or recombinant polypeptide encoding FL. In some embodiments, the FL domain of a fusion polypeptide provided herein comprises an FL polypeptide or a functional fragment thereof. In some embodiments, the function of the FL domain can be assessed by ELISA or an FL performance assay (e.g., a bead-based multiplex assay) (see, e.g., Graddis TJ, et al. J Biol Chem. 1998 Jul 10;273(28):17626-33).

[0068] Thrombopoietin is also referred to herein as megakaryocyte growth and development factor, MGDF, or TPO. In some embodiments, the thrombopoietin domain herein refers to thrombopoietin or a functional fragment thereof, or romiplostim or a functional fragment thereof. Thrombopoietin (TPO) is a glycoprotein encoded by the THPO gene in humans. TPO is produced by the liver and kidney and regulates platelet production by stimulating the production and differentiation of megakaryocytes. TPO can be a ligand for MLP / C_MPL, a product of the myeloproliferative leukemia viral oncogene. Plasma TPO levels can be inversely correlated with the mass of megakaryocytes and platelets, which degrade TPO after binding to specific membrane receptors. In some embodiments, the function of TPO or a TPO domain can be evaluated by a TPO performance assay, such as an ELISA or a bead-based multiplex assay.

[0069] A domain as used herein may refer to a functional analog, mimetic, or synthetic biosimilar compound.

[0070] As used herein, a molecule, peptide, polypeptide, antibody, or antibody fragment may be referred to as "bispecific" or "dual-specific" (including grammatical equivalents). A bispecific molecule has the ability to specifically bind to at least two structurally distinct targets. Specific binding may be the result of two different binding moieties that are structurally distinct at the molecular level (including, but not limited to, different, non-identical amino acid sequences), or may be the result of high affinity (e.g., about 1×10 -6A molecule, peptide, polypeptide, antibody, or antibody fragment referred to as "multi-specific" refers to a molecule having the ability to specifically bind to at least three structurally distinct targets. A "bispecific polypeptide" (including grammatical equivalents) refers to a bispecific molecule that preserves at least one fragment of a polypeptide capable of specifically binding to a target. A "multi-specific polypeptide" (including grammatical equivalents) refers to a multispecific molecule that preserves at least one fragment of a polypeptide that is capable of specifically binding to a target.

[0071] The term "linker" herein may also be referred to as a "linker sequence," "spacer," "tethering sequence," or grammatical equivalents. The "linkers" referred to herein connect two different molecules that themselves possess target binding or catalytic activity, or that are naturally expressed and assembled as separate polypeptides, or that comprise separate domains of the same polypeptide. Several strategies can be used to covalently link molecules. The linkers described herein can be utilized to connect an FL domain and an Fc domain, or can be used to tether a thrombopoietin domain and an Fc domain, or to the N- or C-termini of a polypeptide to generate bispecific or multispecific binding molecules. These include, but are not limited to, polypeptide bonds between the N- and C-termini of proteins or protein domains, disulfide bond-mediated linkages, chemical cross-linking reagent-mediated linkages, and enzymatic coupling-mediated linkages. In some cases, enzymatic coupling involves the use of sortase A to introduce coupling partners, such as, but not limited to, click handles (azides and alkynes). In some cases, the enzymatic coupling involves the use of formylglycine generating enzyme (FGE) combined with Hydrazino-iso-Pictet-Spengler (HIPS) chemistry. In one aspect of this embodiment, the linker is a peptide bond generated by recombinant technology or peptide synthesis. The linker peptide may contain primarily the following amino acid residues: Gly, Ser, Ala, or Thr. The linker peptide should be of an appropriate length to link the two molecules so that they assume the correct conformation relative to each other to retain the desired activity. In one embodiment, the linker is about 1-50 amino acids long or about 1-30 amino acids long. In one embodiment, a linker of 1-20 amino acids long can be used.Useful linkers include glycine-serine polymers, glycine-alanine polymers, alanine-serine polymers, and other flexible linkers, including, for example, (GS), (GSGGS), (GGGGS), and (GGGS), where n is an integer of at least 1. In some embodiments, linkers include rigid linkers, such as, but not limited to, (EAAAK), where n is an integer of at least 1. In some embodiments, linkers include chimeric linkers including, but not limited to, GGGGS and EAAAK motifs. Exemplary linkers can include AAEPKSS, AAEPKSS, AAEPKSSDKTHTCPPCP, GGGG, GGGGGG, HPRGSG, GGGGSGGGGSGGGGS, or GGGGDKTHTCPPCP. Alternatively, various non-proteinaceous polymers can be used as linkers, including, but not limited to, polyethylene glycol (PEG), polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. In some embodiments, a linker of appropriate length and flexibility can synergize activation of multiple receptors, which can be advantageous when cell numbers are very low, such as in situations after radiation exposure.

[0072] The terms "polypeptide" and "protein" are used interchangeably to refer to polymers of amino acid residues and are not limited to a minimum length. Polypeptides, including antibodies and antibody chains as well as other peptides, such as linkers and connecting peptides, can contain amino acid residues, including natural and / or unnatural amino acid residues. The term also includes post-expression modifications of the polypeptide, such as glycosylation, sialylation, acetylation, phosphorylation, and the like. In some aspects, polypeptides can contain modifications relative to the native or native sequence, so long as the protein maintains the desired activity. These modifications can be deliberate, such as by site-directed mutagenesis, or can be accidental, such as by mutation of the host producing the protein or by errors due to PCR amplification. In some embodiments, amino acid sequence variants of the polypeptides provided herein are contemplated. Variants typically differ from the polypeptides specifically disclosed herein in one or more substitutions, deletions, additions, and / or insertions. Such variants may be naturally occurring or may be synthetically produced, for example, by modifying one or more of the above polypeptide sequences of the present disclosure and evaluating one or more biological activities of the polypeptides described herein, and / or using any of several known techniques. For example, it may be desirable to improve the binding affinity and / or other biological properties of the polypeptide. Amino acid sequence variants of a polypeptide can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the polypeptide or by peptide synthesis. Such modifications include, for example, deletion and / or insertion and / or substitution of residues within the amino acid sequence of the polypeptide. Any combination of deletion, insertion, and substitution can be performed to arrive at the final construct, provided that the final construct has the desired characteristics, for example, target binding.

[0073] The percent (%) sequence identity to a reference polypeptide sequence is the percent of amino acid residues in a candidate sequence that are identical to the amino acid residues in the reference polypeptide sequence, after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity, without considering any conservative substitutions as part of the sequence identity. Alignment for determining percent amino acid sequence identity can be achieved in a variety of known ways, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Appropriate parameters for aligning sequences can be determined, including the algorithm required to achieve maximum alignment across the entire length of the sequences being compared. However, for the purposes herein, the percent amino acid sequence identity value is generated using the sequence comparison computer program ALIGN-2. The ALIGN-2 sequence comparison computer program was created by Genentech, Inc., and its source code has been submitted together with user documentation to the US Copyright Office, Washington, DC, 20559, and is registered under US Copyright Registration No. TXU510087. The ALIGN-2 program is publicly available from Genentech, Inc., South San Francisco, CA, or can be compiled from source code. The ALIGN-2 program should be compiled for use on UNIX operating systems, including Digital UNIX V4.0D. All sequence comparison parameters are set by the ALIGN-2 program and do not vary.

[0074] In situations where ALIGN-2 is used for amino acid sequence comparison, the % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B (alternatively, this may be referred to as a given amino acid sequence A having a particular % amino acid sequence identity with or containing a given amino acid sequence B) is calculated as follows: 100 times the fraction X / Y, where X is the number of amino acid residues scored as identical matches by the sequence alignment program ALIGN-2 in that program's alignment of A and B, and Y is the total number of amino acid residues in B. It should be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A. Unless otherwise specified, all % amino acid sequence identity values ​​used herein are obtained as described in the immediately preceding paragraph using the ALIGN-2 computer program.

[0075] Modifications (e.g., substitutions) can be made to improve polypeptide affinity. Such modifications can be made when encoding codons with high mutation rates during somatic maturation (see, e.g., Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and the resulting mutants can be tested for binding affinity. Affinity maturation (e.g., using error-prone PCR, chain shuffling, or oligonucleotide-directed mutagenesis) can be used to improve polypeptide affinity (see, e.g., Hoogenboom et al., Methods in Molecular Biology 178:1-37 (2001)). Alternatively, or in addition, the crystal structure of the target-receptor complex identifies contact points between the target and the receptor. Such contact residues and adjacent residues can be targeted or eliminated as candidates for substitution. Mutants can be screened to determine whether they contain desired properties.

[0076] Amino acid sequence insertions and deletions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions and deletions of single or multiple amino acid residues. An example of a terminal insertion includes a polypeptide with an N-terminal methionyl residue. Other insertional variants of the molecule include fusions to the N- or C-terminus of the polypeptide to enzymes (e.g., to ADEPT) or polypeptides that increase the serum half-life of the polypeptide. An example of an intrasequence insertional variant of a polypeptide molecule includes an insertion of three amino acids in the chain. An example of a terminal deletion includes a polypeptide with a deletion of seven or fewer amino acids at the end of the chain.

[0077] In some embodiments, fusion polypeptides are modified to increase or decrease their glycosylation (e.g., by altering the amino acid sequence to create or remove one or more glycosylation sites). Carbohydrates attached to the Fc region of the polypeptide can be modified. Naturally occurring polypeptides from mammalian cells typically have an N-linkage between Asn and Asn2 in the CH2 domain of the Fc region. 297 (See, e.g., Wright et al., TIBTECH 15:26-32 (1997)). The oligosaccharides can be various carbohydrates, such as mannose, N-acetylglucosamine (GlcNAc), galactose, sialic acid, or fucose attached to a GlcNAc in the stem of the biantennary oligosaccharide structure. Modifications of oligosaccharides in polypeptides can be performed to generate polypeptide variants with, for example, certain improved properties. Polypeptide glycosylation variants can have improved ADCC and / or CDC function. In some embodiments, variants are provided that have carbohydrate structures lacking fucose attached (directly or indirectly) to the Fc region. For example, the amount of fucose in such polypeptides can be 1% to 80%, 1% to 65%, 5% to 65%, or 20% to 40%. The amount of fucose can be determined by the ratio of Asn to ATP. 297 Asn compared to the sum of all sugar structures bound to 297The average amount of fucose in the glycan is determined by calculating the average amount of fucose in the glycan (see, for example, WO 08 / 077546). 297 refers to the asparagine residue located at about position 297 of the Fc region (EU numbering of Fc region residues; see, e.g., Edelman et al., Proc Natl Acad Sci USA. 1969 May;63(1):78-85). However, Asn 297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300, due to slight sequence variations in the polypeptide. Such fucosylation variants may have improved ADCC function (see, e.g., Okazaki et al., J. Mol. Biol. 336:1239-1249 (2004); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004)). Cell lines, e.g., knockout cell lines, and methods for their use can be used to produce defucosylated polypeptides, such as Lecl3 CHO cells and α-1,6-fucosyltransferase gene (FUT8) knockout CHO cells, which are deficient in protein fucosylation (see, e.g., Ripka et al., Arch. Biochem. Biophys. 249:533-545 (1986); Yamane-Ohnuki et al., Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng., 94(4):680-688 (2006)). Other glycosylation mutants are also included (see, e.g., U.S. Patent No. 6,602,684).

[0078] In some embodiments, the fusion polypeptides provided herein have an activity of about 1 μM, 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, or 0.01 nM or less (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 Dissociation constant (K DIn some embodiments, the fusion polypeptides provided herein have an activity of about 100 nM, 50 nM, 40 nM, 30 nM, 20 nM, 10 nM, 5 nM, 2 nM, 1 nM, 0.5 nM, 0.1 nM, 0.05 nM, 0.01 nM, or 0.001 nM or more (e.g., 10 -8 M or less, e.g. 10 -8 M~10 -13 M, e.g. 10 -9 M~10 -13 Dissociation constant (K D ) The target can be an Flt3 receptor target. KD can be measured by any suitable assay. In some embodiments, KD can be measured using a surface plasmon resonance assay (e.g., BIACORE®-2000, -3000, or Octet BIACORE®).

[0079] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of a polypeptide provided herein, thereby generating an Fc region variant. The Fc region herein refers to the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. Fc regions include native-sequence Fc regions and variant Fc regions. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions. The Fc region variant may comprise a mouse Fc region sequence containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0080] In some embodiments, one or more amino acid modifications may be introduced into the Fc region of a polypeptide provided herein, thereby generating an Fc region variant. The Fc region herein refers to the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the constant region. Fc regions include native-sequence Fc regions and variant Fc regions. The Fc region variant may comprise a human Fc region sequence (e.g., a human IgG1, IgG2, IgG3, or IgG4 Fc region) containing an amino acid modification (e.g., substitution) at one or more amino acid positions. The Fc region variant may comprise a mouse Fc region sequence containing an amino acid modification (e.g., substitution) at one or more amino acid positions.

[0081] In some examples, the Fc region of an immunoglobulin is important for many important antibody functions (e.g., effector functions), such as antigen-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), and antibody-dependent cell-mediated phagocytosis (ADCP), which result in target cell killing, albeit by different mechanisms. Thus, in some embodiments, the polypeptides described herein comprise an Fc region selected based on the biological activity of the antibody for its intended use. In a particular example, human IgG can be classified into, for example, four subclasses, IgG1, IgG2, IgG3, and IgG4, each of which comprises an Fc region with a unique profile for binding to one or more Fcγ receptors (activating receptors FcγRI (CD64), FcγRIIA, FcγRIIC (CD32); FcγRIIIA and FcγRIIIB (CD16) and inhibitory receptor FcγRIIB) and for the first component of complement (C1q). Human IgG1 and IgG3 bind to all Fcγ receptors; IgG2 binds to FcγRIIA. H131 binds to FcγRIIA R131 FcγRIIIA V158 IgG4 binds with lower affinity to FcγRI, FcγRIIA, FcγRIIB, FcγRIIC, and FcγRIIIA V158Furthermore, the inhibitory receptor FcγRIIB has lower affinity for IgG1, IgG2, and IgG3 than all other Fcγ receptors. Studies have shown that FcγRI does not bind to IgG2, and FcγRIIIB does not bind to IgG2 or IgG4. Ibid. In general, with regard to ADCC activity, human IgG1≧IgG3>>IgG4≧IgG2.

[0082] In some embodiments, polypeptides of the present disclosure are fused to or include an Fc region and have one or more variants with reduced effector function, making them desirable candidates for applications in which a particular effector function (e.g., complement fixation and ADCC) is unnecessary or deleterious. Such polypeptides may have reduced complement-dependent cytotoxicity (CDC), antibody-dependent cellular cytotoxicity (ADCC), or antibody-dependent cellular phagocytosis (ADCP). In some embodiments, polypeptides of the present disclosure include variants with increased effector function for applications in which increased immunogenicity is beneficial. Such polypeptides may have increased CDC, ADCC, or ADCP, or a combination thereof. Non-limiting examples of in vitro assays for assessing ADCC activity of a molecule of interest are described in U.S. Patent Nos. 5,500,362 and 5,821,337. Alternatively, non-radioactive assays may be used (e.g., ACTI™ and CytoTox 96® non-radioactive cytotoxicity assays). Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC), monocytes, macrophages, and natural killer (NK) cells.

[0083] Fc-containing fusion polypeptides can have increased half-life and improved binding to the neonatal Fc receptor (FcRn) (see, e.g., US 2005 / 0014934). Such polypeptides can include an Fc region with one or more substitutions that improve binding of the Fc region to FcRn, including substitutions at one or more of the following Fc region residues according to the EU numbering system: 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 (see, e.g., U.S. Patent No. 7,371,826). Other examples of Fc region variants are also contemplated (see, e.g., Duncan & Winter, Nature 322:738-40 (1988), U.S. Patent Nos. 5,648,260 and 5,624,821, and WO 94 / 29351).

[0084] In some embodiments, the polypeptides provided herein can be further modified to contain additional non-proteinaceous moieties that are known and available. Suitable moieties for derivatizing polypeptides include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (either homopolymers or random copolymers), and dextran or poly(n-vinylpyrrolidone), polyethylene glycol, polypropylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have manufacturing advantages due to its stability in water. Polymers may be of any molecular weight and may be branched or unbranched. The number of polymers attached to the polypeptide can vary, and if more than one polymer is attached, they can be the same or different molecules.

[0085] The fusion polypeptides described herein can be encoded by nucleic acids. A nucleic acid is a type of polynucleotide containing two or more nucleotide bases. In some embodiments, a nucleic acid is a component of a vector that can be used to introduce a polynucleotide encoding a polypeptide into a cell. As used herein, the term "vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it is linked. One type of vector is a genomic integrated vector or "integrated vector," which can be integrated into the chromosomal DNA of a host cell. Another type of vector is an "episomal" vector, e.g., a nucleic acid capable of extrachromosomal replication. A vector capable of inducing the expression of an operably linked gene is referred to herein as an "expression vector." Suitable vectors include plasmids, bacterial artificial chromosomes, yeast artificial chromosomes, viral vectors, and the like. In expression vectors, regulatory elements such as promoters, enhancers, and polyadenylation signals used to control transcription can be derived from mammalian, microbial, viral, or insect genes. The expression vector may further include the ability to replicate in a host (usually conferred by an origin of replication) and a selection gene to facilitate recognition of transformants. Vectors derived from viruses such as lentivirus, retrovirus, adenovirus, and adeno-associated virus may also be used. Plasmid vectors can be linearized for integration into the genomic region. In some embodiments, the expression vector is a plasmid. In some embodiments, the expression vector is a lentivirus, adenovirus, or adeno-associated virus. In some embodiments, the expression vector is an adeno-associated virus. In some embodiments, the expression vector is a lentivirus.

[0086] As used herein, the terms "homologous," "homology," or "percent homology," when used herein to describe an amino acid sequence or a nucleic acid sequence relative to a reference sequence, can be determined using the formula described by Karlin and Altschul (Proc. Natl. Acad. Sci. USA 87:2264-2268, 1990, as revised in Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993). Such formula is incorporated into the Basic Local Alignment Search Tool (BLAST) program of Altschul et al. (J. Mol. Biol. 215:403-410, 1990). Percent sequence homology can be determined using the latest version of BLAST as of the filing date of this application.

[0087] Nucleic acids encoding the fusion polypeptides described herein can be used to infect, transfect, transform, or otherwise transgenic appropriate cells, thereby enabling the production of fusion polypeptides for commercial or therapeutic use. Standard cell lines and methods for the production of Fc-containing polypeptides from large-scale cell culture are known in the art. See, for example, Li et al., "Cell culture processes for monoclonal antibody production." Mabs. 2010 Sep-Oct;2(5):466-477. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the eukaryotic cell is a mammalian cell. In some embodiments, the mammalian cell is a cell line useful for producing fusion polypeptides, such as a Chinese hamster ovary (CHO) cell, an NS0 mouse myeloma cell, or a PER.C6® cell. In some embodiments, the nucleic acid encoding the fusion polypeptide is integrated into a genomic locus of a cell useful for producing the fusion polypeptide. In certain embodiments, described herein are methods of making a fusion polypeptide, comprising culturing a cell comprising a nucleic acid encoding the fusion polypeptide under in vitro conditions sufficient to allow the production and secretion of the fusion polypeptide.

[0088] In some embodiments, described herein is a master cell bank comprising (a) a mammalian cell line comprising a nucleic acid encoding a fusion polypeptide described herein integrated at a genomic location, and (b) a cryoprotectant. In some embodiments, the cryoprotectant comprises glycerol or DMSO. In some embodiments, the master cell bank comprises (a) a CHO cell line comprising a nucleic acid encoding a fusion polypeptide provided herein, and (b) a cryoprotectant. In some embodiments, the cryoprotectant comprises glycerol or DMSO. In some embodiments, the master cell bank is contained in a suitable vial or container that can withstand freezing with liquid nitrogen.

[0089] Also described herein are methods for producing the fusion polypeptides described herein. Such methods include incubating cells or cell lines containing a nucleic acid encoding the fusion polypeptide in a cell culture medium under conditions sufficient to allow expression and secretion of the fusion polypeptide, and further recovering the polypeptide from the cell culture medium. Harvesting can further include one or more purification steps to remove viable cells, cell debris, non-antibody proteins or polypeptides, undesired salts, buffers, and medium components. In certain embodiments, additional purification steps include centrifugation, ultracentrifugation, Protein A, Protein G, Protein A / G, or Protein L purification, size exclusion chromatography, and / or ion exchange chromatography.

[0090] "Treat," "treatment," or "treating," as used herein, refers to the deliberate intervention in a physiological disease state that results in, for example, a reduction in the severity of the disease or condition, shortening the course of the condition, ameliorating or eliminating one or more symptoms associated with the disease or condition, or providing a beneficial effect to a subject with a disease or condition. Treatment does not require curing the underlying disease or condition.

[0091] A "therapeutically effective amount," "effective dose," "effective amount," or "therapeutically effective dosage" of a drug or therapeutic agent is any amount of drug that, when used alone or in combination with another therapeutic agent, protects a subject from developing a disease or promotes disease regression as evidenced by a decrease in the severity of disease symptoms, an increase in the frequency and duration of disease-free periods, or prevention of disability or impairment due to disease affliction. The ability of a therapeutic agent to promote disease regression can be evaluated using a variety of methods known to those skilled in the art, such as by assaying the activity of the agent in human subjects during clinical trials, in animal model systems predictive of efficacy in humans, or in in vitro assays.

[0092] As used herein, "pharmaceutically acceptable" with respect to a "carrier," "excipient," or "diluent" includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. In some embodiments, the carrier is suitable for intravenous, intramuscular, subcutaneous, parenteral, spinal, or epidermal administration (e.g., by injection or infusion). Depending on the route of administration, the active compound, i.e., polypeptide, may be coated in a material to protect the compound from the action of acids and other natural conditions that may inactivate the compound.

[0093] The pharmaceutical compounds described herein may contain one or more pharmaceutically acceptable salts. A "pharmaceutically acceptable salt" refers to a salt that retains the desired biological activity of the parent compound and does not impart any undesired toxicological effects (see, e.g., Berge, SM et al. (1977) J. Pharm. Sci. 66:1-19). Examples of such salts include acid addition salts and base addition salts. Acid addition salts include those derived from non-toxic inorganic acids such as hydrochloric acid, nitric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, and phosphorous acid, as well as non-toxic organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, aromatic acids, and aliphatic and aromatic sulfonic acids. Base addition salts include those derived from alkaline earth metals such as sodium, potassium, magnesium, and calcium, and non-toxic organic amines such as N,N'-dibenzylethylenediamine, N-methylglucamine, chloroprocaine, choline, diethanolamine, ethylenediamine, and procaine. [Example]

[0094] The following illustrative examples are representative of embodiments of the compositions and methods described herein and are not intended to be limiting in any way.

[0095] Example 1: Expression of fusion polypeptides The objective of this study was to generate and validate an Flt3 Ligand-thrombopoietin fusion polypeptide produced in an expression system. Transfections were performed according to standard protocols. Sequence-verified FLT3L-romiplostim Fc fusion midi DNA was transfected into the Expi293™ Expression System or the FreeStyle™ Expression System. For the Expi293™ Expression System, enhancers I and II were added to the transfected cultures on day 1. Cultures were harvested on day 6, and the supernatant was purified using His60 Ni Superflow™ resin. Ni +2The eluate from the resin was concentrated and dialyzed against buffer (1X PBS + 0.1 M arginine + 2% glycerol).

[0096] The endotoxin level in the polished and purified polypeptide was measured using a Kinetic-QCL™ LAL assay using buffer as the diluent. The endotoxin concentration was 0.5 EU / ml. The final concentration of the FLT3L-romiplostim Fc fusion polypeptide from the Expi293™ expression system was 0.8 mg / ml.

[0097] The dialyzed samples were run on a 4-12% Bis-Tris precast gel. Relevant lanes are as follows:

[0098] A: Unstained protein ladder (NEB; P7717)

[0099] B:FL-Romi Expi293(trademark)

[0100] D:FL-Romi Freestyle(trademark)

[0101] F:FL-Romi Expi293(trademark)

[0102] H:FL-Romi Freestyle(trademark)

[0103] Figure 2 shows stained 4-12% Bis-Tris gels showing the expression of a fusion polypeptide containing an FL domain and a thrombopoietin domain in lanes B, D, F, and H. Lanes B, D, F, and H show stained bands at 100-150 kDa (indicated by arrows), indicating the expression of a fusion polypeptide containing an FL domain and a thrombopoietin domain.

[0104] Dialyzed samples of purified protein were reduced and run on a 4-12% Bis-Tris precast SDS-PAGE gel. Figure 3 shows the purified FLT3-L-1-romiplostim protein on a 4-12% Bis-Tris precast SDS-PAGE gel, both non-reduced and reduced. The SDS-PAGE gel shows protein bands of approximately 100 kDa under non-reducing conditions and approximately 70 kDa under reducing conditions (indicated by arrows), corresponding to the predicted molecular weight of the FL-romiplostim fusion polypeptide under each condition. The first lane shows the unstained protein ladder.

[0105] Example 2: Effect of fusion polypeptides on apoptosis To study the effects of the fusion polypeptides provided herein, human myeloid leukemia cell lines were cultured with the fusion polypeptides and evaluated for rescue from apoptosis by flow cytometry.The OCI-AML5 cell line, a human myeloid leukemia cell line expressing the Flt3 receptor, was used in this study.The OCI-AML5 cell line was established from a patient with acute myeloid leukemia (AML) and is constitutively growth factor-dependent.In some cases, the fusion polypeptides provided herein containing the Flt3 ligand domain can stimulate cell proliferation and promote cell survival by inhibiting or reducing apoptosis through phosphorylation of the MAPK and PI3K / Akt pathways.

[0106] ERK and / or AKT polypeptides OCI-AML5 cells were seeded in serum-free medium without growth factors (500,000 cells / ml / well) and cultured for 16 hours. Cells were grown with the fusion polypeptide for 48 or 72 hours. The positive control was GM-CSF-treated cells, and the negative control was cells treated without cytokines. OCI-AML5 cells were treated with the fusion polypeptide, and the positive control cells were treated with GM-CSF for 48 or 72 hours before analysis. The negative control treatment was medium alone without cytokines. The level of apoptosis was measured using Annexin V flow cytometry.

[0107] Figure 4 shows flow cytometry results of FL-romiplostim Fc fusion polypeptide-treated OCI-AML5 cells. Figure 5 shows the percentage of apoptotic cells from the flow cytometry results. For cells treated with FL-romiplostim Fc fusion polypeptide from the Expi293 expression system, apoptosis was observed in approximately 60.9%, with approximately 38.7% viable cells. For cells treated with FL-romiplostim Fc fusion polypeptide from the Freestyle expression system, apoptosis was observed in approximately 66.9%, with approximately 32.7% viable cells. For cells treated with FL dimer Fc fusion polypeptide, apoptosis was observed in approximately 59.0%, with approximately 40.6% viable cells. For cells treated with FL monomer Fc fusion polypeptide, apoptosis was observed in approximately 53.8%, with approximately 45.8% viable cells. In cells treated with medium + CM-CSF (positive control), apoptosis was approximately 14.8% with approximately 85.0% viable cells. For negative control cells, apoptosis was approximately 90.2% with approximately 9.14% viable cells. In cells treated with human IgG1 Fc domain, approximately 90.8% of cells showed apoptosis and approximately 8.48% of cells were viable.

[0108] FL-romiplostim Fc fusion polypeptide treatment rescues OCI-AML5 cells from apoptosis.

[0109] Example 3: Effect of fusion polypeptides on the MAPK pathway To study the effect of the fusion polypeptide provided herein, human myeloid leukemia cell line was cultured with the fusion polypeptide, and its effect on the activation of MAPK pathway was evaluated.The OCI-AML5 cell line, which is a human myeloid leukemia cell line that expresses Flt3 receptor, was used in this study.In some cases, the fusion polypeptide provided herein that comprises Flt3 ligand domain can stimulate cell proliferation and promote cell survival by inhibiting or reducing apoptosis and phosphorylating ERK and / or AKT polypeptide.

[0110] OCI-AML5 cells were seeded in serum-free medium (1 million / ml / well) and cultured without serum for 16–24 hours. After serum starvation, activation of the MAPK kinase pathway by added cytokines was measured using flow cytometry. GM-CSF-treated cells served as the positive control, and cells treated without cytokines served as the negative control. OCI-AML5 cells were treated with the fusion polypeptide, and positive control cells were treated with GM-CSF for 3–5 minutes at 37°C before analysis. The negative control treatment consisted of medium alone without serum or cytokines. The level of Erk1 / 2 phosphorylation was measured by flow cytometry using a phospho-p44 / 42 MAPK (Erk1 / 2) (Thr202 / Tyr204) antibody.

[0111] Figure 6A shows the percentage of cells positive for Erk1 / 2 phosphorylation from the flow cytometry results. For cells treated with FL-romiplostim Fc fusion polypeptide from the Expi293 expression system, approximately 62% of the cells were positive for Erk1 / 2 phosphorylation. For cells treated with FL-romiplostim Fc fusion polypeptide from the Freestyle expression system, approximately 62% of the cells were positive for Erk1 / 2 phosphorylation. For cells treated with FL dimeric Fc fusion polypeptide, approximately 75% of the cells were positive for Erk1 / 2 phosphorylation. For cells treated with FL monomeric Fc fusion polypeptide, approximately 75% of the cells were positive for Erk1 / 2 phosphorylation. For cells treated with medium + GM-CSF (positive control), approximately 75% of the cells were positive for Erk1 / 2 phosphorylation. For negative control cells, approximately 20% of the cells were positive for Erk1 / 2 phosphorylation. In cells treated with the human IgG1 Fc domain, approximately 30% of the cells were positive for Erk1 / 2 phosphorylation. Figure 6B shows the mean fluorescence intensity of Erk1 / 2 phosphorylation from the flow cytometry results, which generally shows a similar trend to the percentage of positive cells in Figure 6A.

[0112] Figure 7A shows the percentage of cells positive for Erk1 / 2 expression using a p44 / 42 MAPK (Erk1 / 2) antibody from flow cytometry, providing a baseline for Erk1 / 2 expression of approximately 70-75% positive cells. Figure 7B shows the mean fluorescence intensity using a p44 / 42 MAPK (Erk1 / 2) antibody from flow cytometry, which generally follows a similar trend to the percent positive cells in Figure 7A.

[0113] FL-romiplostim fusion polypeptide treatment of OCI-AML5 appears to activate the MAPK pathway.

[0114] Example 4: Effect of fusion polypeptides on the PI3K / Akt pathway To study the effect of the fusion polypeptide provided herein, the human acute megakaryoblastic leukemia cell line M-07e was cultured with the fusion polypeptide and its effect on the activation of the PI3K / Akt pathway was evaluated. The M-07e cell line expresses cMPL / TPO receptor, and the protein expression of Flt3 receptor is very low in M-07e cells. Therefore, the M-07e cell line is ideal for testing the romiplostim function of the FL-romiplostim fusion polypeptide.

[0115] M-07e cells were seeded in serum-free medium (1 million / ml / well) and cultured without serum for 16 hours. After serum starvation, activation of the PI3K / Akt pathway by added cytokines was measured using flow cytometry. Cells treated with GM-CSF were used as a positive control, and cells treated without cytokines were used as a negative control. M-07e cells were treated with the fusion polypeptide, and positive control cells were treated with GM-CSF for 3–5 minutes at 37°C before analysis. Negative control cells were treated with medium without serum or cytokines. Akt phosphorylation was activated with various growth factors for 15 minutes. The level of PI3K / Akt phosphorylation was measured by flow cytometry using a phospho-Akt (Ser473) antibody.

[0116] Figure 8A shows the percentage of cells positive for PI3K / Akt phosphorylation from flow cytometry. For cells treated with FL-romiplostim Fc fusion polypeptide from the Expi293 expression system, approximately 18% of the cells were positive for Akt phosphorylation. For cells treated with FL-romiplostim Fc fusion polypeptide from the Freestyle expression system, approximately 10% of the cells were positive for Akt phosphorylation. For cells treated with romisplostim (Nplate), approximately 20% of the cells were positive for Akt phosphorylation. For cells treated with medium + CM-CSF (positive control), approximately 20% of the cells were positive for Akt phosphorylation. For control cells treated with thrombopoietin mimetic (TPOm) peptide, less than 5% of the cells were positive for Akt phosphorylation. For control cells treated with Flt3 ligand, less than 5% of the cells were positive for Akt phosphorylation. For cells treated with human IgG1 Fc domain, less than 3% of cells were positive for Akt phosphorylation. Figure 8B shows the mean fluorescence intensity of Akt phosphorylation from the flow cytometry results, which generally show a similar trend to the percent positive cells in Figure 8A. Figures 9A and B show the percentage and mean fluorescence intensity of cells positive for PI3K / Akt protein expression using the Akt(pan)(C67E7) antibody from the flow cytometry results, providing a baseline of Akt expression of approximately 80-90% of positive cells.

[0117] Treatment of M-07e cells with FL-romiplostim fusion polypeptide appears to activate the PI3K / Akt pathway.

[0118] Example 5: Effect of fusion polypeptides on the MAPK pathway To study the effect of the fusion polypeptide provided herein, the human acute megakaryoblastic leukemia cell line M-07e was cultured with the fusion polypeptide and its effect on the activation of the MAPK pathway was evaluated. The M-07e cell line expresses cMPL / TPO receptor, and the protein expression of Flt3 receptor is very low in M-07e cells. Therefore, the M-07e cell line is ideal for testing the romiplostim function of the FL-romiplostim fusion polypeptide.

[0119] M-07e cells were seeded in serum-free medium (1 million / ml / well) and cultured without serum or growth factors for 16–24 hours. MAPK pathway activation via added cytokines was measured in serum-starved M-07e cells using flow cytometry. Cells treated with GM-CSF served as the positive control, while cells treated without serum or cytokines served as the negative control. M-07e cells were treated with the fusion polypeptide, and positive control cells were treated with GM-CSF for 3–5 minutes at 37°C before analysis. Negative control treatment consisted of medium without serum or cytokines. ERK phosphorylation levels were measured by flow cytometry using a phospho-p44 / 42 MAPK (ERK1 / 2) (Thr202 / Tyr204) antibody.

[0120] Figures 10A and 10B show the percentage of cells positive for Erk1 / 2 phosphorylation and the mean fluorescence intensity after treatment with thrombopoietin mimetic (TPOm) peptide. The percentage of positive cells increased dose-dependently from less than 5% to approximately 20% from 25 ng / ml to 800 ng / ml TPOm, respectively. The mean fluorescence intensity generally showed a similar trend to the percentage of positive cells in Figure 10A, and increased in a TPOm dose-dependent manner. This indicates that TPOm can activate the MAPK pathway.

[0121] Figure 11A shows the percentage of cells positive for Erk1 / 2 phosphorylation after treatment with romiplostim. The percentage of positive cells is consistently about 60% at different concentrations of romiplostim ranging from 25 ng / ml to 800 ng / ml. This indicates that romiplostim can activate the MAPK pathway. Figure 11B shows the average fluorescence intensity for Erk1 / 2 phosphorylation after treatment with romiplostim, which generally follows the same trend as the percentage of positive cells in Figure 11A.

[0122] Figure 12A shows the percentage of cells positive for ERK phosphorylation from flow cytometry results. Cells treated with isotype, negative control, human IgG1 Fc, and FLT3L dimer Fc fusion had a percentage of cells positive for Erk1 / 2 phosphorylation close to zero. For cells treated with medium + GM-CSF (positive control), more than 75% of cells were positive for Erk1 / 2 phosphorylation. For cells treated with romiplostim, approximately 60% of cells were positive for Erk1 / 2 phosphorylation at 6.67 nM and 13.33 nM romiplostim. For cells treated with FL-romiplostim Fc fusion polypeptide from the Expi293 expression system, approximately 50% of cells were positive for Erk1 / 2 phosphorylation at 3.03 nM and approximately 55% were positive at 6.06 nM. For cells treated with FL-romiplostim Fc fusion polypeptide from the Freestyle expression system, approximately 30% of the cells were positive for Erk1 / 2 phosphorylation at 3.03 nM, and approximately 50% were positive at 6.06 nM. Figure 12B shows the mean fluorescence intensity of ERK phosphorylation from the flow cytometry results.

[0123] Treatment of M-07e cells with FL-romiplostim fusion polypeptide appears to activate the MAPK pathway in a dose-dependent manner.

[0124] Example 6: Effect of fusion polypeptide on M-07e cells To study the effects of the fusion polypeptides provided herein, the M-07e cell line was cultured with the fusion polypeptides and their effect on cell proliferation was evaluated. M-07e cells were seeded in serum-free medium (0.5 million / ml / well) and cultured without serum for 48 hours. After 48 hours of serum starvation, the cells were cultured with FLT3L-romiplostim Fc fusion polypeptide or romiplostim for 72 hours. After 72 hours of incubation, M-07e cell proliferation was measured using an XTT assay.

[0125] Figure 13 shows the results of an XTT assay of the M-07e cell line, which was cultured with a fusion polypeptide and evaluated for its effect on cell proliferation. Absorbance is plotted against the logarithmic scale of the FLT3L-romiplostim Fc fusion polypeptide or romiplostim concentration. Treatment with the FLT3L-romiplostim fusion polypeptide dose-dependently increased M-07e cell proliferation, with an EC50 value of 1.129 nM. Romiplostim treatment showed a similar trend, dose-dependently increasing M-07e cell proliferation, but with an EC50 value of 12.11 nM. Treatment of M-07e cells with the Flt3 Ligand-romiplostim fusion polypeptide resulted in an EC50 value that was more than 10-fold lower than that of romiplostim treatment alone. Fusion polypeptide treatment demonstrated improved cell proliferation compared to romiplostim alone.

[0126] Example 6: Binding of fusion polypeptides to the thrombopoietin receptor cMPL To study the binding ability of the fusion polypeptides provided herein to the thrombopoietin receptor, the fusion polypeptides were contacted with cells expressing the thrombopoietin receptor cMPL. Mouse or human thrombopoietin receptor cMPL was expressed on the surface of the Freestyle™ HEK293 cell line. Mouse or human thrombopoietin receptor cMPL was expressed as a fusion polypeptide with either GPF or mCherry. cMPL receptor expression was confirmed by measuring the fluorescent signal. PD1 protein, which binds to PDL1-expressing cells, was used as a general control. Human IgG1 Fc and secondary antibodies were used as negative controls because they are not expected to bind to cMPL. FLT3L-dimer Fc was also used as a negative control to demonstrate that fusion polypeptide binding was due to romiplostim. cMPL-expressing cells were treated with Nplate (romiplostim) as a positive control. The FLT3L-romiplostim fusion polypeptide with and without a His tag was tested. Binding was analyzed using a fluorescent secondary antibody against the IgG1 domain of the polypeptide. The percentage of cells that bound to the tested polypeptide was quantified.

[0127] Figure 14 shows the results of binding of the tested polypeptides to cMLP-expressing Freestyle™ HEK293 cells. The percentage of bound cells, as determined by secondary antibody fluorescent signal, was plotted for each experimental condition. FLT3L-romiplostim with or without a His tag showed binding comparable to that of mouse cMLP-expressing cells as the positive control, Nplate (romiplostim). The percentage of bound cells was greater than 40 percent for Nplate, FLT3L-romiplostim with a His tag, and FLT3L-romiplostim without a His tag. The best performing condition was FLT3L-romiplostim without a His tag, which had approximately 50% bound cells. FLT3L-romiplostim with or without a His tag showed binding comparable to that of human cMLP-expressing cells as the positive control, Nplate (romiplostim). The percentage of binding cells was greater than 30 percent for Nplate, His-tagged FLT3L-romiplostim, and non-His-tagged FLT3L-romiplostim. The best performing condition was non-His-tagged FLT3L-romiplostim, which had approximately 40 percent binding cells. Binding of PDL1 to PD1 served as a general control and validated the assay format. As expected, no binding was detected using the negative control, FLT3L-dimer Fc, human IgG1 Fc, or secondary alone. This fusion polypeptide appears to bind to both mouse and human cMLP.

[0128] Example 7: Expression of fusion polypeptide Fc mutants Transfection was performed according to standard protocols. Sequence-verified FLT3L-romiplostim Fc mutant fusion midi DNA was transfected into the Expi-CHO expression system. For the expression system, enhancers I and II were added to the transfected cultures on day 1. The cultures were harvested, and the supernatants were purified using a MabSelect Sure column. The fusion polypeptide was further purified by size-exclusion chromatography.

[0129] Dialyzed samples of the purified proteins were reduced and run on a 4-12% Bis-Tris precast SDS-PAGE gel. Figure 15 shows purified FLT3L-romiplostim Fc mutant polypeptides on a 4-12% Bis-Tris precast SDS-PAGE gel. The SDS-PAGE gel shows approximately 150 kDa protein bands for FLT3L-romiplostim LALA, FLT3L-romiplostim N297A, FLT3L-romiplostim N297Q, and FLT3L-romiplostim P329G, which correspond to the predicted molecular weights of the FT3L-romiplostim fusion polypeptides under each condition. The first lane shows the protein ladder.

[0130] Example 8: Pharmacokinetics and Pharmacodynamics (PK / PD) of Fusion Polypeptides To study the pharmacokinetic and pharmacodynamic parameters of the fusion polypeptide Fc mutants provided herein, mice were treated with the fusion polypeptide and evaluated for pharmacokinetics and pharmacodynamics. Mice were treated with subcutaneous or intravenous injections of 0.1 mg / kg or 1 mg / kg of the fusion polypeptide. The vehicle for injection was PBS, 0.1 M arginine-cl, and 2% glycerol. Mice were treated with FLT3L-romiplostim with Fc mutations, romiplostim, FLT3 ligand Fc fusions from BioXcell, or vehicle (negative control). Table 1 shows the experimental design of the study. Blood samples were collected to collect plasma and platelets. Plasma was measured for FLT3 ligand using the human FLT3 ligand / FLT3L DuoSet ELISA. Platelets were measured using a hemocytometer (Hemavet 950FS, Drew Scientific). Dendritic cells were isolated and analyzed using flow cytometry. Peripheral blood mononuclear cells and spleen cells were isolated and analyzed for dendritic cell proliferation using flow cytometry. Bone marrow was isolated and analyzed for megakaryocyte progenitor cell proliferation using flow cytometry.

[0131] [Table 1]

[0132] Figure 16 shows that the bioavailability of the four fusion polypeptide Fc mutants tested was higher than that of FLT3L-romiplostim without a His tag. Mice were subcutaneously injected at 0.1 mg / kg. Buffer alone and N-plate were included as negative controls. The negative control showed a baseline FLT3L plasma concentration of approximately 150 pg / mL on both days 4 and 6. FLT3L-romiplostim without a His tag had slightly elevated FLT3L plasma concentrations at approximately 175 pg / mL on day 4 and 150 pg / mL on day 6. The FLT3L-romiplostim Fc mutants had superior levels of FLT3L plasma concentrations compared to baseline at both time points. On day 4, all four mutants tested, N297A, LALA (L234A and L235A), P329G, and N297Q, had FLT3L plasma concentrations of approximately 400 pg / mL or higher. N297A, LALA, and P329G had FLT3L plasma concentrations of approximately 500 pg / mL. On day 6, all four mutants tested, N297A, LALA (L234A and L235A), P329G, and N297Q, had FLT3L plasma concentrations greater than 200 pg / mL. A FLT3L-Fc fusion from BioXcell was included as a comparative positive control. The FLT3L-Fc fusion had FLT3L plasma concentrations of approximately 600 pg / mL on day 4 and approximately 650 pg / mL on day 6. These data demonstrated improved bioavailability of the FLT3L-romiplostim Fc mutants compared to FLT3L-romiplostim without the His-tagged wild-type Fc.

[0133] Figure 17 shows that the bioavailability of the four tested fusion polypeptide Fc mutants was higher than that of the baseline and positive control FLT3L-Fc fusion. Mice were intravenously injected at 1 mg / kg. Buffer alone and N-plate were included as negative controls. The negative controls showed baseline levels of FLT3L plasma concentrations of approximately 0 pg / mL on both days 4 and 6. The FLT3L-romiplostim Fc mutant had superior levels of FLT3L plasma concentrations compared to baseline at both time points. On day 4, all four tested mutants, N297A, LALA (L234A and L235A), P329G, and N297Q, had FLT3L plasma concentrations of approximately 2000 pg / mL or higher. N297A, LALA, and P329G had FLT3L plasma concentrations of approximately 6000 pg / mL or higher. P329G had a FLT3L plasma concentration of approximately 8000 pg / mL. On day 6, all four mutants tested, N297A, LALA (L234A and L235A), P329G, and N297Q, had FLT3L plasma concentrations slightly above baseline. FLT3L-Fc fusion from BioXcell was included as a comparative positive control. The FLT3L-Fc fusion had FLT3L plasma concentrations of approximately 6000 pg / mL on day 4 and approximately 2000 pg / mL on day 6. These data demonstrate the improved bioavailability of the FLT3L-romiplostim Fc mutants compared to the negative control, which had similar plasma concentrations to the FLT3L-Fc fusion on day 4.

[0134] Platelet counts from blood collected via the retro-orbital route on day 4 and the cardiac route on day 7 were analyzed using a HEMA VET 950FS CBC device. The in vivo functionality of the fusion polypeptide Fc mutants is shown in Figure 18. All fusion polypeptides showed similar increases in platelet counts on days 4 and 7. Buffer and FLT3L-Fc were included as negative controls. The negative controls showed baseline platelet levels of approximately 1,000,000 / μL. The FLT3L-romiplostim Fc mutants had superior platelet levels compared to baseline at both time points. On day 4, all four mutants tested, N297A, LALA (L234A and L235A), P329G, and N297Q, had FLT3L platelet concentrations of approximately 1,500,000 / μL or greater. On day 6, all four mutants tested—N297A, LALA (L234A and L235A), P329G, and N297Q—had FLT3L plasma concentrations slightly above baseline, exceeding 1,000,000 platelets / μL. Despite lower bioavailability on day 7, FLT3L-romiplostim LALA exhibited greater pharmacodynamics than the other fusion polypeptides. Nplate (romiplostim) was used as a positive control. Nplate had platelet concentrations exceeding 2,000,000 platelets / μL from day 4 onward and 3,000,000 platelets / μL on day 7. Negative controls, buffer and FLT3L-Fc fusion from BioXcell, both lacking the romiplostim domain, demonstrated baseline platelet count levels. The positive control, Nplate, demonstrated rapid platelet expansion. These data demonstrated that the romiplostim domain of FLT3L-romiplostim functions as intended as evidenced by increased platelet concentrations on days 4 and 7.

[0135] Splenocytes were isolated and analyzed using flow cytometry. Uncoagulated blood was labeled with antibodies, RBCs were lysed using Cal-Lyse lysis solution, and then analyzed using flow cytometry. Total dendritic cells (DCs) (CD11C high and MHC II high) were plotted as a percentage of CD45+ cells. Figures 19A and 19B show the percentage of CD45+ DCs in both the spleen and blood. Buffer and Nplate (romiplostim) were included as negative controls. For splenic DCs, the negative controls showed baseline levels of CD45+ DC percentage of approximately 2-3%. The FLT3L-romiplostim Fc mutant significantly increased the percentage of splenic DCs compared to baseline. All four mutants tested, N297A, LALA (L234A and L235A), P329G, and N297Q, had splenic DC percentages of approximately 6 percent or higher than CD45+ cells. LALA (L234A and L235A) had the highest percentage of approximately 8 percent among the fusion polypeptide Fc mutants. Despite its lower bioavailability at day 7, FLT3L-romiplostim LALA exhibited greater pharmacodynamics than the other fusion polypeptides. The FLT3L-Fc fusion from BioXcell was used as a positive control. The FLT3L-Fc fusion had a splenic DC percentage of approximately 20 percent CD45+ cells. The negative controls, buffer and Nplate (romiplostim), both lacking the FLT3L domain, exhibited baseline splenic DC percentages of CD45+ cells. The positive control FLT3L-Fc fusion from BioXcell demonstrated an increased splenic DC percentage of CD45+ cells.

[0136] For blood DC cells, the negative control showed a baseline level of approximately 2-4% CD45+ DC percentage. The FLT3L-romiplostim Fc mutants had superior levels of CD45+ blood DC percentage compared to baseline. All four mutants tested, N297A, LALA (L234A and L235A), P329G, and N297Q, had splenic DC percentages of approximately 6% or higher CD45+ cells. N297A, LALA (L234A and L235A), and P329G had the highest percentage of approximately 8% among the fusion polypeptide Fc mutants. Despite low bioavailability at day 7, FLT3L-romiplostim LALA's pharmacodynamics were comparable to those of the other fusion polypeptides. The FLT3L-Fc fusion from BioXcell was used as a positive control. The FLT3L-Fc fusion had a blood DC percentage of approximately 14% of CD45+ cells. The negative controls, buffer and Nplate (romiplostim), both lacked the FLT3L domain and showed baseline blood DC percentages of CD45+ cells. The positive control FLT3L-Fc fusion from BioXcell showed an increase in blood DC percentages of CD45+ cells. These data demonstrated that the FLT3L domain of FLT3L-romplostim functions as intended, resulting in an increase from baseline of 2-4% to approximately 6-8%.

[0137] The FLT3L-romiplostim polypeptide Fc mutant showed improved pharmacokinetic and pharmacodynamic properties compared with FLT3L-romiplostim without the His-Tag polypeptide. The FLT3L-Fc fusion from BioXcell surpassed or was comparable to the FLT3L-romiplostim Fc mutant fusion polypeptide in FLT3L plasma concentrations on days 4 and 7 after DC cell generation in the spleen and blood, but was significantly inferior in mouse cMPL protein binding, human cMPL protein binding, and platelet production. Romiplostim surpassed or was comparable to the FLT3L-romiplostim Fc mutant fusion polypeptide in platelet production, but was significantly inferior in FLT3L plasma concentrations of DC cells from days 4 and 7 after spleen or blood generation. While individual parameters may not have increased compared with the positive control, overall parameters were improved in all stated categories.

[0138] Example 8: Study of the treatment of FLT3L-romiplostim fusion polypeptide in acute radiation syndrome (ARS) The purpose of this study was to determine the rescue effect of the FLT3L-romiplostim P329GFc mutant in acute radiation syndrome (ARS). It is hypothesized that FLT3L-romiplostim may protect against or mitigate ARS toxicity.

[0139] Experimental Details: Mice are injected subcutaneously at 1 mg / kg with the polypeptide 1 day before (protective group) or 1 day after (relaxation group). The vehicle for injection is PBS, 0.1 M arginine-cl, and 2% glycerol. A vehicle-only negative control is included. Mice are irradiated using a partial-body irradiation model. Unanesthetized C57BL / 6J mice (9-11 weeks old, Jackson Laboratories) are restrained in a 50 mL conical tube with the left lower limb exposed outside the tube. The tibia, fibula, ankle, and foot are shielded under lead to provide 2.5% bone marrow shielding, and then moved into the chamber of a CIX-3 orthovoltage irradiator (Xstrahl Inc., Suwanee, GA). The X-ray irradiator is operated at 300 kVp, 10 mA with Thoraeus [4 mm Cu half-value layer (HVL)] filtration, delivered at 1.12 Gy / min. Mice are exposed to 13 Gy of PBI.

[0140] Mice are monitored for survival for 30 days post-irradiation. Mice are weighed to monitor radiation toxicity and provide information on mortality kinetics, cause of death, and drug pharmacodynamics. At termination, 50% of the negative control group is expected to die. Mouse weight is also expected to decrease after 30 days in the negative control group. It is hypothesized that the protective or mitigating group will have a lower percentage of mice dying at 30 days than the negative control. Mouse weight is also not expected to decrease as much as the negative control group at 30 days.

[0141] While preferred embodiments of the present disclosure have been shown and described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will occur to those skilled in the art without departing from the present disclosure. It is understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the present disclosure.

[0142] All publications, patent applications, issued patents, and other documents mentioned herein are incorporated by reference herein as if each individual publication, patent application, issued patent, or other document was specifically and individually indicated to be incorporated by reference in its entirety. Definitions contained in texts incorporated by reference are excluded to the extent they conflict with definitions in this disclosure.

[0143] [Table 2]

Claims

1. A polypeptide comprising a thrombopoietin domain and an Flt3 ligand domain.

2. The polypeptide of claim 1 , wherein the thrombopoietin domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO:

9.

3. 2. The polypeptide of claim 1, wherein the Flt3 ligand domain comprises an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 3 or 5.

4. A polypeptide comprising an amino acid sequence that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO:

1.

5. The polypeptide of claim 1 , further comprising an immunoglobulin Fc polypeptide or a fragment thereof.

6. The polypeptide of claim 5 , wherein the immunoglobulin is immunoglobulin G1 (IgG1).

7. 7. The polypeptide of claim 1, further comprising an EPO leader sequence and / or a TEV cleavage domain.

8. The polypeptide of claim 1 , further comprising a linker.

9. 9. The polypeptide of claim 1, wherein the linker connects an amino acid that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 9 to the immunoglobulin Fc polypeptide or fragment thereof.

10. 10. The polypeptide of claim 1, wherein a linker connects the thrombopoietin domain to an immunoglobulin Fc polypeptide or a fragment thereof.

11. The polypeptide of claim 1 , wherein a linker connects the thrombopoietin domain to a second thrombopoietin domain.

12. 12. The polypeptide of claim 1, wherein a linker connects the TEV cleavage domain to the thrombopoietin domain or a second thrombopoietin domain.

13. 13. The polypeptide of any one of claims 1 to 12, wherein the linker attaches an amino acid that is at least 80%, 85%, 90%, 95%, or 100% identical to SEQ ID NO: 3 or 5 to the immunoglobulin Fc polypeptide or fragment thereof.

14. 14. The polypeptide of any one of claims 1 to 13, wherein a linker connects the Flt3 ligand domain to an immunoglobulin Fc polypeptide or a fragment thereof.

15. 15. The polypeptide of claim 1, wherein the Flt3 ligand domain is Flt3 ligand isoform 1.

16. 16. The polypeptide of claim 1, wherein the Flt3 ligand domain is human Flt3 ligand isoform 1.

17. 17. A polypeptide according to any one of claims 1 to 16, whose amino acid sequence is identical to SEQ ID NO:

1.

18. 18. The polypeptide of any one of claims 4 to 17, wherein the immunoglobulin Fc polypeptide or fragment thereof comprises one or more modifications compared to a wild-type IgG Fc region identified in SEQ ID NO: 7, wherein the one or more modifications affect the immunological properties of the immunoglobulin Fc polypeptide or fragment thereof.

19. 19. The polypeptide of claim 18, wherein the one or more modifications include L234A, L235A, N297A, N297Q, P329Q, or a combination thereof according to EU numbering.

20. 20. The polypeptide of claim 19, wherein the one or more modifications include L234A and L235A according to EU numbering.

21. 20. The polypeptide of claim 19, wherein the one or more modifications include N297A according to EU numbering.

22. 20. The polypeptide of claim 19, wherein the one or more modifications include N297Q according to EU numbering.

23. 20. The polypeptide of claim 19, wherein the one or more modifications include P329Q according to EU numbering.

24. 24. The polypeptide of any one of claims 18 to 23, wherein the immunological properties include antigen-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated phagocytosis (ADCP), or a combination thereof.

25. 25. The polypeptide of any one of claims 1 to 24, wherein administration of the polypeptide to a subject increases the number of platelets in the subject.

26. 25. The polypeptide of any one of claims 1 to 24, wherein administration of the polypeptide to a subject increases the number of dendritic cells in the spleen of the subject.

27. 25. The polypeptide of any one of claims 1 to 24, wherein administration of the polypeptide to a subject increases the number of dendritic cells in the blood of the subject.

28. 28. A polypeptide according to any one of claims 1 to 27, which improves the survival of cells associated with hematopoiesis in treated cells compared to untreated cells.

29. 29. A polypeptide according to any one of claims 1 to 28, which stimulates proliferation of cells associated with hematopoiesis in treated cells compared to untreated cells.

30. 30. A polypeptide according to any one of claims 1 to 29, which activates the MAPK pathway.

31. 31. A polypeptide according to any one of claims 1 to 30, which increases ERK phosphorylation in treated cells compared to untreated cells.

32. 32. A polypeptide according to any one of claims 1 to 31, which increases Erk1 / 2 phosphorylation in treated cells compared to untreated cells.

33. 33. A polypeptide according to any one of claims 1 to 32, which activates the PI3K / Akt pathway.

34. 34. A polypeptide according to any one of claims 1 to 33, which increases Akt phosphorylation in treated cells compared to untreated cells.

35. 35. A polypeptide according to any one of claims 1 to 34, which increases ERK / Akt phosphorylation in treated cells compared to untreated cells.

36. 36. The polypeptide of any one of claims 1 to 35, configured to bind to fms-like tyrosine kinase 3 (FLT3).

37. 37. A composition comprising a polypeptide according to any one of claims 1 to 36 and a pharmaceutically acceptable excipient.

38. 38. The composition of claim 37, formulated for administration either intravenously, subcutaneously or intratumorally.

39. 37. A method of assisting in the treatment of cancer in an individual, comprising administering to said individual a polypeptide described in any one of claims 1 to 36.

40. 40. A method of assisting in the treatment of cancer in an individual, comprising administering to said individual a composition according to claim 37 or 38.

41. 37. A method of modulating an immune response in an individual, comprising administering to said individual a polypeptide described in any one of claims 1 to 36.

42. 42. A method for stimulating the proliferation and activation of dendritic cells, comprising administering to an individual a polypeptide according to any one of claims 1 to 41.

43. 43. The method of any one of claims 39 to 42, wherein the survival of cells associated with hematopoiesis is improved in treated cells compared to untreated cells.

44. 44. The method of any one of claims 39 to 43, wherein proliferation of cells associated with hematopoiesis is stimulated in treated cells compared to untreated cells.

45. 45. The method of any one of claims 39 to 44, wherein the MAPK pathway is activated.

46. 46. ​​The method of any one of claims 39 to 45, wherein ERK phosphorylation in treated cells is increased compared to untreated cells.

47. 47. The method of any one of claims 39 to 46, wherein Erkl / 2 phosphorylation in treated cells is increased compared to untreated cells.

48. 48. The method of any one of claims 39 to 47, wherein the PI3K / Akt pathway is activated.

49. 49. The method of any one of claims 39 to 48, wherein Akt phosphorylation is increased in treated cells compared to untreated cells.

50. 50. The method of any one of claims 39 to 49, wherein ERK / Akt phosphorylation in treated cells is increased compared to untreated cells.

51. 37. A nucleic acid encoding a polypeptide according to any one of claims 1 to 36.

52. 52. An expression vector comprising the nucleic acid of claim 51.

53. 37. A cell comprising a nucleic acid encoding a polypeptide according to any one of claims 1 to 36.

54. 37. A method for producing a polypeptide according to any one of claims 1 to 36, comprising culturing cells under conditions sufficient to express said polypeptide.

55. 37. A polypeptide according to any one of claims 1 to 36 for use in a method of assisting in the treatment of cancer in an individual.

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