EPO receptor agonists and antagonists

Abstract

The present disclosure provides EPO analogs, anti-EPOR antibodies, anti-EPO antibodies, and fragments thereof that specifically bind to hetero-EPOR or homo-EPOR or EPO with high affinity. Engineered EPOs are also provided herein. EPO analogs, anti-EPOR antibodies, anti-EPO antibodies, and / or engineered EPOs can be used to treat patients. There are two main tyrosine kinase receptors for EPO: homodimeric EPOR / EPOR ("homo-EPOR") and heterodimeric EPOR / CD131 receptor ("hetero-EPOR").

Description

[Technical field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 317,943, filed March 8, 2022, which is incorporated by reference herein in its entirety. [Background technology]

[0002] Background of the disclosure Erythropoietin (EPO) induces hematopoiesis by dimerizing the EPO receptor (EPOR) molecule, which leads to the activation of EPO receptor-associated Janus tyrosine kinase 2 (Jak2) and secondary signaling molecules, such as signal transducer and activator of transcription 5 (Stat5; Brines and Cerami, Nat Rev Neurosci, 2005; 6:484-94). EPO acts by binding to EPOR, which is expressed in erythroid progenitor cells and inhibits apoptosis and promotes cell survival, proliferation, and differentiation in the production of mature erythrocytes (Figure 1). However, EPOR expression is not limited to erythroid tissues. EPOR is expressed in several non-hematopoietic tissues and induces tissue protective effects in ischemic injury, promotes wound healing, cardiovascular protection, angiogenesis, neuroprotection, regulation of metabolic homeostasis, and bone remodeling. [Prior art documents] [Non-patent literature]

[0003] [Non-Patent Document 1] Brines and Cerami, Nat Rev Neurosci (2005)6:484~94 Summary of the Invention [Means for solving the problem]

[0004] Disclosure Summary There are two main tyrosine kinase receptors for EPO: homodimeric EPOR / EPOR ("homo-EPOR") and heterodimeric EPOR / CD131 receptor ("hetero-EPOR"). Homo-EPOR signaling is important for erythropoiesis, while hetero-EPOR signaling is known to have tissue protective activity and may be involved in EPO-mediated immunomodulatory functions in immune cells (e.g., myeloid cells, T cells and B cells). Modulation of EPO signaling by hetero-EPOR may provide benefits in various pathological conditions, including, but not limited to, inhibiting or stimulating immune responses, inducing or breaking antigen-specific tolerance, stimulating erythropoiesis without tolerogenic or immunosuppressive effects, providing neuroprotection and tissue protection without stimulating erythropoiesis, and inducing preventative or therapeutic immunity. The present disclosure relates to new erythropoietin (EPO) analogs and new EPO-related antibodies. The EPO analogs disclosed herein may include, for example, eight types. An EPO analog can bind to hetero-EPOR and cannot bind to homo-EPOR, and may be either an agonist or antagonist of hetero-EPOR. Another EPO analog can bind to homo-EPOR and cannot bind to hetero-EPOR, and may be either an agonist or antagonist of homo-EPOR. An EPO analog can bind to both homo-EPOR and hetero-EPOR, and may be an agonist for both, an antagonist for both, or an agonist for one and an antagonist for the other. At least four types of anti-EPO receptor (anti-EPOR) antibodies can be obtained. An anti-EPOR antibody can be an agonist or antagonist of hetero-EPOR, and an anti-homo-EPOR antibody can be an agonist or antagonist of homo-EPOR. At least two anti-CD131 antibodies can be obtained. The anti-CD131 antibodies can be agonists or antagonists of hetero-EPOR. At least three anti-EPO antibodies can be obtained. The anti-EPO antibodies can inhibit EPO binding to homo-EPOR, inhibit EPO binding to hetero-EPOR, or inhibit EPO binding to both homo-EPOR and hetero-EPOR.

[0005] The antibodies disclosed herein may include fragments thereof that specifically bind to homo-EPOR, hetero-EPOR, EPO, CD131, or a combination thereof with high binding affinity (collectively, hetero-EPOR and homo-EPOR are referred to as "EPOR"). The antibodies may be monoclonal and may be human, chimeric, or humanized. Chimeric anti-EPOR and / or anti-EPO antibodies (including fragments thereof) may have non-human (e.g., mouse) complementarity determining regions (CDRs) and / or non-human framework regions, and optionally, one or more human constant domains. Humanized anti-EPOR and / or humanized anti-EPO antibodies (including fragments thereof) may have non-human (e.g., mouse) CDRs and / or human framework regions, and optionally, non-human framework amino acid residues adjacent to the CDRs, and optionally, one or more human constant domains. In some embodiments, the antibodies disclosed herein may be grafted antibodies.

[0006] The humanized antibody disclosed herein may represent an anti-EPOR and / or anti-EPO antibody obtained by grafting CDRs into a human framework for the heavy chain and / or a human framework for the light chain with a selected number of framework residues from a mouse antibody. The anti-EPOR and / or anti-EPO antibodies disclosed herein also include those obtained from an affinity maturation library made from an anti-EPOR or anti-EPO antibody. The anti-EPOR and / or anti-EPO antibody may also comprise a heavy chain variable region having 99%, 95%, 90%, 80% or 70% sequence identity with one of the heavy chains, and a light chain variable region having 99%, 95%, 90%, 80% or 70% sequence identity with one of the light chains. The anti-EPOR antibody can bind to homo-EPOR or hetero-EPOR with an affinity of about 0.1 pM to about 300 nM, about 1.0 nM to about 10.0 nM, about 50 nM to about 100 nM, or about 1.0 to about 100 nM. The anti-EPOR antibody can bind to homo-EPOR or hetero-EPOR with an affinity of at least about 100 nM, at least about 50 nM, at least about 10 nM, at least about 5 nM, or at least about 1.0 nM. The anti-EPO antibody can bind to EPO with an affinity of about 1.0 nM to about 10 nM, about 50 nM to about 100 nM, or about 1.0 to about 100 nM. The anti-EPO antibody can bind to EPO with an affinity of at least about 100 nM, at least about 50 nM, at least about 10 nM, at least about 5.0 nM, or at least about 1.0 nM.

[0007] The anti-EPOR and / or anti-EPO antibodies described herein may include modifications that provide the antibody with desired properties. For example, modifications can increase the serum half-life of the antibody, or modifications can decrease the serum half-life. Modifications can also increase or decrease the effector function of the antibody. Modifications can decrease immunogenicity or reduce other unwanted side effects or adverse events caused by the antibody.

[0008] EPO analogs that are antagonists to hetero-EPOR, anti-hetero-EPOR antibodies that are antagonists to hetero-EPOR, anti-CD131 antibodies that are antagonists to hetero-EPOR, and / or anti-EPO antibodies that inhibit EPO binding to hetero-EPOR can be used to overcome immunosuppressive or tolerogenic conditions in subjects.For example, these EPO analogs, anti-hetero-EPOR antibodies, anti-CD131 antibodies, and / or anti-EPO antibodies can be used to overcome tumor immunosuppressive microenvironment, boost immune response to vaccines, and / or enhance immune response during acute inflammatory response to disease (e.g., infectious disease from microorganisms or viruses).

[0009] EPO analogs that are agonists for hetero-EPOR, anti-CD131 antibodies that are agonists for hetero-EPOR, and / or anti-EPOR antibodies that are agonists for hetero-EPOR can be used to induce negative immune modulation (e.g., immunosuppression or tolerogenic state) in subjects.For example, these EPO analogs, anti-CD131 antibodies that are agonists for hetero-EPOR, and / or anti-hetero-EPOR antibodies can be used to suppress transplant rejection, induce antigen-specific immune tolerance, reduce immune responses in autoimmune diseases, reduce systemic chronic inflammation, and reduce damage to nervous tissue and other tissues during injury or other stress.These EPO analogs, anti-CD131 antibodies that are agonists for hetero-EPOR, and / or anti-hetero-EPOR antibodies can also be administered with an antigen to induce an immune tolerogenic state for the antigen.

[0010] EPO analogues that are agonists for homo-EPOR and do not bind to hetero-EPOR, and are not antagonists for hetero-EPOR, and / or anti-EPO antibodies that inhibit EPO binding to hetero-EPOR, and / or anti-CD131 antibodies that inhibit EPO binding to hetero-EPOR, and / or anti-hetero-EPOR antibodies that are antagonists for hetero-EPOR, can be used with or without erythropoietin stimulating agents (ESAs) for cancer patients who need ESA treatment.Any cancer patients who need ESA can be provided with ESA in combination with these EPO analogues, and / or anti-EPOR antibodies, and / or anti-EPO antibodies.

[0011] Modulation of signal transduction from homo-EPOR or hetero-EPOR can be performed using RNA or small molecules. Stimulation of signal transduction from homo-EPOR or hetero-EPOR can be achieved by delivery of mRNA of positive regulator, siRNA of negative regulator, small molecule that upregulates positive regulator, or small molecule that downregulates negative regulator. Inhibition of signal transduction from homo-EPOR or hetero-EPOR can be achieved by delivery of mRNA of negative regulator, siRNA of positive regulator, small molecule that upregulates negative regulator, or small molecule that downregulates positive regulator.

[0012] In some aspects, provided herein is a composition comprising an antibody or functional fragment thereof, wherein (i) the antibody or functional fragment thereof selectively binds to a target comprising erythropoietin (EPO) protein, an EPO receptor subunit, a CD131 subunit, or a combination thereof; (ii) binding of the antibody or functional fragment thereof to the target (a) prevents formation of an EPO protein-hetero-EPO receptor complex, wherein the hetero-EPO receptor comprises the EPO receptor subunit and the CD131 subunit, (b) prevents formation of a hetero-EPO receptor between the EPO receptor subunit and the CD131 subunit, or (c) prevents activation of a hetero-EPO receptor comprising the EPO receptor subunit and the CD131 subunit; and (iii) the antibody or functional fragment thereof comprises an antigen-binding domain.

[0013] In some aspects, provided herein is a method for treating cancer, comprising administering a composition or a derivative thereof to a subject having or at risk of having cancer, wherein the composition or derivative thereof inhibits hetero-erythropoietin (EPO) receptor activity in the subject.

[0014] In some aspects, provided herein is a composition comprising an antibody or functional fragment thereof, wherein (i) the antibody or functional fragment thereof selectively binds to a target comprising erythropoietin (EPO) protein, an EPO receptor subunit, a CD131 subunit, or a combination thereof; (ii) binding of the antibody or functional fragment thereof to the target (a) promotes formation of an EPO protein-hetero-EPO receptor complex, wherein the hetero-EPO receptor comprises the EPO receptor subunit and the CD131 subunit, (b) promotes formation of a hetero-EPO receptor between the EPO receptor subunit and the CD131 subunit, or (c) promotes activation of a hetero-EPO receptor comprising the EPO receptor subunit and the CD131 subunit; and (iii) the antibody or functional fragment thereof comprises an antigen-binding domain.

[0015] In some aspects, provided herein are compositions for administration to a subject having cancer or a chronic infectious disease condition, wherein the composition or a derivative thereof inhibits erythropoietin (EPO) receptor activity in myeloid cells of the subject.

[0016] In some aspects, provided herein are compositions comprising an engineered erythropoietin (EPO) protein, wherein the engineered EPO protein inhibits hetero-erythropoietin (EPO) receptor activity in myeloid cells. In some embodiments, the engineered EPO protein comprises at least one amino acid substitution including K20A, N24Q, N24A, N24S, N38Q, N38A, N38S, K45A, K52A, Q58A, E62R, E62A, Q65A, L69A, E72A, R76E, R76A, L80A, N83Q, N83A, N83S, S84A, S85A, K97A, K116A, G151A, R103A, K45D, N147K, R150E, Q65A, E72R, N83A, K140A, K152A, or K154A.

[0017] In some aspects, a composition comprising an engineered erythropoietin (EPO) protein, wherein the engineered EPO protein is selected from the group consisting of K20A, N24Q, N24A, N24S, N38Q, N38A, N38S, K45A, K52A, Q58A, E62R, E62A, Q65A, L69A, E72A, R76E, R76A, L80A, N83Q, N83A, N83S, S84A, Provided herein are compositions comprising at least one amino acid substitution including S85A, K97A, K116A, G151A, R103A, K45D, N147K, R150E, Q65A, E72R, N83A, K140A, K152A, or K154A, wherein the engineered EPO protein inhibits hetero-erythropoietin (EPO) receptor activity in myeloid cells.

[0018] In some aspects, provided herein is a composition comprising an engineered erythropoietin (EPO) protein, wherein the engineered EPO protein promotes hetero-erythropoietin (EPO) receptor activity to reduce an immune response, and the hetero-EPO receptor comprises an EPO receptor subunit and a CD131 subunit. In some embodiments, the engineered EPO protein comprises at least one amino acid modification and / or at least one amino acid substitution comprising K20A, N24Q, N24A, N24S, N38Q, N38A, N38S, K45A, K52A, Q58A, E62R, E62A, Q65A, L69A, E72A, R76E, R76A, L80A, N83Q, N83A, N83S, S84A, S85A, K97A, K116A, G151A, R103A, K45D, N147K, R150E, Q65A, E72R, N83A, K140A, K152A, or K154A.

[0019] In some aspects, a composition comprises an engineered erythropoietin (EPO) protein, wherein the engineered EPO protein has at least one amino acid modification and / or one of K20A, N24Q, N24A, N24S, N38Q, N38A, N38S, K45A, K52A, Q58A, E62R, E62A, Q65A, L69A, E72A, R76E, R76A, L80A, N83Q, N83A, N83S, S84A, S85A, K9 Provided herein are compositions comprising at least one amino acid substitution, including 7A, K116A, G151A, R103A, K45D, N147K, R150E, Q65A, E72R, N83A, K140A, K152A, or K154A, wherein the engineered EPO protein promotes hetero-erythropoietin (EPO) receptor activity, and the hetero-EPO receptor comprises an EPO receptor subunit and a CD131 subunit.

[0020] In some aspects, provided herein are compositions comprising an engineered erythropoietin (EPO) protein, wherein the engineered EPO protein promotes homo-erythropoietin (EPO) receptor activity and has a reduced effect on hetero-EPO receptor activity, wherein the homo-EPO receptor comprises at least two EPO receptor subunits, and wherein the hetero-EPO receptor comprises an EPO receptor subunit and a CD131 subunit. In some embodiments, the engineered EPO protein comprises at least one amino acid substitution including K20A, N24Q, N24A, N24S, N38Q, N38A, N38S, K45A, K52A, Q58A, E62R, E62A, Q65A, L69A, E72A, R76E, R76A, L80A, N83Q, N83A, N83S, S84A, S85A, K97A, K116A, G151A, R103A, K45D, N147K, R150E, Q65A, E72R, N83A, K140A, K152A, or K154A.

[0021] In some aspects, a composition comprising an engineered erythropoietin (EPO) protein, wherein the engineered EPO protein is selected from the group consisting of K20A, N24Q, N24A, N24S, N38Q, N38A, N38S, K45A, K52A, Q58A, E62R, E62A, Q65A, L69A, E72A, R76E, R76A, L80A, N83Q, N83A, N83S, S84A, S85A, K97A, K116A, G151A, R103A, K45D, N147K, R150E, Q65A Provided herein are compositions comprising at least one amino acid substitution, including at least one amino acid substitution, including E72R, N83A, K140A, K152A, or K154A, wherein the engineered EPO protein promotes homo-erythropoietin (EPO) receptor activity and has no substantial effect on hetero-EPO receptor activity, wherein the homo-EPO receptor comprises at least two EPO receptor subunits, and wherein the hetero-EPO receptor comprises an EPO receptor subunit and a CD131 subunit.

[0022] In some aspects, a composition comprising an engineered erythropoietin (EPO) protein, wherein the engineered EPO protein is selected from the group consisting of K20A, N24Q, N24A, N24S, N38Q, N38A, N38S, K45A, K52A, Q58A, E62R, E62A, Q65A, L69A, E72A, R76E, R76A, L80A, N83Q, N83A, N83S, S84A, S85A, K97A, K116A, G151A, R103A, K45D, N147K, R150E, Q65A Provided herein are compositions comprising at least one amino acid substitution, including at least one amino acid substitution, including E72R, N83A, K140A, K152A, or K154A, wherein the engineered EPO protein promotes hetero-erythropoietin (EPO) receptor activity and has no substantial effect on homo-EPO receptor activity, wherein the homo-EPO receptor comprises at least two EPO receptor subunits, and wherein the hetero-EPO receptor comprises an EPO receptor subunit and a CD131 subunit.

[0023] In some aspects, provided herein are compositions for administration to a subject having cancer or a chronic infectious disease condition, the compositions comprising a compound, or a pharma- ceutically acceptable salt, solvate, or stereoisomer thereof, that inhibits erythropoietin (EPO) receptor activity in myeloid cells of the subject.

[0024] In some aspects, provided herein are compositions for administration to a subject having cancer or a chronic infectious disease condition, the composition comprising a compound, or a pharma- ceutically acceptable salt, solvate, or stereoisomer thereof, that inhibits erythropoietin (EPO) receptor activity, such that immune checkpoint blockade resistance is reversed in the subject.

[0025] In some aspects, provided herein is a composition for administration to a subject, comprising a compound that promotes hetero-erythropoietin (EPO) receptor activity, or a pharma- ceutically acceptable salt, solvate, or stereoisomer thereof, wherein the hetero-EPO receptor comprises an EpoR subunit and a CD131 subunit, such that immune tolerance to an antigen is increased in the subject, and wherein the compound has no substantial effect on homo-EPO receptor activity, and wherein the homo-EPO receptor comprises at least two EPO receptor subunits.

[0026] In some aspects, provided herein is a composition for administration to a subject having cancer, comprising an RNA interference (RNAi) molecule, wherein the RNAi binds to an RNA molecule selected from the group consisting of an mRNA molecule encoding an erythropoietin (EPO) protein, an mRNA molecule encoding an EPO receptor subunit, an mRNA molecule encoding a CD131 subunit, and any combination thereof, and wherein upon administration of the RNAi to the subject, tumor burden in the subject is reduced.

[0027] In some aspects, provided herein is a composition for administration to a subject having cancer, comprising an RNA interference (RNAi) molecule, wherein the RNAi binds to an RNA molecule selected from the group consisting of an mRNA molecule encoding an erythropoietin (EPO) protein, an mRNA molecule encoding an EPO receptor subunit, an mRNA molecule encoding a CD131 subunit, and any combination thereof, and wherein upon administration of the RNAi to the subject, the immune response of the subject is increased by inducing more effector T (Teff) cells.

[0028] In some aspects, provided herein is a method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising any one of the single-stranded siRNAs described herein, at a dose and schedule sufficient to reduce expression levels of erythropoietin (EPO) protein, EPO receptor subunit, or CD131 subunit.

[0029] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0030] A better understanding of the features and advantages of the present disclosure will be obtained by reference to the following detailed description, which illustrates exemplary embodiments of the disclosure and the accompanying drawings. [Brief description of the drawings]

[0031] [Figure 1] FIG. 1 is an overview of EPO- and homo-EPOR-mediated erythropoiesis in erythroid progenitor cells.

[0032] [Diagram 2]FIG. 2 is a summary of immune tolerance mediated by EPO and hetero-EPOR in dendritic cells and macrophages.

[0033] [Diagram 3] Figures 3A-3B show gene expression analysis of genes in EpoR+ (EpoR+) versus EpoR- (EpoR-). Figure 3A shows a volcano plot of up- and down-regulated genes in EpoR+XCR1+CD8α+cDC1 versus EpoR-XCR1+CD8α+cDC1. XCR1: XC-chemokine receptor 1. CD8α: Cluster of differentiation 8α. cDC1: conventional type 1 dendritic cell. Figure 3B shows a heatmap representing RNA-seq gene expression of the top up- and down-regulated genes in EpoR+ versus EpoR-.

[0034] [Figure 4] Figures 4A-4C show the effect of hetero-EPOR knockout in dendritic cells (DCs). Figure 4A shows hetero-EPOR expression in DCs. Figure 4B shows the number of donor TCRβ+ T cells in mice (C57BL / 6J), Batf3 knockout mice (Batf3- / -), mice with CD11cCre (CD11cCre), mice with EPORflox / flox (EPORflox / flox), and mice with hetero-EPOR knockout in dendritic cells (EPORΔCD11c). TCRβ: T cell receptor β. Batf3: basic leucine zipper ATF-like transcription factor 3. CD11c: cluster of differentiation 11c. P values ​​from two-tailed t-test of independent means. *P<.05; **P<.01; ***P<.001; ns, not significant (P>.05). Figure 4C shows the percentage of heart survival in C57BL / 6J, Batf3- / -, EPORflox / flox, and EPORΔCD11c mice after heart transplantation (TX). WT TX (C57BL / 6J) vs. Batf3- / -TX: P<.001; WT TX (C57BL / 6J) vs. EpoRΔXCR1 TX: P<.001, log-rank; Mantel-Cox test.

[0035] [Diagram 5] Figures 5A-5B show antigen (Ag)-specific regulatory T cell (Treg) induction by EPOR in dendritic cells (cDC1). Figure 5A shows the percentage of FoxP3+Treg in transgenic mice expressing mouse alpha and beta chain T cells (OTII mice) with (i) expression of hetero-EPOR in cDC1 (EpoR+cDC) or (ii) no expression of hetero-EPOR in untreated (UNT) or EPO-treated (+EPO) cDC1 (EpoR-cDC). FoxP3: Forkhead box P3. P values ​​from two-tailed t-test of independent means. *P<.05; **P<.01; ***P<.001; ns, not significant (P>.05). Figure 5B shows flow cytometry data measuring Ag-specific Tregs from C57BL / 6 mice treated with total lymph node irradiation and antithymocyte serum (TLI / ATS) or untreated (UNT) or mice with a knockout of EPOR in dendritic cells (EPORΔCD11c).

[0036] [Figure 6] Figures 6A-6B show tumor burden in mice with hetero-EPOR deleted from myeloid cells. Figure 6A shows lung tumor size (Lewis lung carcinoma) of (i) wild type (WT) mice, (ii) wild type mice with PD-L1 treatment (WT+αPD-L1) and (iii) mice with hetero-EPOR knockout in macrophages (EpoRΔLysM). PD-L1: Programmed cell death ligand 1. Figure 6B shows tumor size (mammary adenocarcinoma) of (i) WT mice and (ii) mice with hetero-EPOR knockout in dendritic cells (EPORΔCD11c).

[0037] [Figure 7]Figures 7A-7C show tumor burden in mice with hetero-EPOR deletion dendritic cells. Figure 7A shows expression of EPOR-tdT in various immune cells of Zbtb46gfp / +EpoRtdTomato / + mice. Figure 7B shows tumor size (colon cancer) in (i) mice with hetero-EPOR deletion in dendritic cells (EpoRΔXCR1) versus (ii) mice without hetero-EPOR deletion (EPORflox / flox). Figure 7B shows tumor size in (i) mice with mTOR deletion in dendritic cells (mTORΔXCR1) versus (ii) mice without mTOR deletion (mTORflox / flox). mTOR: mammalian target of rapamycin. Data are mean ± standard error of the mean (sem). *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001, two-way ANOVA. FIG. 7C shows photographs of EPORΔXCR1 and EPORflox / flox mice at day 14 (left) and tumor size in mice with mTOR deletion in EPORΔXCR1 vs. EPORflox / flox at day 14 (right). Scale bars are as indicated. Mean tumor size. P values ​​from two-tailed t-test for independent means. ***P<.001.

[0038] [Figure 8]Figures 8A-8C show altered resistance to immune checkpoint blockade in cold tumors of mice bearing macrophages with EPOR deletion (EporΔLysM). Figure 8A shows the experimental scheme for administering anti-programmed cell death-1 antibody (αPD-1) to cold hepatocellular carcinoma (HCC)-bearing mice. A spontaneous model of cold HCC was generated by delivering plasmids pCMV-SB13, pT3-EF1a-C-Myc-IRES-luciferase, and pX330-Trp53-targeting sgRNA to the liver of mice using hydrodynamic tail vein injection (HDTV) in vivo. Two weeks later, mice of C57BL / 6 wild type (WT) and EporΔLysM strains were treated with either 2 mg / kg αPD-1 or IgG isotype control via intraperitoneal injection every 3 days for a total of 5 doses. Trp53: cellular tumor antigen p53. C-myc: c-Myc oncoprotein. Luc: luciferase. Figure 8B shows the tumor growth kinetics of wild-type mice treated with IgG isotype (WT IgG isotype), wild-type mice treated with αPD-1 (WT αPD-1), mice with macrophage-specific knockout of hetero-EPOR treated with IgG isotype (EporΔLysM IgG isotype), and mice with macrophage-specific knockout of hetero-EPOR treated with αPD-1 (EporΔLysMαPD-1), analyzed by measuring luciferin-based bioluminescence. Figure 8C shows the survival curves of WT IgG isotype, WT αPD-1, EporΔLysM IgG isotype, and EporΔLysMαPD-1.

[0039] [Figure 9]Figures 9A-9C show changes in cold tumors resistant to immune checkpoint blockade in mice with EPO knockout in dendritic cells. Figure 9A shows the experimental scheme of treating melanoma mice with αPD-1 (programmed cell death-1). Figure 9B shows the tumor size (melanoma) of (i) control mice, (ii) control mice treated with αPD-1 (control + αPD-1), (iii) mice with hetero-EPOR deletion in dendritic cells (EpoRΔXCR1) and (iv) mice with hetero-EPOR deletion in dendritic cells treated with αPD-1 (EpoRΔXCR1 + αPD-1). *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001, two-way ANOVA. FIG. 9C shows flow cytometry data measuring perforin, granzyme B, interferon-gamma (IFNγ), and tumor necrosis factor alpha (TNFα) in mice without hetero-EPOR deletion (EPORflox / flox) and mice with hetero-EPOR deletion in dendritic cells (EpoRΔXCR1).

[0040] [Figure 10] FIG. 10 shows percent survival data from The Cancer Genome Atlas Liver Hepatocellular Carcinoma (TCGA-LIHC) for patients with hepatocellular carcinoma who have (i) low levels of EPO versus (ii) high levels of EPO.

[0041] [Figure 11]Figures 11A-11C show the effect of EPO on tumor development in mice with regressing hepatocellular carcinoma (HCC). Figure 11A shows the experimental scheme for establishing a regressing HCC model. 3x106 allogeneic Hepa1-6 cells were orthotopically implanted into C57BL / 6 mice. Two Hepa1-6 stable cell lines were created using lentivirus with either EPO overexpression (Hepa1-6_EpoOE) or empty vehicle (Hepa1-6_EV). Figure 11B shows tumors from hepatocellular carcinoma mice treated with Hepa1-6_EV or Hepa1-6_EpoOE harvested at 14 and 21 days after injection. Figure 11C shows quantification of tumor volume and complete regression (CR) rate measurement of HCC mice treated with Hepa1-6_EV or Hepa1-6_EpoOE.

[0042] [Figure 12] Figures 12A-12B show colon tumor growth in mice with or without liver metastasis. Figure 12A shows the change in colon tumor volume in wild-type mice with or without liver metastasis. Figure 12B shows the change in colon tumor volume in mice with EPOR deletion in macrophages (EpoRΔLysM) with or without liver metastasis.

[0043] [Figure 13]Figures 13A-13C show the effect of liposomes targeted to macrophages loaded with siRNA targeting EPOR (siEpor) in mice bearing hepatocellular carcinoma (Hepa1-6_EpoOE). Figure 13A shows the experimental scheme of liposome treatment in two HCC models. Hepa1-6_EpoOE: 3x106 EPO-overexpressing Hepa1-6 cells were orthotopically transplanted into C57BL / 6 mice. One week later, mice were treated with liposomes containing either 50μg siEpor (siRNA targeting EPOR) or siNTC (non-targeting control) RNA via intravenous injection every 4 days for a total of 3 doses. HDTV: A spontaneous model of cold HCC was created by delivering plasmids pCMV-SB13, pT3-EF1a-C-Myc, and pX330-Trp53-targeted sgRNA to the liver of mice using hydrodynamic tail vein injection (HDTV) in vivo. Two weeks later, mice were treated with liposomes containing 50 μg of either siEpor or siNTC RNA via intravenous injection every 4 days for a total of 6 doses. Figure 13B shows tumors (left) and tumor volumes (right) recovered from hepatocellular carcinoma mice treated with liposomes containing either siEpor or siNTC. Figure 13C shows livers (left) and liver weights (right) recovered from mice with cold HCC treated with liposomes containing either siEpor or siNTC.

[0044] [Figure 14]Figures 14A-14C show the effect of liposomes targeted to macrophages loaded with siRNA targeting heterodimer EPOR. Figure 14A shows the physical properties of liposomes targeted to macrophages. Figure 14B shows flow cytometry analysis showing macrophages as the major cell type taking up liposomes. C57BL / 6 mice implanted with Hepa1-6_EpoOE were administered liposomes loaded with 50 μg of fluorescein isothiocyanate (FITC)-conjugated siRNA. After 24 hours, tumors were harvested and dissociated into single cell suspensions. Flow cytometry analysis was performed to measure the percentage of FITC+ cells in different myeloid cell types. Figure 14C shows the knockdown efficiency of EPOR in tumor-infiltrating macrophages. 3×106 Epo-overexpressing Hepa1-6 cells were implanted orthotopically into C57BL / 6 mice. One week later, mice were treated with liposomes containing 50 μg of either siRNA targeting EPOR (siEpor) or non-targeting control siRNA (siNTC) via intravenous injection every 4 days for a total of 3 doses. Tumors were harvested 3 weeks after injection and dissociated into single cell suspensions. Macrophages were isolated using magnetic-activated cell sorting, and RNA was extracted for real-time PCR quantification.

[0045] [Figure 15A] Figures 15A-15B show EPOR expression in myeloid cells from fresh human cancer specimens of breast cancer (Figure 15A) and metastatic sites of left axillary lymph node metastases of breast cancer (Figure 15B). Figure 15A shows EPOR expression levels analyzed by flow cytometry. CD45+ cancer-infiltrating lymphocytes were gated as live-dead aqua-CD45+. Histograms showed EPOR expression in individual myeloid cell subsets. Left: breast cancer. Right: surrounding healthy tissue. Bottom: EPOR expression in dendritic cells gated as CD11c+HLA-DR+CD14-CD16-. Figure 15B shows EPOR expression in tumor-infiltrating lymphocytes of breast cancer left axillary lymph node (LN) metastatic sites. Top: EPOR expression in dendritic cells. Bottom: EPOR expression in HLA-DR- cells. [Figure 15B] Same as above.

[0046] [Figure 16] Figures 16A-16B show EPOR expression in myeloid cells at metastatic sites of fresh human liver metastases paired with peripheral blood samples. Samples were collected from three individual patients with different original tumor types. Figure 16A shows EPOR expression levels in CD45+ tumor-infiltrating lymphocytes at liver metastatic sites analyzed by flow cytometry. CD45+ cancer-infiltrating lymphocytes were gated as live-dead aqua-CD45+. Right: EPOR expression in peripheral blood samples of liver metastases patients compared to healthy donor blood. The percentage of EPOR+ cells is shown in red rectangles. Figure 16B shows the percentage of EPOR+ cells in blood of liver metastases patients, healthy donor blood, and blood of liver cancer or cirrhosis. Statistical analysis was performed using unpaired two-tailed t-tests. *P<0.05; **P<0.01; ***P<0.001 and ****P<0.0001.

[0047] [Figure 17] FIG. 17 shows examples of EPO blocking efficiency of the hybridoma clones listed in Table 11.

[0048] [Figure 18] Figures 18A-18B show TLI / ATS-induced tolerance to allogeneic bone marrow (BM) and heart transplantation. Figure 18A shows the experimental scheme of performing heart transplantation on mice, treating mice with TLI / ATS, performing bone marrow transplantation, and checking allogeneic BM chimerism and heart engraftment. Figure 18B shows engraftment of heart grafts in wild-type and Baf3- / - mice (left), and BM chimerism in wild-type and Baf3- / - mice at 34 days after BM transplantation (TX) (right).

[0049] [Figure 19]Figures 19A-19E show TLI / ATS-induced local apoptosis and extramedullary erythropoiesis in combination with dendritic cell (DC) enrichment and systemic upregulation of EPO. Figure 19A shows representative images of TUNEL staining in sections of untreated spleen (UNT) and TLI / ATS-treated spleen. Figure 19B shows cellular composition analysis of changes in different cell populations in untreated spleen (UNT), ATS-treated spleen, TLI-treated spleen, and TLI / ATS-treated spleen. Pie charts show the average frequency of the indicated populations from one representative experiment (n=4). T cells (TCRβ+CD19-), B cells (TCRβ-CD19+), erythroid progenitors (TER119+CD71+), DCs (CD11chighMHCIIhigh), CD11b+ myeloid cells are subdivided into LyG+, Ly6C+ and F4 / 80+ (RPM, red pulp macrophages). Figure 19C shows the gating strategy of erythroid progenitors in untreated spleens and TLI-treated spleens. Figure 19D shows extramedullary erythropoiesis in TLI-treated spleens and TLI-treated bone marrow. Figure 19E shows the systemic increase of EPO in peripheral blood serum measured by enzyme-linked immunosorbent assay (ELISA).

[0050] [Fig. 20A-D]Figures 20A-20H show RNA-seq analysis of CD8α+cDC1 sorted from TLI / ATS conditioned mice versus untreated (UNT) mice. Figure 20A shows total spleen cell counts in untreated or TLI / ATS treated mice. Figure 20B shows the frequency of CD11chighMHCIIhighDC in liver cells (DAPI-) of untreated or TLI / ATS treated mice. Figure 20C shows the gating strategy for CD8α+CD11b- and CD11b+CD8α-cDC (left) and the frequency of CD8a+CD11b-DC in UNT versus TLI / AT CD11highMHCIIhighDC (right). Representative samples from TLI / AT conditioned mice are shown. For Figures 20A-20C, the numbers in the plots indicate the percentage of positively stained cells within the respective gates. Data are mean ± sem, ***p<0.001 and ****p<0.001 determined by unpaired Student's t-test, number of mice per group as indicated. Results represent one of at least three similar experiments. Figure 20D is a principal component analysis (PCA) plot showing separate clustering of CD8α+DCs in UNT vs. TLI / AT. Figure 20E shows a heatmap representing RNA-seq gene expression of the top 30 upregulated (P≦0.01 and fold change ≧log2) genes in TLI / AT conditioned vs. UNT groups. Biological replicates (n=2, each pooled from 3-5 mice) for each group are shown separately. Heatmaps were generated from differential expression analysis using DESeq2 based on R studio software. Figure 20F shows Gene Set Enrichment Analysis (GSEA) analysis using signature gene sets in the Molecular Signatures Database (MSigDB) after TLI / AT. NES: Normalized Enrichment Score. FDR: False Discovery Rate. Right half of graph: Upregulated pathways. Left half of graph: Downregulated pathways. Figure 20G shows real-time PCR of the indicated genes in splenic CD8α+cDC1 and CD11b+cDC2.CD8α+cDC1 (upper panel) and CD11b+cDC2 (lower panel) were sorted by flow cytometry from (i) UNT, (ii) ATS, (iii) TLI, (iv) TLI / ATS conditioned mice the day after the last dose of TLI. The gating strategy is shown in Figure 20C. Data are mean ± SEM, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.001, ns (not significant) determined by unpaired Student's T-test. Figure 20H shows EPOR expression of CD8α+cDC1 in UNT, TLI, and TLI / ATS treated EPOR-tdT mice. In the TLI and TLI / ATS groups, spleens were harvested the day after the last dose of TLI. [Fig. 20E-F] Same as above. [Fig. 20G-H] Same as above.

[0051] [Figure 21] Figure 21 shows TLI-ATS-induced chimerism in B cells, T cells, granulocytes, and macrophages (MΦ) of wild-type (WT), EPOR floxed / floxed mice, mice with dendritic cell (DC)-specific hetero-EPOR gene deletion (EPORΔCD11c), and Baft3 knockout (KO) mice. Percentage of donor-type cells among T cells (TCRβ+), B cells (B220+), and granulocytes (Ly6G+) in the host's blood 14 days after BM transplantation. Bars indicate the mean percentage of donor cells. P values ​​by two-tailed t-test of independent means. *P<05; **P<01; ***P<001; ns, not significant.

[0052] [Figure 22]Figures 22A-22B show the abrogation of both bone marrow chimerism establishment and maintenance by administration of diphtheria toxin (DT) injection to FoxP3-DTR (forkhead box P3-diphtheria toxin receptor) recipient mice. Figure 22A shows the experimental scheme of two groups of FoxP3-DTR mice with different treatments of DT. FoxP3-DTR mice were conditioned with TLI / ATS and DT was administered by intravenous (iv) injection either on day 3 after allogeneic BM (group A; top) or on day 15 after BM chimerism establishment (group B; bottom). DT was given every 2 days. Bone marrow chimerism was examined on days 14 and 29 in both groups. FIG. 22B shows the percentages of donor (MHCI-H2kb)-derived T cells (TCRβ+), B cells (B220+), macrophages (MΦ; CD64+), and granulocytes (Ly6G+) in Group A (left three bars in all four graphs) and Group B (right three bars in all four graphs).

[0053] [Diagram 23]Figures 23A-23D show the requirement of CD8α+cDC1 for antigen-specific CD4+FoxP3+Treg induction and expansion. C57BL / 6 (wild type) or Batf3- / - mice, or EPORΔCD11c recipient mice were either untreated (UNT) or conditioned with TLI. Macrophage-negative selected OTII cells (cells expressing ovalbumin (Ova)-specific αβTCR) were injected intravenously (iv) 1 day after the last dose of TLI, and Ova-expressing bone marrow cells were injected iv another day later. Five days later, FoxP3 expression was examined by flow cytometry in adoptively transferred OTII cells defined as TCR-vα2+CD4+. Figures 23A and 23C show plots for the gating strategy of FoxP3+Treg in TCR-vα2+CD4+OTII cells. The graphs show the percentage of FoxP3+ Treg among TCRvα2+CD4+ viable OTII cells from the spleens of C57BL / 6 (FIG. 23A), mice with Batf knockout (KO) (FIG. 23A), mice with hetero-EPOR deleted in dendritic cells (EPORΔCD11c) (FIG. 23C) either untreated (UNT) or treated with TLI. Figures 23B and 23D show histograms of FoxP3 expression in adoptively transferred OTII cells. The graphs show the mean fluorescence intensity (MFI) of FoxP3 or UNT vs. TLI treated C57BL / 6 mice or mice with Batf3 KO (FIG. 23B) or EPORΔCD11c mice (FIG. 23D). Data are means ± SEM, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.001, ns (not significant) determined by unpaired Student's T-test, numbers of mice per group are as indicated.

[0054] [Figure 24]Figures 24A-24D show the induction of CD4+FoxP3-CD73+Folate Receptor 4+(FR4+) anergic T cells upon allo-BM challenge, which depends on the presence of Tregs. Figure 24A shows BM chimerism without (w / o) and with diphtheria toxin (DT) in B cells, T cells, granulocytes, and macrophages (MΦ). DT was injected from day -1 to day 1. Figure 24B shows the analysis of Tregs and anergic T cells for intercellular IFNγ expression at day 5 after allo-BM challenge with or without DT. Figure 24C shows the statistical analysis of Figure 24B. Figure 24D shows the correlation between FoxP3+Treg frequency (X-axis) and CD4+FoxP3-CD73+FR4+anergic T cell frequency (Y-axis). Linear regression was determined by Prism. Data are means ± SEM, *p<0.05, **p<0.01, ***p<0.001 and ****p<0.001, ns (not significant) determined by unpaired Student's T-test, numbers of mice per group are as indicated.

[0055] [Diagram 25] Figures 25A-25C show 5-chloromethylfluorescein diacetate+ (CMFDA+) allogeneic bone marrow uptake by CD8α+ cDC1 after TLI. Figure 25A is a plot showing phagocytosis of viable allogeneic BM cells in recipient CD11highMHCIIhighDC (left) and comparison of the frequency of CMFDA+CD8α+ and CD8α- cDC among CD11highMHCIIhigh recipient DCs 12 hours after BM injection, respectively (right). Figure 25B shows the percentage of CMFDA+ cells in gated CD11highMHCIIhighCD8α+ cDC1 untreated (UNT) or treated with TLI. Figure 27C shows CD103 and DEC-205 expression in CD8α+CMFDA+ cDC1. Distribution by TLI (pink) overlaid on UNT (black).

[0056] [Figure 26]Figures 26A-26B show the expression of EPO-EPOR downstream signaling molecules in CD8α+cDC1 and CD11b+cDC2, and the percentage of donor cells from various mouse strains. Figure 26A shows the expression of EPO-EPOR downstream signaling molecules in CD8α+cDC1 and CD11b+cDC2 from untreated (UNT) mice, mice treated with TLI, and mice treated with TLI and ATS, as measured by MFI. Intracellular phospho-flow was performed 1 day after the last dose of TLI. Figure 26B shows the percentage of donor cells of B cells, T cells, granulocytes, and macrophages with C57 / 6J, Baftf3- / -, CD11cCre, EPORflox / flox, mTORflox / flox, EPORΔXCR1, and mTORΔXCR1.

[0057] [Figure 27] Figures 27A-27C show the effect of XCR1-specific deletion of EpoR or mTOR on tumor Ag-specific CD8+ T cells in tumor-draining lymph nodes (tdLNs). Figure 27A shows the experimental scheme to analyze OT-I (CD8+ T cells expressing T cell antigen receptor) in control mice, mice with EpoR knockout in dendritic cells (EpoRΔXCR1), and mice with mTOR knockout in dendritic cells (mTORΔXCR1). Figure 27B shows the measurement of CD44, SLAMF6, PD-1, and Tim3 expressing cells, as well as the measurement of proliferating cells via flow cytometry (cell trace violet (CTV)). Figure 27C shows the percentage of proliferated OT-I in control, EpoRΔXCR1, and mTORΔXCR1 mice. ***P<0.001; ns=not significant.

[0058] [Figure 28A]Figures 28A-28B show analysis of antibodies in the supernatants of hybridoma clones. Figure 28A shows the percentage of cell staining for 293T cells expressing EPOR, CD131, or both, binding kinetics data (EPOR-CD131-Fc, EPOR-Fc, and CD131-Fc), and percentage data for blocking of EPO / EPOR interaction for 17 clones with unique antibody sequences. Figure 28B shows the expression of human EPOR (hEPOR) and human CD131 (hCD131) measured by flow cytometry using Phycoerythrin (PE)-labeled anti-EPOR and Alexa Fluor® 647 (AF647)-labeled anti-CD131, respectively. [Figure 28B] Same as above.

[0059] [Figure 29] Figures 29A-29D show the mean or median fluorescence intensity (MFI) of human leukemia UT-7 cells, 293T cells expressing EPOR (293T / EPOR), 293T cells expressing CD131 (293T / CD131), and 293T cells expressing both EPOR and CD131 (293T / EPOR / CD131) stained with purified antibodies. Figure 29A shows the MFI of 293T / EPOR cells labeled with purified hybridoma clones M2 and M41 across different antibody concentrations. Figure 29B shows the MFI of 293T / EPOR / CD131 cells labeled with purified hybridoma clones M2 and M41 across different antibody concentrations. Figure 29C shows the MFI of 293T / CD131 cells labeled with purified hybridoma clones M2 and M41 across different antibody concentrations. FIG. 29D shows the MFI of UT-7 cells labeled with purified hybridoma clones M2 and M41 across different antibody concentrations.

[0060] [Diagram 30]Figure 30 shows phosphorylated STAT5 analyzed by flow-based assay. UT-7 cells, 293T cells expressing EPOR (293T / EPOR), and 293T cells expressing both EPOR and CD131 (293T / EPOR / CD131) were incubated with anti-EPOR antibody (hybridoma clone M2; upper panel or hybridoma clone M41; lower panel) after stimulation with (EPO+M2 or EPO+M41) or without recombinant human EPO (no EPO control). The same cells without anti-EPOR antibody incubation after EPO stimulation were used as control (EPO, no Ab control).

[0061] [Diagram 31] Figures 31A-31B show SDS-PAGE analysis of IME001, IME003, IME004, carbamylated EPO (CEPO), and recombinant human EPO (rhEPO). Figure 31A shows SDS-PAGE of expression vectors IME001 and IME003 with EPO fused at the N-terminus of human IgG4 or human serum albumin, and expression vector IME004 with EPO fused at the C-terminus of human albumin. Figure 31B shows SDS-PAGE of BSA control, rhEPO with or without Lyc-C digestion, and CEPO with or without Lyc-C digestion.

[0062] [Fig. 32A-C]Figures 32A-32D show cell staining assays measuring the receptor binding activity of IME001, IME003, and IME004. Figure 32A shows flow cytometry analysis of 293T cells (left) or 293T / EPOR cells (right) stained with anti-EPOR PE conjugate. Figure 32B shows flow cytometry analysis of 293T / EPOR cells incubated with 1 μg / ml (left), 0.1 μg / ml (center), or 0.01 μg / ml (right) IME001 and stained with anti-human Fc PE conjugate. Figure 32C shows flow cytometry analysis of 293T / EPOR cells incubated with IME003 (upper panel) or IME004 (lower panel), biotinylated anti-HSA (human serum albumin), and streptavidin PE conjugate at 10 μg / ml (left), 1 μg / ml (middle), or 0.1 μg / ml (right). Figure 32D shows binding of IME003 EPOR-Fc, IME003 IME 020, IME004 EPOR-Fc, and IME004 IME020 at various concentrations of IME003 / IME004. [Fig. 32D] Same as above.

[0063] [Diagram 33] Figures 33A-33B show analysis of STAT5 phosphorylation in 293T / EPOR cells stimulated with various EPO proteins. Figure 33A shows Western blot analysis using human phospho-STAT5a / b (Y694 / Y699) of 293T / EPOR cells untreated (control) or stimulated with CEPO, IME001, IME003 or IME004. Figure 33B shows the results of phospho-STAT5 enzyme-linked immunosorbent assay (ELISA) using lysates of 293T / EPOR cells untreated (untreated control) or stimulated with IME001, IME003, IME004, IME005, IME008 or IME013.

[0064] [Diagram 34] FIG. 34 shows the amino acid and nucleic acid sequences of human EPO, including the signal peptide sequence.

[0065] [Fig. 35A-C] Figures 35A-35E show EpoR expression in peripheral lymph node (pLN) migratory cDC1. Figure 35A shows flow cytometry analysis of EpoR expression in pLN migratory and resident cDC1 from EpoRtdt / +, Zbtb46gfp / +EpoRtdT / +, CCR7- / -EpoRtdT / +, and Batf3- / -EpoRtdT / + mice. Figure 35B shows histograms of EpoR expression in migratory and resident cDC1 of individual mouse strains. Figure 35C shows flow analysis of EPOR expression in individual inguinal, axillary, brachial, or superficial cervical lymph nodes. Figure 35D shows flow cytometry analysis of EPOR and CD103 expression in pLN migratory cDC. FIG. 35E shows the experimental scheme of EpoR-tdT-cre mice mating with Rosa26-lox-Stop-lox-EYFP mice and flow cytometry analysis of pLN migratory cDC1 (MHCIIhighCD11interXCR1+) for EYFP expression. [Fig. 35D-E] Same as above.

[0066] [Diagram 36] Figure 36 shows flow cytometry analysis of peripheral LN migratory EpoR+XCR1+cDC1 expressing DEC205+ and CCR7+. Figure 36 also shows histograms comparing PD-L1, Tim3, Axl and CD131 expression in EpoRhigh and EpoRlow migratory cDC1s.

[0067] [Figure 37]Figures 37A-37C show the effect of peripheral LN (pLN) migratory EpoR+XCR1+cDC1 on the induction of Ag-specific Tregs toward DEC205-Ova and Ova-expressing cells. Figure 37A shows flow cytometry analysis of pLN migratory and resident EpoR+cDC1 and EpoR-cDC1. Figure 37B shows flow cytometry and quantification of FoxP3 expression of CellTrace™ Violet (CTV)-labeled naive CD45.1+OT-II cells cultured with DEC-205-Ova with or without TGFβ treatment. FIG. 37C shows flow cytometry and quantification of FoxP3 expression of CellTrace™ Violet (CTV)-labeled naive CD45.1+ OT-II cells cultured with CD45.2+ cDC1, purified macrophages, and Gray-irradiated Act-mOVA thymocytes (CD45.2+) with or without TGFβ treatment or with or without EPO treatment.

[0068] [Figure 38] Figures 38A-38C show in vitro antigen (Ag)-specific regulatory T cell (Treg) induction by carbamylated EPO (CEPO) treatment. Figure 38A shows flow cytometry analysis of FoxP3 expression and proliferation (CellTrace™ Violet (CTV)) of CD11cIntMHCIIHighXCR1+cDC1 with EPO or CEPO treatment. Figure 38A also shows quantification of FoxP3+Treg percentage in surviving OTII with untreated (UNT) or EPO or CEPO treatment. Figure 38B shows an experimental scheme to study the effect of EPO or CEPO on antigen-specific tolerance by mice with mTOR knockout in dendritic cells (mTORΔXCR1), mice with EPOR knockout in dendritic cells (EPORΔXCR1), and littermate controls.

[0069] [Figure 39]Figures 39A-39C show the expression of EPOR in migratory cDCs carrying apoptotic cells. Figure 39A shows the experimental scheme of mice injected with cDC1 in the third mammary fat pad. Figure 39B shows flow cytometry analysis of EPOR expression in cDC1 in the third mammary fat pad. Figure 39C shows flow cytometry analysis of EPOR expression in draining lymph nodes (inguinal LNs) injected with PKH67-labeled CD45.1+ dexamethasone (DEX)-induced apoptotic thymocytes.

[0070] [Diagram 40] Figures 40A-40B show the effect of EPO on peripheral Ag-specific tolerance in draining lymph nodes to cell-associated Ag (Ova). Figure 40A shows the experimental scheme of iv injection of 5x105 purified macrophages and CellTrace™ Violet (CTV)-labeled naive CD45.1+ OT-II cells on day -1. On day 0, dexamethasone (DEX)-induced apoptotic Act-mOVA thymocytes were injected sc into the third mammary fat pad. 50 IU of EPO was given ip over the course of 4 consecutive days. Figure 40B shows flow cytometry analysis and quantification of FoxP3 expression in CD45.1+ OT-II of draining lymph nodes (inguinal LN) with or without EPO.

[0071] [Diagram 41] Figures 41A-41C show the binding activity of IME003 and IME004. Figure 41A shows the binding of IME003 and IME004 to IME083 or IME020 at various concentrations of IME003 and IME004. Figure 41B shows the binding of IME061 / IME062, IME061 / IME063, IME061 / IME064, IME063 / IME084 to IME003 at various concentrations of IME003. Figure 41C shows the binding of IME061 / IME062, IME061 / IME063, IME061 / IME064, IME063 / IME084 to IME004 at various concentrations of IME004.

[0072] [Figure 42A] Figures 42A-42D show the amino acid and nucleic acid sequences of the human EPOR extracellular domain (ECD) or human CD131 ECD, human CD131 D3D4 domain, and human EPOR(F93A) domain, including the signal peptide sequence in red. Figure 42A shows the amino acid and nucleic acid sequences of the human EPOR ECD in IME020 and IME061. Figure 42B shows the amino acid and nucleic acid sequences of the human CD131 ECD in IME062. Figure 42C shows the amino acid and nucleic acid sequences of the human CD131 D3D4 domain in IME063. Figure 42D shows the amino acid and nucleic acid sequences of the human EPOR(F93A) domain in IME083 and IME034. [Figure 42B] Same as above. [Figure 42C] Same as above. [Fig.42D] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0073] Detailed Description of the Disclosure Although various embodiments of the present disclosure are described herein, it will be obvious to those skilled in the art that such embodiments are provided only as examples.Many modifications and changes to the embodiments described herein, as well as variations and replacements thereof, will be apparent to those skilled in the art without departing from the present disclosure.It is understood that various alternatives to the embodiments described herein can be used in implementing the present disclosure.It is also understood that all embodiments of the present disclosure can be combined with any one or more of the other embodiments described herein that are consistent with the embodiment, as necessary.

[0074] When elements are presented in list form (e.g., Markush groups), it is understood that each possible subgroup of the elements is also disclosed, and that any one or more elements can be removed from the list or group.

[0075] Unless expressly indicated to the contrary, in any method described or claimed herein that includes two or more acts or steps, the order of the method acts or steps is not necessarily limited to the order in which the method acts or steps are recited, but the present disclosure includes embodiments in which the order is so limited.

[0076] In general, when an embodiment in the specification or claims is referred to as comprising one or more features, it is further understood that the disclosure also encompasses embodiments that consist of or consist essentially of such features.

[0077] It is also understood that any embodiment of the present disclosure, for example any embodiment found within the prior art, may be explicitly excluded from the claims, regardless of whether a specific exclusion is recited herein.

[0078] Reference herein to a peptide, polypeptide or protein, such as an antibody or fragment thereof, is further understood to include pharma- ceutically acceptable salts thereof, unless specifically stated otherwise or the context clearly indicates otherwise. Such salts may have a positive net charge, a negative net charge, or no net charge.

[0079] Headings are included herein for reference and to aid in locating certain sections. The headings are not intended to limit the scope of the embodiments and concepts described in the section under that heading, and the embodiments and concepts may be applicable in other sections throughout this disclosure.

[0080] All patent and non-patent literature cited in this specification are hereby incorporated by reference in their entirety to the same extent as if each patent or non-patent literature was specifically and individually indicated to be incorporated by reference in its entirety.

[0081] Besides erythroid progenitors, growing evidence suggests broad EPOR expression in non-erythroid cells, such as hematopoietic stem cells (HSCs), megakaryocytes, B cells, T cells, macrophages (MΦ), endothelial cells, and neurons (Broxmeyer, J Exp Med 2013:210:205-208). Notably, the immunomodulatory role of EPO is increasingly recognized (Cantarelli et al., Am J Transplant 2019:19:2407-2414; Peng et al., Cell Death Dis 2020:11:79). Involvement of EPO signaling suppresses inflammatory responses by inhibiting NFκB-induced immune pathways (Nairz et al., Immunity 2011:34:61-74). EPO also primes MΦs for efficient efferocytosis, thereby preventing autoimmunity (Luo et al., Immunity 2016:44:287-302).

[0082] EPO is cardioprotective in ischemia-reperfusion injury and myocardial infarction. EPO improves cardiac function related to neovascularization mediated by stimulating coronary endothelial cells to activate endothelial nitric oxide (NO) synthase (eNOS) and NO production (Teng et al., Basic Res. Cardiol. 2011:106:343-354).

[0083] EPO stimulates neovascularization and angiogenesis by activating endothelial cells (ECs) and endothelial progenitor cells (EPCs) in physiological and pathological conditions, such as ischemic cardiovascular disease and tumors. Activation of EPO leads to EC and EPC recruitment, proliferation, migration, and differentiation (Annese et al., Experimental Cell Research, 2019: 374(2):266-273).

[0084] In the central nervous system, EPO and EPOR are expressed by neurons, glial cells and cerebrovascular endothelium. EPO has been shown to be neurotrophic and neuroprotective in vitro and in animal models of neuronal damage associated with trauma, stroke, ischemia, inflammation and epileptic seizures. The beneficial effects of EPO have also been demonstrated in clinical studies of stroke, schizophrenia and progressive multiple sclerosis. EPO protects neurons both directly by preventing apoptosis and indirectly by modulating inflammatory processes and stimulating neurogenesis and angiogenesis (Wang et al., Stroke 2004:35:1732-7).

[0085] EPO regulation of metabolism extends beyond oxygen delivery and contributes to the maintenance of white adipose tissue and metabolic homeostasis. EPO is protective in diet-induced obesity, improves glucose tolerance, reduces insulin resistance, and regulates fat mass accumulation, especially in male mice (Alnaeeli and Noguchi, Adipocyte 2015:4:153-157). EPO promotes an anti-inflammatory phenotype by modulating the proinflammatory response of macrophage infiltration in white adipose tissue, inhibiting the expression of inflammatory cytokines, and reducing macrophage infiltration (Alnaeeli et al., Diabetes Metab. Res. Rev. 2014:63:2415-2431).

[0086] It has been shown that some of the cytoprotective effects of EPO are mediated through its binding to heterodimers containing the standard EPOR and common beta receptor (βcR or CD131; Brines et al., Proc Natl Acad Sci USA 2004; 101: 14 907-14 912). Interestingly, carbamylated EPO binds to these heteroreceptors and shows tissue protective effects, but it does not bind to the classical EPOR and does not stimulate erythropoiesis. βcR is not required for erythropoiesis. It is hypothesized that βcR combined with EPOR expressed by non-hematopoietic cells constitutes a tissue protective receptor, thus creating a tissue protective heteroreceptor.

[0087] The expression levels of EPO and EPOR are regulated. EPO production is induced under hypoxic conditions mediated by HIF (Semenza, Blood 2009:114(10):2015-9). EPOR expression is regulated by transcription factors Sp1, GATA1, and TAL1. Binding of EPO to EPOR in erythroid progenitor cells increases the expression of transcription factors GATA1 and TAL1, which in turn transactivates EPOR expression (Suresh et al., Front Physiol. 2020:10:1534). EPOR is also regulated at the protein level. P85 promotes endocytosis and degradation of EPOR. Prolyl hydroxylase D3 (PHD3) mediates prolyl hydroxylation of EPOR, leading to proteasomal degradation. TFR2 and Scribble facilitate the reuse of EPOR (Bhoopalan et al., F1000Res. 2020; 9: F1000 Faculty Rev-1153).

[0088] We recently found that EPOR plays an important role in the induction of tumor immune tolerance by myeloid cells, including dendritic cells (DCs) and macrophages (MΦs) in a wide range of primary and metastatic tumors, including liver metastasis-induced systemic antigen-specific immune tolerance (Figure 2). EPOR is also essential for myeloid cell-mediated tolerance in transplants of allogeneic organs, such as kidney, liver, lung, and heart (Figure 2).

[0089] definition Unless otherwise defined by their usage herein or clearly indicated otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs.

[0090] As used in this specification and the appended claims, the indefinite articles "a" and "an," and the definite article "the," can include plural referents as well as singular referents unless specifically stated otherwise or the context clearly dictates otherwise.

[0091] The term "about" or "approximately" refers to an acceptable error for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined. In certain embodiments, the term "about" or "approximately" refers to within one standard deviation. In some embodiments, when no specific limit of error (e.g., standard deviation for an average value given in a chart or table of data) is given, the term "about" or "approximately" refers to a range that would encompass the given value, and also a range that would be included by rounding up or down the given value, taking into account significant figures. In certain embodiments, the term "about" or "approximately" refers to within ±10%, 5%, 4%, 3%, 2% or 1% of the stated value. Whenever the term "about" or "approximately" precedes a series of two or more numbers, or the first number in a series of two or more ranges of numbers, the term "about" or "approximately" applies to each one of the numbers in that series or range of numbers.

[0092] The term "antibody" may refer to a protein that is functionally defined as a binding protein and structurally defined as comprising an amino acid sequence recognized as being derived from a framework region of an immunoglobulin (Ig)-encoding gene. An antibody may comprise one or more polypeptides substantially encoded by an immunoglobulin gene or a fragment of an immunoglobulin gene. Recognized immunoglobulin genes may include kappa, lambda, alpha, gamma, delta, epsilon and mu constant region genes, as well as a myriad of immunoglobulin variable region genes. Light chains may be classified as either kappa or lambda. Heavy chains may be classified as gamma, mu, alpha, delta or epsilon, which in turn define the immunoglobulin classes IgG, IgM, IgA, IgD and IgE, respectively. In some embodiments, these may be further subdivided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2.

[0093] A typical gamma immunoglobulin (antibody) structural unit is known to comprise a tetramer. Each tetramer may be composed of two identical pairs of polypeptide chains, each pair having one "light" chain (about 25 kD) and one "heavy" chain (about 50-70 kD). The N-terminus of each chain may define a variable region of about 100-110 or more amino acids that are primarily responsible for antigen recognition. The variable light chain (V L ) and variable heavy chain (V H ) may refer to these light and heavy chains, respectively.

[0094] Antibodies can exist as intact immunoglobulins or as a number of well-characterized fragments. Thus, for example, pepsin digests antibodies below the disulfide linkage in the hinge region to produce Fab' dimers, F(ab)' 2 F(ab)', which itself is a light chain joined to the VH-CH1-hinge by a disulfide bond. 2 is reduced under mild conditions to break the disulfide linkages in the hinge region, thereby forming (Fab') 2 The dimer may be converted to a Fab' monomer, which is essentially a Fab with a portion of the hinge region (see Fundamental Immunology, WE Paul, ed., Raven Press, NY (1993) for a more detailed description of other antibody fragments). Although various antibody fragments have been defined in terms of the digestion of an intact antibody, one of skill in the art will understand that the fragments may be synthesized de novo, either chemically or by utilizing recombinant DNA methodology. Thus, the term antibody, as used herein, may also include antibody fragments either produced by the modification of whole antibodies or synthesized using recombinant DNA methodology. Preferred antibodies include single chain antibodies (antibodies that exist as a single polypeptide chain), such as VFv antibodies (sFv or scFv), in which the variable heavy and variable light regions are joined together (directly or through a peptide linker) to form a continuous polypeptide.H -V L Single chain Fv antibodies can include dimers. Single chain Fv antibodies can include V2s linked either directly or via a peptide-encoded linker. H and V L A covalently linked V that can be expressed from a nucleic acid containing a coding sequence H -V L It is a heterodimer (see, e.g., Huston, et al. Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988, which is incorporated herein by reference in its entirety). H and V L are connected to each other as a single polypeptide chain, but V H and V L The domains are non-covalently associated. Alternatively, the antibody can be another fragment. Other fragments can be made, including using recombinant techniques. For example, Fab molecules can be displayed on phage when one of the heavy or light chains is fused to g3 capsid protein and the complementary chain is excreted into the periplasm as a soluble molecule. The two chains can be encoded in the same or different replicons, and the two antibody chains in each Fab molecule are post-translationally assembled, and the dimer is incorporated into the phage particle via linkage of g3p to one of the chains (see, e.g., U.S. Patent No. 5,733,743, which is incorporated herein by reference in its entirety). scFv antibodies, as well as several other structures that convert naturally aggregated, but chemically isolated, polypeptide light and heavy chains derived from antibody V regions into molecules that fold into three-dimensional structures substantially similar to the structure of an antigen-binding site, are known to those of skill in the art (see, e.g., U.S. Pat. Nos. 5,091,513, 5,132,405, and 4,956,778, all of which are incorporated herein by reference in their entirety). Particularly preferred antibodies may include all those displayed on phage or produced by recombinant techniques using vectors, where the chains are soluble proteins, e.g., scFv, Fv, Fab, (Fab') 2Antibodies may also include diabodies and minibodies.

[0095] The antibody may also include a heavy chain dimer, for example an antibody from a camelid. H The regions in the heavy chains that normally contact the light chains are altered to hydrophilic amino acid residues in camelids because they do not have to make hydrophobic interactions with the light chains. H The domain is V HH is called a domain.

[0096] In camelids, the diversity of the antibody repertoire is largely determined by V H or V HH The region can be determined by complementarity determining regions (CDRs) 1, 2, and 3. HH The CDR3 in the region can be characterized by a relatively long length, averaging 16 amino acids (Muyldermans et al., 1994, Protein Engineering 7(9): 1129, which is incorporated herein by reference in its entirety). This is in contrast to the CDR3 regions of antibodies of many other species. For example, the CDR3 in the mouse V H The CDR3 of may have an average of 9 amino acids.

[0097] Libraries of camelid-derived antibody variable regions that maintain the in vivo diversity of camelid variable regions can be generated, for example, by the methods disclosed in U.S. Patent Application Publication No. US20050037421, published February 17, 2005, which is incorporated herein by reference in its entirety.

[0098] The terms "functional fragment," "antigen-binding portion," "antigen-binding fragment," "antigen-binding domain," or "antibody fragment" may be used interchangeably herein to refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Exemplary antigen-binding fragments include, but are not limited to, Fab, Fab', (Fab') 2, Fv, scFv, dsFv, variable heavy domain, variable light domain, variable NAR domain, bispecific scFv, bispecific Fab 2 , Trispecific Fab 3 , AVIMER®, minibodies, diabodies, maxibodies, camelids, VHHs, intrabodies, fusion proteins comprising antibody portions (e.g., domain antibodies), single-chain binding polypeptides, scFv-Fc, or Fab-Fc.

[0099] In some instances, the antibody or functional fragment thereof may comprise an isolated antibody or functional fragment thereof, a purified antibody or functional fragment thereof, a recombinant antibody or functional fragment thereof, a modified antibody or functional fragment thereof, or a synthetic antibody or functional fragment thereof. It will be understood that the antibodies described herein may be modified as described herein or as known in the art. In some instances, the antibodies and functional fragments described herein may be partially or wholly synthetically produced. The antibody or functional fragment thereof may be a polypeptide or protein having a binding domain, which may be an antigen binding domain or may be homologous thereto. In some instances, the antibody or functional fragment thereof may be produced in a suitable in vivo animal model and then isolated and / or purified.

[0100] The term "Fc region" can be used to define the C-terminal region of an immunoglobulin heavy chain. The "Fc region" can be a native sequence Fc region or a variant Fc region. The Fc region of an immunoglobulin can generally comprise two constant domains, CH2 and CH3.

[0101] "Antibody" may include, but is not limited to, a monoclonal antibody, a polyclonal antibody, a chimeric antibody, a bispecific antibody, a multispecific antibody, a heteroconjugate antibody, a humanized antibody, a human antibody, a deimmunized antibody, a variant thereof, a fusion thereof, an immunoconjugate thereof, an antigen-binding fragment thereof, a functional fragment thereof, and / or any other modified configuration of an immunoglobulin molecule containing an antigen recognition site of the required specificity, such as glycosylation variants of the antibody, amino acid sequence variants of the antibody, and / or covalently modified antibodies.

[0102] The antibody may be a human antibody. A human antibody may be an antibody having an amino acid sequence corresponding to that of an antibody produced by a human and / or produced using any of the techniques for producing human antibodies known in the art or disclosed herein. This definition of a human antibody includes an antibody that comprises at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody that comprises a mouse light chain and a human heavy chain polypeptide. Human antibodies can be generated using a variety of techniques known in the art. In one embodiment, human antibodies are selected from phage libraries, where the phage libraries express human antibodies (Vaughan et al., 1996, Nature Biotechnology, 14:309-314; Sheets et al., 1998, PNAS USA, 95:6157-6162; Hoogenboom and Winter, 1991, J. Mol. Biol., 227:381; Marks et al., 1991, J. Mol. Biol., 222:581). Human antibodies can also be produced by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. This approach is described in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016. Alternatively, human antibodies can be prepared by immortalizing human B-lymphocytes that produce antibodies against target antigens (such B-lymphocytes can be retrieved from subjects or immunized in vitro). See, for example, Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985); Boerner et al., 1991, J. Immunol., 147 (1):86-95; and U.S. Patent No. 5,750,373.

[0103] As used herein, the term "binding specificity" of an antibody or "antibody specificity" may refer to the identity of the antigen to which the antibody binds, preferably, the identity of the epitope to which the antibody binds.

[0104] As used herein, the term "chimeric polynucleotide" can mean that a polynucleotide contains regions that are wild type and regions that are mutated. It can also mean that a polynucleotide contains wild type regions from one polynucleotide and wild type regions from another, related polynucleotide.

[0105] As used herein, the term "complementarity determining region" or "CDR" may refer to the art-recognized term exemplified by Kabat and Chothia. CDR is also generally known as hypervariable region or hypervariable loop (Chothia and Lesk (1987) J Mol. Biol. 196: 901; Chothia et al. (1989) Nature 342: 877; EA Kabat et al., Sequences of Proteins of Immunological Interest (National Institutes of Health, Bethesda, Md.) (1987); and Tramontano et al. (1990) J Mol. Biol. 215: 175, all of which are incorporated herein by reference in their entirety). "Framework region" or "FR" may refer to the region of V domain adjacent to CDR. The positions of the CDRs and framework regions can be determined using various definitions well known in the art, e.g., Kabat, Chothia, the international ImMunoGeneTics database (IMGT), and AbM (see, e.g., Johnson et al., supra; Chothia & Lesk, 1987, Canonical structures for the hypervariable regions of immunoglobulins. J. Mol. Biol. 196, 901-917; Chothia C. et al., 1989, Conformations of immunoglobulin hypervariable regions. Nature 342, 877-883; Chothia C. et al., 1992, structural repertoire of the human VH segments J. Mol. Biol. 227, 799-817; Al-Lazikani et al., J. Mol. Biol 1997, 273(4)).Definitions of antigen binding sites are also described in: Ruiz et al., IMGT, the international ImMunoGeneTics database. Nucleic Acids Res., 28, 219-221 (2000); and Lefranc, M.-P. IMGT, the international ImMunoGeneTics database. Nucleic Acids Res. Jan 1;29(1):207-9 (2001); MacCallum et al, Antibody-antigen interactions: Contact analysis and binding site topography, J. Mol. Biol., 262 (5), 732-745 (1996); and Martin et al, Proc. Natl Acad. Sci. USA, 86, 9268-9272 (1989); Martin, et al, Methods Enzymol., 203, 121-153, (1991); Pedersen et al, Immunomethods, 1, 126, (1992); and Rees et al, In Sternberg MJE (ed.), Protein Structure Prediction. Oxford University Press, Oxford, 141-172 1996, all of which are incorporated herein by reference in their entireties).

[0106] As used herein, the term "affinity" may refer to the equilibrium constant for the reversible binding of two agents, and is defined as the binding affinity (K D In some cases, K D k may refer to the rate constant for dissociation of an antibody from the antibody or antigen-binding fragment / antigen complex. off k may refer to the rate constant for association of an antibody, antigen-binding domain, or antigen-binding fragment to an antigen, onThe binding affinity may be expressed as a ratio to the binding affinity of the antibody, antigen-binding domain, or antigen-binding fragment thereof. Binding affinity may be determined using methods known in the art, including, for example, surface plasmon resonance (SPR; Biacore), Kinexa biosensor, scintillation proximity assay, enzyme-linked immunosorbent assay (ELISA), ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence transfer, yeast display, or any combination thereof. Binding affinity may also be screened using a suitable bioassay. The binding affinity (K D) is less than 600nM, less than 590nM, less than 580nM, less than 570nM, less than 560nM, less than 550nM, less than 540nM, less than 530nM, less than 520nM, less than 510nM, less than 500nM, less than 490nM, less than 480nM, less than 470nM, less than 460nM, less than 450nM, less than 440nM, less than 430nM, less than 420nM, less than 410nM, less than 400nM, less than 390nM, less than 380nM, less than 370nM, less than 360nM, less than 350nM, less than 340nM, less than 330nM, less than 320nM, less than 310nM, less than 300nM full, less than 290nM, less than 280nM, less than 270nM, less than 260nM, less than 250nM, less than 240nM, less than 230nM, less than 220nM, less than 210nM, less than 200nM, less than 190nM, less than 180nM, less than 170nM, less than 160nM, less than 150nM, less than 140nM, less than 130nM, less than 120nM, less than 110nM, less than 100nM, less than 90nM, less than 80nM, less than 70nM, less than 50nM, less than 50nM, less than 49nM, less than 48nM, less than 47nM, less than 46nM, less than 45nM, less than 44nM, less than 43nM, 42n Less than M, Less than 41nM, Less than 40nM, Less than 39nM, Less than 38nM, Less than 37nM, Less than 36nM, Less than 35nM, Less than 34nM, Less than 33nM, Less than 32nM, Less than 31nM, Less than 30nM, Less than 29nM, Less than 28nM, Less than 27nM, Less than 26nM, Less than 25nM, Less than 24nM, Less than 23nM, Less than 22nM, Less than 21nM, Less than 20nM, Less than 19nM, Less than 18nM, Less than 17nM, Less than 16nM, Less than 15nM, Less than 14nM, Less than 13nM, Less than 12nM, Less than 11nM, Less than 10nM, Less than 9nM, Less than 8nM, Less than 7nM, Less than 6nM less than, less than 5nM, less than 4nM, less than 3nM, less than 2nM, less than 1nM, less than 990pM, less than 980pM, less than 970pM, less than 960pM, less than 950pM, less than 940pM, less than 930pM, less than 920pM, less than 910pM, less than 900pM, less than 890pM, less than 880pM, less than 870pM, less than 860pM, less than 850pM, less than 840pM, less than 830pM, less than 820pM, less than 810pM, less than 800pM, less than 790pM, less than 780pM, less than 770pM, less than 760pM, less than 750pM, less than 740pM, less than 730pM,Less than 720pM, Less than 710pM, Less than 700pM, Less than 690pM, Less than 680pM, Less than 670pM, Less than 660pM, Less than 650pM, Less than 640pM, Less than 630pM, Less than 620pM, Less than 610pM, Less than 600pM, Less than 590pM, Less than 580pM, Less than 570pM, Less than 560pM, Less than 550pM, Less than 540pM, Less than 530pM, Less than 520pM, Less than 510pM, Less than 500pM, Less than 490pM, Less than 480pM, Less than 470pM, Less than 460pM, Less than 450pM, Less than 440pM, 43 It may be less than 0 pM, less than 420 pM, less than 410 pM, less than 400 pM, less than 390 pM, less than 380 pM, less than 370 pM, less than 360 pM, less than 350 pM, less than 340 pM, less than 330 pM, less than 320 pM, less than 310 pM, less than 300 pM, less than 290 pM, less than 280 pM, less than 270 pM, less than 260 pM, less than 250 pM, less than 240 pM, less than 230 pM, less than 220 pM, less than 210 pM, less than 200 pM, less than 190 pM, less than 180 pM, less than 170 pM, or any integer therebetween.

[0107] An antibody can selectively bind to a target if it can bind to the target with greater affinity, avidity, more readily, and / or for a longer period of time than it binds to other substances. For example, an anti-EPO antibody or functional fragment thereof that selectively binds to an EPO protein is an antibody or functional fragment thereof that can bind to this target with greater affinity, avidity, more readily, and / or for a longer period of time than it binds to a protein that is not an EPO protein.

[0108] As used herein, the term "EPO analog" may refer to a polypeptide having modifications of its polypeptide structure or a polypeptide having a shorter, longer, and / or different amino acid sequence compared to wild-type human erythropoietin, all of which bind to hetero-EPOR or homo-EPOR with high affinity. An EPO analog may be an antagonist or agonist of hetero-EPOR or homo-EPOR. An EPO analog may block the activity of hetero-EPOR or the activity of homo-EPOR. An EPO analog may activate hetero-EPOR without activating homo-EPOR. An EPO analog may activate homo-EPOR without activating hetero-EPOR. An EPO analog may inhibit hetero-EPOR without inhibiting homo-EPOR. An EPO analog may inhibit homo-EPOR without inhibiting hetero-EPOR.

[0109] Whenever the term "at least" or "greater" precedes a first number in a series of two or more numbers, the term "at least" or "greater" applies to every one of the numbers in the series.

[0110] The term "heterologous" can refer to an amino acid or nucleotide sequence that is not found in nature in association with the amino acid or nucleotide sequence with which it is related.

[0111] As used herein, the term "immunotherapy" can refer to a particular therapy with drugs or other agents that aim to modulate components of the immune system, such as antibodies or immune cells, or stimulate, inhibit, or otherwise modulate the immune system. For example, "immunotherapy" can refer to checkpoint inhibitor therapy, adoptive cell therapy, and / or autologous or allogeneic CAR T cell therapy.

[0112] Whenever the term "less than" or "less than" precedes the first number of a series of two or more numbers, the term "less than" or "less than" applies to each and every one of the numbers in the series.

[0113] The term "polynucleotide" may refer to a polymer composed of nucleotide units. Polynucleotides may include naturally occurring nucleic acids, such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), as well as nucleic acid analogs. Nucleic acid analogs may include non-naturally occurring bases, nucleotides that participate in linkages with other nucleotides other than naturally occurring phosphodiester bonds, or / and bases attached through linkages other than phosphodiester bonds. Non-limiting examples of nucleotide analogs may include phosphorothioates, phosphorodithioates, phosphorotriesters, phosphoramidates, boranophosphates, methyl phosphonates, chiral-methyl phosphonates, 2-O-methyl ribonucleotides, peptide nucleic acids (PNAs), and the like. Such polynucleotides may be synthesized, for example, using an automated DNA synthesizer. The term "nucleic acid molecule" may refer to larger polynucleotides. The term "oligonucleotide" may refer to shorter polynucleotides. In certain embodiments, an oligonucleotide may contain about 50 or fewer nucleotides. Where a nucleotide sequence is represented by a DNA sequence (i.e., A, T, G, C), this is understood to also include RNA sequences in which "U" replaces "T" (i.e., A, U, G, C).

[0114] The term "polypeptide" may refer to a polymer composed of natural or / and unnatural amino acid residues, naturally occurring structural variants thereof, or / and synthetic non-naturally occurring analogs thereof linked via peptide bonds. Synthetic polypeptides can be synthesized, for example, using an automated polypeptide synthesizer. Polypeptides can also be produced recombinantly in cells expressing a nucleic acid sequence encoding the polypeptide. The term "protein" may refer to larger polypeptides. The term "peptide" may refer to shorter polypeptides. In certain embodiments, peptides may contain about 50 or less, about 40 or less, or about 30 or less amino acid residues. Polypeptides may include antibodies and fragments thereof. Conventional notation is used herein to depict polypeptide sequences, with the left-hand end of a polypeptide sequence being the amino (N)-terminus and the right-hand end of a polypeptide sequence being the carboxyl (C)-terminus.

[0115] A polypeptide may include one or more modifications that may occur during the process of synthesis or cellular production of the polypeptide, such as one or more post-translational modifications, whether or not the one or more modifications are intentional. Modifications may include, but are not limited to, glycosylation (e.g., N-linked glycosylation and O-linked glycosylation), lipidation, phosphorylation, sulfation, acetylation (e.g., acetylation of the N-terminus), amidation (e.g., amidation of the C-terminus), hydroxylation, methylation, formation of intramolecular or intermolecular disulfide bonds, formation of lactams between two side chains, formation of pyroglutamate, carbamylation, and ubiquitination. As another example, a polypeptide may be attached to a natural polymer (e.g., a polysaccharide) or a synthetic polymer (e.g., polyethylene glycol [PEG]), may be lipidated (e.g., C-terminal glycosylation, etc.). 8 ~C 20 PEGylation can be performed by PEGylation of a polypeptide, such as a PEG-2 ester, acylated with an acyl group, or labeled with a detectable agent (e.g., a radionuclide, a fluorescent dye, or an enzyme). Pegylation can increase protease resistance, stability and half-life, increase solubility, and reduce aggregation of the polypeptide.

[0116] The term "conservative substitution" can refer to the replacement of an amino acid in a polypeptide with a functionally, structurally or chemically similar natural or non-natural amino acid. In certain embodiments, the following groups each contain natural amino acids that are conservative substitutions for one another: 1) glycine (Gly / G), alanine (Ala / A); 2) isoleucine (Ile / I), leucine (Leu / L), methionine (Met / M), valine (Val / V); 3) phenylalanine (Phe / F), tyrosine (Tyr / Y), tryptophan (Trp / W); 4) serine (Ser / S), threonine (Thr / T), cysteine ​​(Cys / C); 5) asparagine (Asn / N), glutamine (Gln / Q); 6) Aspartic acid (Asp / D), glutamic acid (Glu / E); and 7) Arginine (Arg / R), Lysine (Lys / K), Histidine (His / H).

[0117] In further embodiments, the following groups each contain naturally occurring amino acids that are conservative substitutions for one another: 1) Non-polar: Ala, Val, Leu, Ile, Met, Pro (proline / P), Phe, Trp; 2) Hydrophobic: Val, Leu, Ile, Phe, Tyr, Trp; 3) Aliphatic: Ala, Val, Leu, Ile; 4) Aromatic: Phe, Tyr, Trp, His; 5) Uncharged polar or hydrophilic: Gly, Ala, Pro, Ser, Thr, Cys, Asn, Gln, Tyr (tyrosine is sometimes considered a hydrophobic amino acid with a polar side chain); 6) Aliphatic hydroxyl or sulfhydryl containing: Ser, Thr, Cys; 7) Amide-containing: Asn, Gln; 8) Acidic: Asp, Glu; 9) Basic: Lys, Arg, His; and 10) Small size: Gly, Ala, Ser, Cys.

[0118] In other embodiments, the amino acids may be grouped as shown below: 1) Hydrophobicity: Val, Leu, Ile, Met, Phe, Trp, Tyr; 2) Aromatic: Phe, Tyr, Trp, His; 3) Neutral hydrophilic: Gly, Ala, Pro, Ser, Thr, Cys, Asn, Gln; 4) Acidic: Asp, Glu; 5) Basic: Lys, Arg, His; and 6) Residues that affect main-chain orientation: Pro, Gly.

[0119] A polypeptide that has one or more modifications relative to a parent polypeptide may be referred to as an "analog," "derivative," or "variant" of the parent polypeptide, as appropriate.

[0120] The present disclosure encompasses pharma- ceutically acceptable salts of the polypeptides, including those that have a positive net charge, those that have a negative net charge, and those that have no net charge.

[0121] The term "pharmaceutically acceptable" may refer to a substance (e.g., an active ingredient or additive) that is suitable for use in contact with the tissues and organs of a subject without undue irritation, allergic reaction, immunogenicity and toxicity, and is effective for its intended use commensurate with a reasonable benefit / risk ratio. A "pharmaceutically acceptable" additive or carrier of a pharmaceutical composition is also compatible with the other components of the composition. The term "pharmaceutically acceptable" may refer to a material, such as a carrier or diluent, that does not abrogate the biological activity or properties of the compound and is relatively non-toxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained. A pharma- ceutically acceptable additive may refer to any pharma- ceutically acceptable ingredient in a pharmaceutical composition that has no therapeutic activity and is non-toxic to the subject to which it is administered, such as a disintegrant, binder, filler, solvent, buffer, tonicity agent, stabilizer, antioxidant, surfactant, carrier, diluent, excipient, preservative, or lubricant used in the formulation of a pharmaceutical product.The pharmaceutical composition may be formulated in a conventional manner using one or more pharma- ceutically acceptable inactive ingredients that can facilitate administration of the compound to an organism and facilitate processing of the active compound into a preparation that can be used pharma- ceutically.Suitable formulations depend on the route of administration selected, and summaries of pharmaceutical compositions can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, HA and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, NY, 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins 1999), which are incorporated herein by reference. In some embodiments, pharmaceutical compositions can be formulated by dissolving the active agent (e.g., the EPOR agonist or antagonist described herein) in an aqueous solution for injection into diseased tissue or diseased cells. In some embodiments, pharmaceutical compositions can be formulated by dissolving an active agent (e.g., an EPOR agonist or antagonist described herein) in an aqueous solution for direct injection into diseased tissue or cells.

[0122] The term "stringent hybridization conditions" may refer to hybridization in 5xSSC, 50% formamide, at a temperature of 42°C, and washing of the filters in 0.2xSSC, at 60°C (1xSSC is 0.15M NaCl, 0.015M sodium citrate). Stringent hybridization conditions include low ionic strength and high temperature for washing, e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50° C.; denaturing agents, e.g., formamide, e.g., 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer pH 6.5 with 750 mM sodium chloride, 75 mM sodium citrate; or 50% formamide at 42° C. Hybridization using formamide, 5x SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5x Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate, with washes in 0.2x SSC (sodium chloride / sodium citrate) at 42°C and in 50% formamide at 55°C, followed by a high stringency wash consisting of 0.1x SSC containing EDTA at 55°C.

[0123] The term "subject" may refer to animals including, but not limited to, mammals, such as primates (e.g., humans, chimpanzees, or monkeys), rodents (e.g., rats, mice, guinea pigs, gerbils, or hamsters), lagomorphs (e.g., rabbits), swines (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), or felines (e.g., cats). Additional examples of mammals may include, but are not limited to, any member of the class Mammalia: humans, non-human primates, such as chimpanzees, and other ape and monkey species; livestock animals, such as cows, horses, sheep, goats, pigs; domestic animals, such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice, and hamsters, and the like. In some cases, the mammal is a human. In some instances, the subject is an adult, a child, or an infant. In some cases, the subject may be an animal. In some cases, the animal may include humans and non-human animals. In one embodiment, the non-human animal may be a non-human mammal as described herein. In some instances, the subject is a companion animal. In some instances, the subject is a cat, a dog, or a rodent.

[0124] The term "substantially homologous" or "substantially identical" in the context of two polypeptides or polynucleotides may refer to two or more sequences or subsequences that have at least about 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% amino acid or nucleic acid residue sequence identity when compared and aligned for maximum correspondence as measured using a sequence comparison algorithm or by visual inspection. The term "substantially homologous" or "substantially identical" may mean at least about 70% amino acid or nucleic acid residue sequence identity. The term "substantially homologous" or "substantially identical" may mean at least about 85% amino acid or nucleic acid residue sequence identity. Substantial homology or identity may exist over a region of the sequence that is at least about 20, 30, 40, 50, 100, 150, or 200 residues in length. The sequences can be substantially homologous or identical over the entire length of either or both of the comparison biopolymers.

[0125] Optimal alignment of sequences for comparison can be performed, for example, by the local homology algorithm of Smith and Waterman, Adv. Appl. Math., 2:482 (1981); by the homology alignment algorithm of Needleman and Wunsch, J. Mol. Biol., 48:443 (1970); by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 85:2444 (1988); by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, Madison, Wisconsin); or by visual inspection.

[0126] One example of a useful algorithm is PILEUP. PILEUP uses progressive pairwise alignment to create a multiple sequence alignment from a group of related sequences to show the relationship and percent sequence identity. It also plots a tree or dendrogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle, J. Mol. Evol., 35:351-360 (1987). The method used is similar to that described by Higgins and Sharp, CABIOS, 5:151-153 (1989). The program can align up to about 300 sequences, each with a maximum length of about 5,000 nucleotides or amino acids. The multiple alignment procedure begins with a pairwise alignment of the two most similar sequences, resulting in a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive pairwise alignments. The program is run by designating specific sequences and their amino acid or nucleotide coordinates for the region of sequence comparison, as well as designating program parameters. For example, a reference sequence can be compared to other test sequences to determine sequence identity relative percentage using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps. Another algorithm that is useful for creating multiple alignments of sequences is Clustal W (see, for example, Thompson et al., Nucleic Acids Research, 22:4673-4680

[1994] ).

[0127] Another example of an algorithm suitable for determining percent sequence identity and percent sequence similarity is the BLAST algorithm, which is described in Altschul et al., J. Mol. Biol., 215:403-410 (1990). Software for performing BLAST analysis is publicly available through the National Center for Biotechnology Information. This algorithm involves first identifying high-scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence that match or meet some positive threshold score T when aligned with words of the same length in the database sequence. T is referred to as the neighbor word score threshold (Altschul 1990). These initial neighbor word hits serve as seeds to initiate searches to find longer HSPs that contain them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is ​​used to calculate the cumulative score. Extension of the word hits in each direction is halted when: the cumulative alignment score falls by an amount X from its maximum achieved value; when the cumulative score becomes zero or less due to the accumulation of one or more negative scoring residue alignments; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults, e.g., a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands.For amino acid sequences, the BLASTP program uses as defaults, for example, a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 89:10915

[1989] ).

[0128] In addition to calculating percent sequence identity, the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul, Proc. Natl. Acad. Sci. USA, 90:5873-5787

[1993] ). One measure of similarity provided by the BLAST algorithm is the minimum sum probability [P(N)], which provides an indication of the probability that a match between two nucleotide or amino acid sequences would occur by chance. In certain embodiments, a polynucleotide is considered to be similar to a reference sequence if the minimum sum probability in the comparison of the test polynucleotide to the reference polynucleotide is less than about 0.1, 0.01, or 0.001.

[0129] A polypeptide may be substantially homologous or identical to a second polypeptide if the two polypeptides differ only by conservative amino acid substitutions. Two nucleic acid sequences may be substantially homologous or identical if the two polynucleotides hybridize to each other under stringent conditions, or highly stringent conditions, as described herein.

[0130] The term "therapeutically effective amount" may refer to an amount of a compound that, when administered to a subject, is sufficient to prevent, reduce the risk of developing, delay the onset, slow the progression, or cause regression of the medical condition being treated, or to alleviate to some extent the medical condition or one or more symptoms or complications of that condition. The term "therapeutically effective amount" may also refer to an amount of a compound that is sufficient to elicit the biological or medical response of a cell, tissue, organ, system, animal, or human that is desired by a researcher, veterinarian, physician, or clinician.

[0131] The terms "treat", "treating" and "treatment" may include alleviating, ameliorating, or arresting one or more symptoms or complications associated with a medical condition or condition, alleviating, ameliorating, or eradicating one or more causes of a condition, preventing additional symptoms, inhibiting a disease or condition, e.g., halting the onset of a disease or condition, relieving a disease or condition, causing regression of a disease or condition, alleviating a condition caused by a disease or condition, or prophylactically and / or therapeutically arresting the symptoms of a disease or condition. In some embodiments, treating a disease or condition may include reducing the size of diseased tissue or diseased cells. In some embodiments, treating a disease or condition in a subject may include increasing the survival of the subject. In some embodiments, treating a disease or condition may include reducing or improving the severity of a disease, delaying the onset of a disease, inhibiting the progression of a disease, reducing the length of hospitalization or hospitalization for a subject, improving the quality of life of a subject, reducing the number of symptoms associated with a disease, reducing or improving the severity of symptoms associated with a disease, reducing the duration of symptoms associated with a disease, preventing the recurrence of symptoms associated with a disease, inhibiting the onset or onset of symptoms of a disease, or inhibiting the progression of symptoms associated with a disease. In some embodiments, treating a cancer may include reducing the size of a tumor or increasing the survival of a patient with cancer. Reference to "treatment" of a medical condition may include prevention of the condition. The terms "prevent", "preventing" and "prevention" may include eliminating one or more symptoms or complications associated with a medical condition or condition, reducing the risk of it occurring, and delaying its onset.

[0132] Erythropoietin (EPO) analogs or engineered EPO In some aspects, at least eight EPO analogs are provided herein that can be made or engineered. In some embodiments, EPO analogs can also be referred to as engineered EPOs. EPO analogs or engineered EPOs can bind to hetero-EPORs and cannot bind to homo-EPORs, and can be either agonists or antagonists of hetero-EPORs. Other EPO analogs or engineered EPOs can bind to homo-EPORs and cannot bind to hetero-EPORs, and can be either agonists or antagonists of homo-EPORs. EPO analogs or engineered EPOs can bind to both homo-EPORs and hetero-EPORs, and can be agonists for both, antagonists for both, or agonists for one and antagonists for the other. The term EPO analogs or engineered EPOs can include the EPO shown in SEQ ID NO:1.

[0133] Erythropoietin (EPO) is a pleiotropic cytokine glycoprotein that was first identified as a regulator of red blood cell production in response to hypoxia. The mature human 165 amino acid long EPO protein sequence is shown in SEQ ID NO:1. The amino acid sequence and nucleic acid sequence of human EPO, including the signal peptide sequence, are shown in FIG. 34. The amino acid residue position numbers in the engineered EPO variants and analogs described herein may not include the amino acid residue position numbers of the signal peptide. In some embodiments, the amino acid residue positions of the engineered EPO variants and analogs described herein may be determined by alignment with SEQ ID NO:1.

[0134] EPO contains four alpha helices (A, B, C, and D) that form a compact globular structure. Human recombinant erythropoietin (expressed in mammalian cells) contains three N-linked and one O-linked oligosaccharide chain, which together constitute about 40% of the total molecular weight of the glycoprotein. N-linked glycosylation occurs at asparagine residues (Asn) located at positions 24, 38, and 83, whereas O-linked glycosylation occurs at serine residue (Ser) located at position 126.

[0135] Three of the helices (A, C, and D) are involved in two binding sites with homo-EPOR. Helix B is involved in the interaction with hetero-EPOR. The interaction interface of EPO and homo-EPOR, which contains a high affinity site (site 1) and a low affinity site (site 2), has been mapped in the crystal structure (Syed et al, Nature. 1998:395(6701):511-6). Site 1 is characterized by a central hydrophobic binding pocket flanked at opposite ends by hydrophilic interactions involving amino acid residues S9, R10, E13, L16, L17, K20, T44, K45, V46, N47, F48, Y49, K52, R131, I133, K140, R143, N147, R150, G151, K154, and L155. Mutations K20E, T44I, K45I, V46A, F48G, R143A, R150A, R150Q, L155A, and L155N were shown to result in less than 5-fold reduction in in vitro biological activity, while mutations K45I, N147K, R150E, and G151A were shown to result in less than 50-fold reduction in activity. Mutations at site 1 result in a significant reduction in affinity for homo-EPOR. Site 2 includes amino acid residues L5, D8, R10, V11, R14, Y15, Q78, D96, K97, V99, S100, R103, S104, T107, L108, and R110. Mutations V11S, R14A, R14E, Y15I, K97A, K97E, S104A, L108A, and R110E were shown to have less than 5-fold reduced in vitro bioactivity, while mutations R14Q, S100E, S100T, R103A, R103E, R103H, R103N, R103Q, S104I, and L108K were shown to have less than 50-fold reduced activity. EPO analogs or engineered EPOs with mutations at site 2 may maintain high binding affinity to homo-EPOR but lose signaling activity. These EPO variants with mutations at sites 1 or 2 but no mutations in helix B should have activity with hetero-EPOR.

[0136] Helix B is not involved in binding to homo-EPOR. Hetero-EPOR has an EPOR chain and a CD131 chain. CD131 may be a homodimer resulting in a heterohexameric receptor with EPO receptor chains and higher order dodecameric complexes. Helix B of EPO may be important for EPOR / CD131 (hetero-EPOR) binding.

[0137] Carbamylated EPO (CEPO) is a chemically modified EPO analog in which the Lys residues present in helices A, C, and D are modified by carbamylation. Helix B does not have a Lys residue and is therefore not modified. CEPO has been shown to be equally active as EPO with hetero-EPOR, but not with homo-EPOR. Other modifications of the Lys residues in helices A, C, and D can be used to create EPO analogs or engineered EPOs that interact with hetero-EPOR but not with homo-EPOR. For example, PEG can be attached to the Lys residues in EPO using well-known PEGylation reagents to create chemically modified EPO analogs that improve serum half-life and the preference for activating hetero-EPOR and not activating homo-EPOR. The PEG can be a low molecular weight PEG (e.g., 5000 daltons), and the Lys reactive groups on the PEG can be used to modify all or most or all of the Lys residues in helices A, C, and D. Similarly, other chemical modifications can be made in EPO that attach other moieties to the Lys residues, resulting in other chemical derivatives that can bind to hetero-EPORs and cannot bind to homo-EPORs. In some embodiments, one or more Lys residues on the EPO analogs or engineered EPOs described herein can be carbamylated. In some embodiments, all Lys residues on the EPO analogs or engineered EPOs described herein can be carbamylated. In some embodiments, the Lys residues on the EPO analogs or engineered EPOs described herein can be non-carbamylated.

[0138] Peptide analogs of helix B also showed similar activity against CEPO. Activation of hetero-EPOR leads to phosphorylation of the intracellular domain of CD131, but not EPOR. Activation of both homo-EPOR and hetero-EPOR leads to JAK2 and STAT5 activation. For example, QEQLERALNSS (SEQ ID NO: 2), an 11 amino acid linear peptide that mimics the three-dimensional structure of the outer aqueous face of the helix B peptide, is such a peptide analog that activates hetero-EPOR. This peptide can be cyclized to make a cyclic peptide because the N-terminal residue is glutamine. The cyclic peptide also activates hetero-EPOR.

[0139] EPO has previously been expressed as a functional Fc fusion protein to enhance its in vivo half-life (Schriebl et al, Protein Expr Purif. 2006, 49(2):265-75; Shi et al, PLoS One, 2013 8(8):e72673). Other methods, including albumin fusion, PEGylation, or engineering more glycosylation sites, can improve in vivo PK properties (Joung et al, Protein Expr Purif. 2009:68(2):137-45; Elliott et al, Nat Biotechnol. 2003:21(4):414-21). The EPO variants described herein can be expressed as Fc fusion proteins and tested for receptor specificity. They can also be expressed as albumin fusions or in other modalities (e.g., PEGylation).

[0140] In some aspects, human EPO analogs can be created or engineered to bind hetero-EPOR (as antagonists) and not to homo-EPOR. In some embodiments, these EPO analogs or engineered EPOs can be expressed as Fc fusion proteins. Surface residues in helix B (Q58, E62, Q65, L69, E72, R76, A79, L80, N83, S84, and S85) can play important roles in interacting with hetero-EPOR and can be mutated / substituted. For example, the nucleic acid encoding helix B can be mutated using alanine scanning and / or saturation mutagenesis. Mutations in EPO that can bind hetero-EPOR and cause reduced activation of hetero-EPOR (but still bind hetero-EPOR) can be combined with the above-described mutations that reduce EPO analog binding to homo-EPOR. The resulting EPO analog or engineered EPO can antagonize the hetero-EPOR and can have reduced binding to, or be unable to bind to, the homo-EPOR.

[0141] In some aspects, the human EPO analog or engineered EPO described herein may contain at least one amino acid substitution or mutation.In some embodiments, the human EPO analog or engineered EPO described herein may contain at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, or at least ten amino acid substitutions.For example, the human EPO analog or engineered EPO described herein may contain at least one amino acid substitution or mutation at amino acid residues K20, N24, N38, K45, K52, Q58, E62, Q65, L69, E72, R76, L80, N83, S84, S85, K97, R103, K116, K140, N147, R150, G151, K152, or K154, or a combination thereof. In some embodiments, the human EPO analogs or engineered EPOs described herein may contain at least one amino acid substitution or mutation at amino acid residues K20, N24, N38, K45, K52, Q58, E62, Q65, L69, E72, R76, L80, N83, S84, S85, K97, R103, K116, K140, N147, R150, G151, K152, or K154, or a combination thereof. In this embodiment, at least one amino acid, including K20, N24, N38, K45, K52, Q58, E62, Q65, L69, E72, R76, L80, N83, S84, S85, K97, R103, K116, K140, N147, R150, G151, K152, or K154, or a combination thereof, may be substituted or mutated to any other amino acid (e.g., A, R, N, D, C, Q, E, G, H, I, L, K, M, F, P, S, T, W, Y, or V). In some embodiments, the amino acid residue position may be determined by alignment with SEQ ID NO:1.For example, a human EPO analog or engineered EPO described herein can contain at least one amino acid substitution including K20A, N24Q, N24A, N24S, N38Q, N38A, N38S, K45A, K52A, Q58A, E62R, E62A, Q65A, L69A, E72A, R76E, R76A, L80A, N83Q, N83A, N83S, S84A, S85A, K97A, K116A, G151A, R103A, K45D, N147K, R150E, Q65A, E72R, N83A, K140A, K152A, or K154A, or a combination thereof. In some embodiments, human EPO analogs or engineered EPOs containing at least one amino acid substitution or mutation as described herein can be agonists or antagonists of hetero-EPOR.In some embodiments, human EPO analogs or engineered EPOs containing at least one amino acid substitution or mutation as described herein can be agonists or antagonists of homo-EPOR.

[0142] In some embodiments, the human EPO analog or engineered EPO may include R103A. In some embodiments, the human EPO analog or engineered EPO may include K45D. In some embodiments, the human EPO analog or engineered EPO may include N147K. In some embodiments, the human EPO analog or engineered EPO may include R150E. In some embodiments, the human EPO analog or engineered EPO may include Q58A. In some embodiments, the human EPO analog or engineered EPO may include E62R. In some embodiments, the human EPO analog or engineered EPO may include E62A. In some embodiments, the human EPO analog or engineered EPO may include Q65A. In some embodiments, the human EPO analog or engineered EPO may include L69A. In some embodiments, the human EPO analog or engineered EPO may include E72R. In some embodiments, the human EPO analog or engineered EPO may include E72A. In some embodiments, the human EPO analog or engineered EPO may include R76E. In some embodiments, the human EPO analog or engineered EPO may include R76A. In some embodiments, the human EPO analog or engineered EPO may include L80A. In some embodiments, the human EPO analog or engineered EPO may include N83A. In some embodiments, the human EPO analog or engineered EPO may include S84A. In some embodiments, the human EPO analog or engineered EPO may include S85A. In some embodiments, the human EPO analog or engineered EPO may include K97A. In some embodiments, the human EPO analog or engineered EPO may include K116A. In some embodiments, the human EPO analog or engineered EPO may include K140A. In some embodiments, the human EPO analog or engineered EPO may include G151A. In some embodiments, the human EPO analog or engineered EPO may include K152A. In some embodiments, the human EPO analog or engineered EPO can contain K154A.In some embodiments, the human EPO analog or engineered EPO may include K45D. In some embodiments, the human EPO analog or engineered EPO may include N147K. In some embodiments, the human EPO analog or engineered EPO may include R150E. In some embodiments, the human EPO analog or engineered EPO may include K45D and R103A. In some embodiments, the human EPO analog or engineered EPO may include N147K and R103A. In some embodiments, the human EPO analog or engineered EPO may include R150E and R103A. In some embodiments, the human EPO analog or engineered EPO may include Q65A and E72R. In some embodiments, the human EPO analog or engineered EPO may include Q65A, E72R, and N83A. In some embodiments, the human EPO analog or engineered EPO may include K140A and K152A. In some embodiments, the human EPO analog or engineered EPO may include K140A, K152A, and K154A. In some embodiments, the human EPO analog or engineered EPO may include N24Q, N38Q, and N83Q. In some embodiments, the human EPO analog or engineered EPO may include E62A, Q65A, E72A, and R76A. In some embodiments, the human EPO analog or engineered EPO may include N24A, N38A, and N83A. In some embodiments, the human EPO analog or engineered EPO may include N24S, N38S, and N83S. In some embodiments, the human EPO analog or engineered EPO may include R103A and G151A. In some embodiments, the human EPO analog or engineered EPO may include K20A, K45A, and K52A. In some embodiments, the human EPO analog or engineered EPO may include K20A, K45A, K52A, K140A, K152A, and K154A. In some embodiments, the human EPO analog or engineered EPO may include K97A and K116A.In some embodiments, the human EPO analog or engineered EPO may include K20A, K45A, K52A, K97A, K116A, K140A, K152A, and K154A. In some embodiments, the human EPO analog or engineered EPO may include Q58A, Q65A, and E72R. In some embodiments, the human EPO analog or engineered EPO may include L80A, N83A, S84A, and S85A. In some embodiments, the human EPO analog or engineered EPO may include Q58A, Q65A, E72R, L80A, N83A, S84A, and S85A. In some embodiments, the human EPO analog or engineered EPO may include Q58A and L69A. In some embodiments, the human EPO analog or engineered EPO may include Q58A and L80A. In some embodiments, the human EPO analog or engineered EPO may include L69A and L80A. In some embodiments, the human EPO analog or engineered EPO may include Q58A, L69A, and L80A.

[0143] In some embodiments, the human EPO analog or engineered EPO has a sequence similar to that of any one of SEQ ID NOs: 1973-2019, which sequence is at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88% , at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity. In some embodiments, the human EPO analog or engineered EPO may comprise the amino acid sequence of any one of SEQ ID NOs: 1973-2019.

[0144] In some embodiments, the human EPO analog or engineered EPO has a sequence similar to that of any one of SEQ ID NOs: 2020-2064, at least 70%, at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 100%, at least 101%, at least 102%, at least 103%, at least 104%, at least 105%, at least 106%, at least 107%, at least 108%, at least 109%, at least 110%, at least 111%, at least 112%, at least 113%, at least 114%, at least 115%, at least 116%, at least 117%, at least 118%, at least 119%, at least 120%, at least 122%, at least 124%, at least 126%, at least 128%, at least 129%, at least 130%, at least 131%, at least 132%, at least 133%, at least 134%, at least 135%, at least 136%, at least 137%, at least 138%, at least 139%, at least 140%, at least 141%, at least 142%, at least 143%, at least 144%, at least The human EPO analog or engineered EPO may have a nucleotide sequence comprising a sequence having 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% sequence identity to any one of SEQ ID NOs: 2020-2064.

[0145] In some embodiments, an EPO analog or engineered EPO containing one or more amino acid substitutions described herein has a β-blocking activity of less than about 600 nM, less than about 590 nM, less than about 580 nM, less than about 570 nM, less than about 560 nM, less than about 550 nM, less than about 540 nM, less than about 530 nM, less than about 520 nM, less than about 510 nM, less than about 500 nM, less than about 490 nM, less than about 480 nM, less than about 470 nM, less than about 460 nM, less than about 450 nM, less than about 440 nM, less than about 430 nM, less than about 420 nM, less than about 410 nM, less than about 400 nM, less than about 300 nM, less than about 350 nM, less than about 360 nM, less than about 370 nM, less than about 380 nM, less than about 390 nM, less than about 400 nM, less than about 410 nM, less than about 420 nM, less than about 430 nM, less than about 440 nM, less than about 450 nM, less than about 460 nM, less than about 450 nM, less than about 440 nM, less than about 430 nM, less than about 420 nM, less than about 410 nM, less than about 400 nM, less than about 370 nM, less than about 390 nM, less than about 450 nM, less than about 460 nM, less than about 450 nM, less than about 440 nM, less than about 430 nM, less than about 420 nM Less than about 90 nM, less than about 380 nM, less than about 370 nM, less than about 360 nM, less than about 350 nM, less than about 340 nM, less than about 330 nM, less than about 320 nM, less than about 310 nM, less than about 300 nM, less than about 290 nM, less than about 280 nM, less than about 270 nM, less than about 260 nM, less than about 250 nM, less than about 240 nM, less than about 230 nM, less than about 220 nM, less than about 210 nM, less than about 200 nM, less than about 190 nM, less than about 180 nM, less than about 170 nM, less than about 160 nM, less than about 150 nM, less than about 140 nM, less than about 130 nM, less than about 120 nM less than about 110 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 50 nM, less than about 50 nM, less than about 49 nM, less than about 48 nM, less than about 47 nM, less than about 46 nM, less than about 45 nM, less than about 44 nM, less than about 43 nM, less than about 42 nM, less than about 41 nM, less than about 40 nM, less than about 39 nM, less than about 38 nM, less than about 37 nM, less than about 36 nM, less than about 35 nM, less than about 34 nM, less than about 33 nM, less than about 32 nM, less than about 31 nM, less than about 30 nM, less than about 29 nM, less than about 28 nM, less than about 27 nM, less than about 26 nM M, less than about 25 nM, less than about 24 nM, less than about 23 nM, less than about 22 nM, less than about 21 nM, less than about 20 nM, less than about 19 nM, less than about 18 nM, less than about 17 nM, less than about 16 nM, less than about 15 nM, less than about 14 nM, less than about 13 nM, less than about 12 nM, less than about 11 nM, less than about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 990 pM, less than about 980 pM, less than about 970 pM, less than about 960 pM, less than about 950 pM, less than about 940 pM,Less than about 930 pM, less than about 920 pM, less than about 910 pM, less than about 900 pM, less than about 890 pM, less than about 880 pM, less than about 870 pM, less than about 860 pM, less than about 850 pM, less than about 840 pM, less than about 830 pM, less than about 820 pM, less than about 810 pM, less than about 800 pM, less than about 790 pM, less than about 780 pM, less than about 770 pM, less than about 760 pM, less than about 750 pM, less than about 740 pM, less than about 73 Less than about 0 pM, less than about 720 pM, less than about 710 pM, less than about 700 pM, less than about 690 pM, less than about 680 pM, less than about 670 pM, less than about 660 pM, less than about 650 pM, less than about 640 pM, less than about 630 pM, less than about 620 pM, less than about 610 pM, less than about 600 pM, less than about 590 pM, less than about 580 pM, less than about 570 pM, less than about 560 pM, less than about 550 pM, less than about 540 pM, less than about 530 pM less than about 520 pM, less than about 510 pM, less than about 500 pM, less than about 490 pM, less than about 480 pM, less than about 470 pM, less than about 460 pM, less than about 450 pM, less than about 440 pM, less than about 430 pM, less than about 420 pM, less than about 410 pM, less than about 400 pM, less than about 390 pM, less than about 380 pM, less than about 370 pM, less than about 360 pM, less than about 350 pM, less than about 340 pM, less than about 330 pM, It can bind to homo-EPOR with a binding affinity of less than 20 pM, less than about 310 pM, less than about 300 pM, less than about 290 pM, less than about 280 pM, less than about 270 pM, less than about 260 pM, less than about 250 pM, less than about 240 pM, less than about 230 pM, less than about 220 pM, less than about 210 pM, less than about 200 pM, less than about 190 pM, less than about 180 pM, less than about 170 pM, or about any integer therebetween.

[0146] In some embodiments, an EPO analog or engineered EPO containing one or more amino acid substitutions described herein has a β-blocking activity of less than about 600 nM, less than about 590 nM, less than about 580 nM, less than about 570 nM, less than about 560 nM, less than about 550 nM, less than about 540 nM, less than about 530 nM, less than about 520 nM, less than about 510 nM, less than about 500 nM, less than about 490 nM, less than about 480 nM, less than about 470 nM, less than about 460 nM, less than about 450 nM, less than about 440 nM, less than about 430 nM, less than about 420 nM, less than about 410 nM, less than about 400 nM, less than about 300 nM, less than about 350 nM, less than about 360 nM, less than about 370 nM, less than about 380 nM, less than about 390 nM, less than about 400 nM, less than about 410 nM, less than about 420 nM, less than about 430 nM, less than about 440 nM, less than about 450 nM, less than about 460 nM, less than about 450 nM, less than about 440 nM, less than about 430 nM, less than about 420 nM, less than about 410 nM, less than about 400 nM, less than about 370 nM, less than about 390 nM, less than about 450 nM, less than about 460 nM, less than about 450 nM, less than about 440 nM, less than about 430 nM, less than about 420 nM Less than about 90 nM, less than about 380 nM, less than about 370 nM, less than about 360 nM, less than about 350 nM, less than about 340 nM, less than about 330 nM, less than about 320 nM, less than about 310 nM, less than about 300 nM, less than about 290 nM, less than about 280 nM, less than about 270 nM, less than about 260 nM, less than about 250 nM, less than about 240 nM, less than about 230 nM, less than about 220 nM, less than about 210 nM, less than about 200 nM, less than about 190 nM, less than about 180 nM, less than about 170 nM, less than about 160 nM, less than about 150 nM, less than about 140 nM, less than about 130 nM, less than about 120 nM less than about 110 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 50 nM, less than about 50 nM, less than about 49 nM, less than about 48 nM, less than about 47 nM, less than about 46 nM, less than about 45 nM, less than about 44 nM, less than about 43 nM, less than about 42 nM, less than about 41 nM, less than about 40 nM, less than about 39 nM, less than about 38 nM, less than about 37 nM, less than about 36 nM, less than about 35 nM, less than about 34 nM, less than about 33 nM, less than about 32 nM, less than about 31 nM, less than about 30 nM, less than about 29 nM, less than about 28 nM, less than about 27 nM, less than about 26 nM M, less than about 25 nM, less than about 24 nM, less than about 23 nM, less than about 22 nM, less than about 21 nM, less than about 20 nM, less than about 19 nM, less than about 18 nM, less than about 17 nM, less than about 16 nM, less than about 15 nM, less than about 14 nM, less than about 13 nM, less than about 12 nM, less than about 11 nM, less than about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 990 pM, less than about 980 pM, less than about 970 pM, less than about 960 pM, less than about 950 pM, less than about 940 pM,Less than about 930 pM, less than about 920 pM, less than about 910 pM, less than about 900 pM, less than about 890 pM, less than about 880 pM, less than about 870 pM, less than about 860 pM, less than about 850 pM, less than about 840 pM, less than about 830 pM, less than about 820 pM, less than about 810 pM, less than about 800 pM, less than about 790 pM, less than about 780 pM, less than about 770 pM, less than about 760 pM, less than about 750 pM, less than about 740 pM, less than about 730 pM pM, less than about 720 pM, less than about 710 pM, less than about 700 pM, less than about 690 pM, less than about 680 pM, less than about 670 pM, less than about 660 pM, less than about 650 pM, less than about 640 pM, less than about 630 pM, less than about 620 pM, less than about 610 pM, less than about 600 pM, less than about 590 pM, less than about 580 pM, less than about 570 pM, less than about 560 pM, less than about 550 pM, less than about 540 pM, less than about 530 pM , less than about 520 pM, less than about 510 pM, less than about 500 pM, less than about 490 pM, less than about 480 pM, less than about 470 pM, less than about 460 pM, less than about 450 pM, less than about 440 pM, less than about 430 pM, less than about 420 pM, less than about 410 pM, less than about 400 pM, less than about 390 pM, less than about 380 pM, less than about 370 pM, less than about 360 pM, less than about 350 pM, less than about 340 pM, less than about 330 pM, less than about 32 The hetero-EPOR can bind with a binding affinity of less than 0 pM, less than about 310 pM, less than about 300 pM, less than about 290 pM, less than about 280 pM, less than about 270 pM, less than about 260 pM, less than about 250 pM, less than about 240 pM, less than about 230 pM, less than about 220 pM, less than about 210 pM, less than about 200 pM, less than about 190 pM, less than about 180 pM, less than about 170 pM, or about any integer therebetween.

[0147] In some embodiments, the EPO analog or engineered EPO described herein may have a lower binding affinity to hetero-EPOR compared to wild-type or native EPO protein.For example, the EPO analog or engineered EPO that comprises one or more amino acid substitutions described herein may have a lower hetero-EPOR binding affinity than that of the wild-type or native EPO protein that does not comprise one or more amino acid substitutions described herein. In some embodiments, an EPO analog or engineered EPO containing one or more amino acid substitutions described herein has a hetero-EPOR binding affinity that is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about The hetero-EPOR binding affinity may be about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% lower.

[0148] In some embodiments, the EPO analog or engineered EPO described herein may have higher binding affinity to hetero-EPOR than wild-type or native EPO protein.For example, the EPO analog or engineered EPO that comprises one or more amino acid substitutions described herein may have higher hetero-EPOR binding affinity than the wild-type or native EPO protein that does not comprise one or more amino acid substitutions described herein. In some embodiments, an EPO analog or engineered EPO containing one or more amino acid substitutions described herein has a hetero-EPOR binding affinity that is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about The hetero-EPOR binding affinity may be about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% higher.

[0149] In some embodiments, the EPO analog or engineered EPO described herein may have the same level of binding affinity to hetero-EPOR compared to wild-type or native EPO protein.For example, the EPO analog or engineered EPO with one or more amino acid substitutions described herein may have the same or similar hetero-EPOR binding affinity as the wild-type or native EPO protein that does not contain one or more amino acid substitutions described herein.In some embodiments, the EPO analog or engineered EPO described herein may have the same level of binding affinity to homo-EPOR compared to wild-type or native EPO protein.For example, the EPO analog or engineered EPO with one or more amino acid substitutions described herein may have the same or similar homo-EPOR binding affinity as the wild-type or native EPO protein that does not contain one or more amino acid substitutions described herein.

[0150] In some embodiments, the EPO analog or engineered EPO described herein may have a lower binding affinity to homo-EPOR compared to wild-type or native EPO protein.For example, the EPO analog or engineered EPO that comprises one or more amino acid substitutions described herein may have a lower homo-EPOR binding affinity than that of the wild-type or native EPO protein that does not comprise one or more amino acid substitutions described herein. In some embodiments, an EPO analog or engineered EPO containing one or more amino acid substitutions described herein has a homo-EPOR binding affinity that is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about The homo-EPOR binding affinity may be about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% lower.

[0151] In some embodiments, the EPO analog or engineered EPO described herein may have higher binding affinity to homo-EPOR than wild-type or native EPO protein.For example, the EPO analog or engineered EPO that comprises one or more amino acid substitutions described herein may have higher homo-EPOR binding affinity than the wild-type or native EPO protein that does not comprise one or more amino acid substitutions described herein. In some embodiments, an EPO analog or engineered EPO containing one or more amino acid substitutions described herein has a homo-EPOR binding affinity that is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at least about The homo-EPOR binding affinity may be about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% higher.

[0152] In some embodiments, the EPO analogs or engineered EPOs described herein can bind to the homo-EPO receptor with a higher binding affinity than the binding affinity to the hetero-EPO receptor. In some embodiments, the EPO analogs or engineered EPOs described herein can bind to the homo-EPO receptor with a lower binding affinity than the binding affinity to the hetero-EPO receptor. In some embodiments, the EPO analogs or engineered EPOs described herein can bind to the hetero-EPO receptor with a higher binding affinity than the binding affinity to the homo-EPO receptor. In some embodiments, the EPO analogs or engineered EPOs described herein can bind to the hetero-EPO receptor with a lower binding affinity than the binding affinity to the homo-EPO receptor.

[0153] In some embodiments, the EPO analogs or engineered EPOs described herein can promote the activity of homo-EPOR or increase the level of its activity. In some embodiments, the EPO analogs or engineered EPOs described herein can have no effect on the level of homo-EPOR activity. In some embodiments, the EPO analogs or engineered EPOs described herein can inhibit the activity of homo-EPOR or decrease the level of its activity. In some embodiments, homo-EPOR activity can include, but is not limited to, phosphorylation of the intracellular domain of homo-EPOR, Janus tyrosine kinase 2 (Jak2), or signal transduction and activator of transcription 5 (Stat5). In some embodiments, homo-EPOR activity may include, but is not limited to, activation of Jak2, the Jak2 pathway, the Stat5 pathway, mitogen-activated protein kinase (MAPK), the MAPK pathway, extracellular signal-regulated kinase (ERK), the ERK pathway, phosphatidylinositol 3-kinase (PI3K), the PI3K pathway, v-Akt murine thymoma viral oncogene / protein kinase-B (Akt / PKB), the Akt / PKB pathway, mammalian target of rapamycin (mTOR), or the mTOR pathway.

[0154] In some embodiments, the EPO analog or engineered EPO described herein can promote the activity of hetero-EPOR or increase the level of its activity. In some embodiments, the EPO analog or engineered EPO described herein can have no effect on the level of hetero-EPOR activity. In some embodiments, the EPO analog or engineered EPO described herein can inhibit the activity of hetero-EPOR or decrease the level of its activity. In some embodiments, hetero-EPOR activity can include, but is not limited to, phosphorylation of the intracellular domain of hetero-EPOR, Janus tyrosine kinase 2 (Jak2), or signal transduction and activator of transcription 5 (Stat5). In some embodiments, hetero-EPOR activity may include, but is not limited to, activation of Jak2, the Jak2 pathway, the Stat5 pathway, mitogen-activated protein kinase (MAPK), the MAPK pathway, extracellular signal-regulated kinase (ERK), the ERK pathway, phosphatidylinositol 3-kinase (PI3K), the PI3K pathway, v-Akt murine thymoma viral oncogene / protein kinase-B (Akt / PKB), the Akt / PKB pathway, mammalian target of rapamycin (mTOR), or the mTOR pathway.

[0155] In some embodiments, the EPO analog or engineered EPO described herein may not affect the level of Jak2, Stat5, mTOR, MAPK, ERK, PI3K, Akt / PKB activation or the phosphorylation of the intracellular domain of homo-EPOR or hetero-EPOR compared to wild-type or native EPO protein.For example, when the EPO analog or engineered EPO containing one or more amino acid substitutions described herein is introduced into a cell or a population of cells, the cell or population of cells may have the same or similar activation level of Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB, or mTOR as that of a cell or a population of cells into which a wild-type or native EPO protein not containing one or more amino acid substitutions described herein is introduced.The activation or phosphorylation of homo-EPOR, hetero-EPOR, Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB, or mTOR can be measured using any method known in the art. Exemplary methods for measuring activation levels of Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB, or mTOR include, but are not limited to, Western blot, flow cytometry assay, cell proliferation assay, apoptosis assay, or enzyme-linked immunosorbent assay (ELISA).

[0156] In some embodiments, the EPO analogs or engineered EPOs described herein can increase or promote activation of Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB, or mTOR. For example, a cell or population of cells into which an EPO analog or engineered EPO containing one or more amino acid substitutions described herein has been introduced can have a higher activation level of Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB, or mTOR than a cell or population of cells into which a wild-type or native EPO protein that does not contain one or more amino acid substitutions described herein has been introduced.In some embodiments, a cell or population of cells into which an EPO analog or engineered EPO containing one or more amino acid substitutions described herein has activation levels of Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB, or mTOR that are at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24% higher than a cell or population of cells into which a wild-type or native EPO protein that does not contain one or more amino acid substitutions described herein. , at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, %, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% higher activation levels of Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB, or mTOR.

[0157] In some embodiments, the EPO analogs or engineered EPOs described herein can reduce or inhibit activation of Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB, or mTOR. For example, a cell or population of cells into which an EPO analog or engineered EPO containing one or more amino acid substitutions described herein has been introduced can have a lower activation level of Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB, or mTOR than a cell or population of cells into which a wild-type or native EPO protein that does not contain one or more amino acid substitutions described herein has been introduced.In some embodiments, a cell or population of cells into which an EPO analog or engineered EPO containing one or more amino acid substitutions described herein has activation levels of Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB, or mTOR that are at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24% higher than a cell or population of cells into which a wild-type or native EPO protein that does not contain one or more amino acid substitutions described herein. , at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, %, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% lower activation levels of Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB, or mTOR.

[0158] In some embodiments, the EPO analog or engineered EPO described herein acts as an agonist for homo-EPOR and can selectively bind to homo-EPOR.In some embodiments, the EPO analog or engineered EPO that is an agonist for homo-EPOR can have a higher binding affinity for homo-EPOR than for hetero-EPOR.For example, the EPO analog or engineered EPO that comprises one or more amino acid substitutions described herein can be an agonist for homo-EPOR and has a higher homo-EPOR binding affinity than hetero-EPOR binding affinity. In some embodiments, an EPO analog or engineered EPO containing one or more amino acid substitutions described herein is an agonist for homo-EPOR and has a binding affinity that is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27% higher than the hetero-EPOR binding affinity. , at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% higher homo-EPOR binding affinity.

[0159] In some embodiments, the EPO analogs or engineered EPOs described herein act as agonists for homo-EPOR and may have binding specificity or selectivity for homo-EPOR. In some embodiments, the EPO analogs or engineered EPOs that are agonists for homo-EPOR may have higher binding specificity or selectivity for homo-EPOR than for hetero-EPOR. For example, the EPO analogs or engineered EPOs that contain one or more amino acid substitutions described herein may be agonists for homo-EPOR and may have higher homo-EPOR binding specificity or selectivity than hetero-EPOR binding specificity or selectivity.In some embodiments, an EPO analog or engineered EPO containing one or more amino acid substitutions described herein is an agonist for homo-EPOR and has a hetero-EPOR binding specificity or selectivity that is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%. , at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% higher homo-EPOR binding specificity or selectivity.

[0160] In some embodiments, the EPO analog or engineered EPO described herein acts as an antagonist to homo-EPOR and can selectively bind to homo-EPOR.In some embodiments, the EPO analog or engineered EPO that is an antagonist to homo-EPOR can have a higher binding affinity to homo-EPOR than to hetero-EPOR.For example, the EPO analog or engineered EPO that comprises one or more amino acid substitutions described herein can be an antagonist to homo-EPOR and have a higher homo-EPOR binding affinity than hetero-EPOR binding affinity. In some embodiments, an EPO analog or engineered EPO containing one or more amino acid substitutions described herein is an antagonist for homo-EPOR and has a binding affinity at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69%, at %, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% higher homo-EPOR binding affinity.

[0161] In some embodiments, the EPO analog or engineered EPO described herein acts as an antagonist to homo-EPOR and may have binding specificity or selectivity to homo-EPOR. In some embodiments, the EPO analog or engineered EPO that is an antagonist to homo-EPOR may have higher binding specificity or selectivity to homo-EPOR than to hetero-EPOR. For example, the EPO analog or engineered EPO that includes one or more amino acid substitutions described herein may be an antagonist to homo-EPOR and may have higher homo-EPOR binding specificity or selectivity than hetero-EPOR binding specificity or selectivity.In some embodiments, an EPO analog or engineered EPO containing one or more amino acid substitutions described herein is an antagonist for homo-EPOR and has a hetero-EPOR binding specificity or selectivity that is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 6 %, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% higher homo-EPOR binding specificity or selectivity.

[0162] In some embodiments, the EPO analog or engineered EPO described herein acts as an agonist for hetero-EPOR and can selectively bind to hetero-EPOR.In some embodiments, the EPO analog or engineered EPO that is an agonist for hetero-EPOR can have a higher binding affinity for hetero-EPOR than for homo-EPOR.For example, the EPO analog or engineered EPO that comprises one or more amino acid substitutions described herein can be an agonist for hetero-EPOR and have a higher binding affinity for hetero-EPOR than for homo-EPOR. In some embodiments, an EPO analog or engineered EPO containing one or more amino acid substitutions described herein is an agonist for hetero-EPOR and has a binding affinity that is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27% higher than the homo-EPOR binding affinity. , at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% higher hetero-EPOR binding affinity.

[0163] In some embodiments, the EPO analogs or engineered EPOs described herein act as agonists for hetero-EPOR and may have binding specificity or selectivity for hetero-EPOR. In some embodiments, the EPO analogs or engineered EPOs that are agonists for hetero-EPOR may have higher binding specificity or selectivity for hetero-EPOR than for homo-EPOR. For example, the EPO analogs or engineered EPOs that contain one or more amino acid substitutions described herein may be agonists for hetero-EPOR and may have higher binding specificity or selectivity for hetero-EPOR than for homo-EPOR.In some embodiments, an EPO analog or engineered EPO containing one or more amino acid substitutions described herein is an agonist for hetero-EPOR and has a binding specificity or selectivity that is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%. , at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% higher hetero-EPOR binding specificity or selectivity.

[0164] In some embodiments, the EPO analog or engineered EPO described herein acts as an antagonist to hetero-EPOR and can selectively bind to hetero-EPOR.In some embodiments, the EPO analog or engineered EPO that is an antagonist to hetero-EPOR can have a higher binding affinity to hetero-EPOR than to homo-EPOR.For example, the EPO analog or engineered EPO that comprises one or more amino acid substitutions described herein can be an antagonist to hetero-EPOR and have a higher binding affinity to hetero-EPOR than to homo-EPOR.In some embodiments, an EPO analog or engineered EPO containing one or more amino acid substitutions described herein is an antagonist for hetero-EPOR and has a binding affinity that is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least about 69 %, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% higher hetero-EPOR binding affinity.

[0165] In some embodiments, the EPO analog or engineered EPO described herein acts as an antagonist to hetero-EPOR and can have binding specificity or selectivity to hetero-EPOR. In some embodiments, the EPO analog or engineered EPO that is an antagonist to hetero-EPOR can have higher binding specificity or selectivity to hetero-EPOR than to homo-EPOR. For example, the EPO analog or engineered EPO that contains one or more amino acid substitutions described herein can be an antagonist to hetero-EPOR and can have higher hetero-EPOR binding specificity or selectivity than homo-EPOR binding specificity or selectivity.In some embodiments, an EPO analog or engineered EPO containing one or more amino acid substitutions described herein is an antagonist for hetero-EPOR and has a binding specificity or selectivity that is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 51%, at least about 52%, at least about 53%, at least about 54%, at least about 55%, at least about 56%, at least about 57%, at least about 58%, at least about 59%, at least about 60%, at least about 61%, at least about 62%, at least about 63%, at least about 64%, at least about 65%, at least about 66%, at least about 67%, at least about 68%, at least %, at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% higher hetero-EPOR binding specificity or selectivity.

[0166] In some embodiments, the EPO analogs or engineered EPOs described herein may have a half-life in human plasma of 1 hour to 5 days. In some embodiments, the EPO analogs or engineered EPOs described herein may have a half-life of about 1 hour to about 120 hours. In some embodiments, the EPO analogs or engineered EPOs described herein may have a half-life of about 1 hour to about 5 hours, about 1 hour to about 10 hours, about 1 hour to about 12 hours, about 1 hour to about 24 hours, about 1 hour to about 36 hours, about 1 hour to about 48 hours, about 1 hour to about 60 hours, about 1 hour to about 72 hours, about 1 hour to about 84 hours, about 1 hour to about 96 hours, about 1 hour to about 120 hours, about 5 hours to about 10 hours, about 5 hours to about 12 hours, about 5 hours to about 24 hours, about 5 hours to about 36 hours, about 5 hours about 48 hours, about 5 hours to about 60 hours, about 5 hours to about 72 hours, about 5 hours to about 84 hours, about 5 hours to about 96 hours, about 5 hours to about 120 hours, about 10 hours to about 12 hours, about 10 hours to about 24 hours, about 10 hours to about 36 hours, about 10 hours to about 48 hours, about 10 hours to about 60 hours, about 10 hours to about 72 hours, about 10 hours to about 84 hours, about 10 hours to about 96 hours, about 10 hours to about 120 hours, about 12 hours to about 24 hours, about 12 hours to about 36 hours, about 12 hours to about 48 hours , about 12 hours to about 60 hours, about 12 hours to about 72 hours, about 12 hours to about 84 hours, about 12 hours to about 96 hours, about 12 hours to about 120 hours, about 24 hours to about 36 hours, about 24 hours to about 48 hours, about 24 hours to about 60 hours, about 24 hours to about 72 hours, about 24 hours to about 84 hours, about 24 hours to about 96 hours, about 24 hours to about 120 hours, about 36 hours to about 48 hours, about 36 hours to about 60 hours, about 36 hours to about 72 hours, about 36 hours to about 84 hours, about 36 hours to about 96 hours, It may have a half-life of 36 hours to about 120 hours, about 48 hours to about 60 hours, about 48 hours to about 72 hours, about 48 hours to about 84 hours, about 48 hours to about 96 hours, about 48 hours to about 120 hours, about 60 hours to about 72 hours, about 60 hours to about 84 hours, about 60 hours to about 96 hours, about 60 hours to about 120 hours, about 72 hours to about 84 hours, about 72 hours to about 96 hours, about 72 hours to about 120 hours, about 84 hours to about 96 hours, about 84 hours to about 120 hours, or about 96 hours to about 120 hours.In some embodiments, the EPO analogs or engineered EPOs described herein may have a half-life of about 1 hour, about 5 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, or about 120 hours. In some embodiments, the EPO analogs or engineered EPOs described herein may have a half-life of at least about 1 hour, about 5 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, or about 96 hours. In some embodiments, the EPO analogs or engineered EPOs described herein may have a half-life of up to about 5 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, or about 120 hours.

[0167] The present disclosure also encompasses engineered EPORs that include the extracellular domain (ECD) of EPOR. The ECD of EPOR includes two domains D1 and D2, and these two domains are necessary for EPO binding. In some embodiments, an Fc fusion protein of ECD EPOR-Fc can bind to EPO. In some embodiments, an Fc fusion protein of ECD EPOR-Fc can block EPOR activation. In some embodiments, an Fc fusion protein of ECD EPOR-Fc can include a mutation. For example, an Fc fusion protein of ECD EPOR-Fc can include a mutation at amino acid residue F93. In some embodiments, an Fc fusion protein of ECD EPOR-Fc can include an F93A mutation. In some embodiments, an Fc fusion protein of ECD EPOR-Fc that includes an F93A mutation cannot bind to EPO. For example, a monomeric EPOR ECD that includes an F93A mutation or a dimeric EPOR-Fc that includes an F93A mutation cannot bind to EPO.

[0168] The present disclosure also encompasses engineered hetero-EPORs that contain the extracellular domain (ECD) of CD131. The ECD of CD131 contains four domains D1, D2, D3, and D4. The D1 and D2 domains are responsible for dimerization distal to the cell membrane. Without wishing to be bound by theory, the D3 and D4 domains may be regions that interact with the EPOR to form the hetero-EPOR. In some embodiments, knob-in-hole technology can be used to create heterodimeric Fc fusion proteins with the EPOR ECD and CD131 ECD. Non-limiting examples of designs of heterodimeric Fc fusion proteins with the EPOR ECD and CD131 ECD are shown in Table 3-3, and sequences are shown in Figures 42A-42D. In some embodiments, EPO binding may require the D3 and D4 domains of CD131. For example, monomeric or dimeric EPOR with F93A substitution cannot bind EPO, but monomeric EPOR with F93A mutation and hetero-EPOR of CD131 monomer bind EPO. EPO binding to hetero-EPOR appears to be specific to CDC131 subunit. In some embodiments, heterodimeric EPOR(F93A) / CD131-Fc can be used to specifically block hetero-EPOR but not homo-EPOR.

[0169] Anti-EPOR, anti-CD131, and anti-EPO antibodies In some aspects, provided herein are antibodies, antigen-binding fragments thereof, or functional fragments thereof that can selectively bind to a target. In some embodiments, the antibodies, antigen-binding fragments thereof, or functional fragments thereof described herein can bind to an antigen of a target protein or an epitope on an antigen of a target protein.

[0170] In some embodiments, the antibody may be a monospecific antibody, binding to a single epitope. For example, a monospecific antibody may have multiple immunoglobulin variable domain sequences, each of which binds to the same epitope. In some embodiments, the antibody may be a bispecific antibody. A bispecific antibody may have specificity for no more than two antigens. A bispecific antibody may be characterized by a first immunoglobulin variable domain sequence having binding specificity for a first epitope, and a second immunoglobulin variable domain sequence having binding specificity for a second epitope. In some embodiments, the first and second epitopes may be on the same antigen, e.g., on the same protein (or subunit of a multimeric protein). In some embodiments, the first and second epitopes may overlap. In some embodiments, the first and second epitopes do not overlap. In some embodiments, the first and second epitopes may be on different antigens, e.g., on different proteins (or different subunits of a multimeric protein). In some embodiments, a bispecific antibody may comprise a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence having binding specificity for a second epitope. In some embodiments, a bispecific antibody may comprise a half antibody having binding specificity for a first epitope, and a half antibody having binding specificity for a second epitope. In some embodiments, a bispecific antibody may comprise a half antibody or a fragment thereof having binding specificity for a first epitope, and a half antibody or a fragment thereof having binding specificity for a second epitope. In some embodiments, a bispecific antibody may comprise an scFv or Fab or a fragment thereof having binding specificity for a first epitope, and an scFv or Fab or a fragment thereof having binding specificity for a second epitope.

[0171] In some embodiments, the antibody may be a multispecific or multifunctional antibody. For example, the multispecific or multifunctional antibody may comprise a plurality of immunoglobulin variable domain sequences, a first immunoglobulin variable domain sequence of the plurality of immunoglobulin variable domain sequences having binding specificity for a first epitope, and a second immunoglobulin variable domain sequence of the plurality of immunoglobulin variable domain sequences having binding specificity for a second epitope. In some embodiments, the first and second epitopes are on the same antigen, e.g., on the same protein (or subunit of a multimeric protein). In some embodiments, the first and second epitopes may overlap. In some embodiments, the first and second epitopes may not overlap. In some embodiments, the first and second epitopes may be on different antigens, e.g., on different proteins (or different subunits of a multimeric protein). In some embodiments, the multispecific antibody may comprise a third, fourth, or fifth immunoglobulin variable domain. In some embodiments, the multispecific antibody may be a bispecific antibody, a trispecific antibody, or a tetraspecific antibody. In some embodiments, the multispecific antibody may optionally further comprise one or more additional binding domains that selectively bind to IgE, FcεRIα, FcεRII, tumor-associated antigen (FAA), or a combination thereof. Any bispecific or multispecific antibody described herein may be isolated, purified, recombinant, synthetic, or any combination thereof. The bispecific or multispecific antibody described herein may be produced via any suitable method and may be recombinant, synthetic, or any combination thereof. In one aspect, a liquid or lyophilized composition may be provided herein that comprises one or more of the bispecific or multispecific antibodies described herein. In one embodiment, the composition may comprise a population of bispecific or multispecific antibodies. In another embodiment, the composition may comprise a population of two, three, four, five, six, seven, eight, nine, ten, or more of the bispecific or multispecific antibodies described above.The bispecific or multispecific antibodies described herein can be utilized in in vitro assays to identify and / or purify one or more tumor cells, for example, from a mixed culture (e.g., a biological sample such as a biopsy or blood sample). The bispecific or multispecific antibodies described herein can be utilized in in vivo animal models to test the therapeutic efficacy of the bispecific or multispecific antibodies against tumors.

[0172] In some embodiments, antibodies may include diabodies and single chain molecules, as well as antigen-binding fragments of antibodies (e.g., Fab, F(ab')2, and Fv). For example, an antibody molecule may include a heavy (H) chain variable domain sequence (abbreviated herein as VH), and a light (L) chain variable domain sequence (abbreviated herein as VL). In some embodiments, an antibody may include a heavy chain and a light chain (referred to herein as a half antibody). In another example, an antibody may include two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequences, thereby forming two antigen-binding sites, e.g., Fab, Fab', F(ab')2, Fc, Fd, Fd', Fv, single chain antibodies (e.g., scFv), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be generated by modification of whole antibodies or synthesized de novo using recombinant DNA technology. These functional antibody fragments may retain the ability to selectively bind to their respective antigens. Antibodies and antibody fragments may be from any class of antibody, including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and any subclass of antibody (e.g., IgG1, IgG2, IgG3, and IgG4). Antibody preparations may be monoclonal or polyclonal. Antibodies may also be human, humanized, CDR-grafted, or in vitro generated. Antibodies may have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3, or IgG4. Antibodies may also have a light chain selected from, for example, kappa or lambda. The term "immunoglobulin" (Ig) is used interchangeably herein with the term "antibody."

[0173] Non-limiting examples of antigen-binding fragments of antibodies may include Fab fragments (monovalent fragments consisting of the VL, VH, CL and CH1 domains); F(ab')2 fragments (bivalent fragments containing two Fab fragments linked by a disulfide bridge at the hinge region); Fd fragments consisting of the VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; diabody (dAb) fragments consisting of the VH domain; camel or camelized variable domains; single chain Fvs (scFvs) (see, for example, Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883); and single domain antibodies. These antibody fragments may be obtained using conventional techniques known to those skilled in the art, and the fragments may be screened for utility in the same manner as intact antibodies. For example, single chain antibodies (scFv) can be engineered (see, e.g., Colcher, D. et al. (1999) Ann NY Acad Sci 880:263-80; and Reiter, Y. (1996) Clin Cancer Res 2:245-52). In some embodiments, single chain antibodies can be dimerized or multimerized to create multivalent antibodies with specificities for different epitopes of the same target protein. In some embodiments, antibodies can include intact molecules and functional fragments thereof. The constant region of an antibody can be altered or mutated to modify one or more properties of the antibody (e.g., to increase or decrease one or more of Fc receptor binding, antibody glycosylation, number of cysteine ​​residues, effector cell function, or complement function). Methods for altering antibody constant regions are known in the art.In some embodiments, antibodies with altered function, e.g., altered affinity for an effector ligand, e.g., an FcR on a cell, or the C1 component of complement, can be generated by replacing at least one amino acid residue in the constant portion of the antibody with a different residue (see, e.g., EP 388,151 A1, U.S. Pat. No. 5,624,821, and U.S. Pat. No. 5,648,260, the contents of all of which are incorporated herein by reference).

[0174] In some embodiments, the antibodies, antigen-binding fragments thereof, or functional fragments thereof described herein can comprise a non-antibody scaffold. Non-limiting examples of non-antibody scaffolds include affibodies, affilins, anticalins, atrimers, avimers, bicyclic peptides, Cys-knots, DARPins, FN3 scaffolds (e.g., adnectins, sentinins, pronectins, Tn3), fynomers, Kunitz domains, or O-bodies.

[0175] In some embodiments, the antibodies, antigen-binding fragments thereof, or functional fragments thereof described herein may be derivatized or linked to another functional molecule (e.g., another peptide or protein). As used herein, a "derivatized" antibody is an antibody that has been modified. Methods of derivatization may include, but are not limited to, the addition of a fluorescent moiety, a radioactive nucleotide, a toxin, an enzyme, or an affinity ligand, such as biotin. For example, an antibody may be functionally linked to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and / or a protein or peptide that can mediate the association of the antibody or antibody portion with another molecule (e.g., a streptavidin core region or a polyhistidine tag), for example, by using chemical coupling, genetic fusion, non-covalent association, or other methods. A type of derivatized antibody can be generated by crosslinking two or more antibodies (of the same type or different types, e.g., to create a bispecific antibody). Suitable cross-linkers may include those that are heterobifunctional, having two distinct reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester), or those that are homobifunctional (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, Ill.

[0176] In some embodiments, the antibody may also be a single domain antibody. Single domain antibodies may include antibodies whose complementarity determining regions are part of a single domain polypeptide. Non-limiting examples may include heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from traditional four-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any in the art or any future single domain antibody. Single domain antibodies may be derived from any species, including but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, and cow. In some embodiments, the single domain antibody may be a naturally occurring single domain antibody known as a heavy chain antibody devoid of light chains. Such single domain antibodies are disclosed, for example, in WO94 / 04678. In some embodiments, this variable domain derived from a heavy chain antibody naturally devoid of light chains is known herein as a VHH or nanobody. Such VHH molecules may be derived from antibodies raised in Camelidae species, such as camel, llama, dromedary, alpaca and guanaco. Other non-Camelidae species may produce heavy chain antibodies that are naturally devoid of light chains.

[0177] The VH and VL regions can be subdivided into regions of hypervariability called "complementarity determining regions" (CDRs) interspersed with more conserved regions called "framework regions" (FR or FW). The extent of framework regions and CDRs has been precisely defined by several methods (see Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917; and the AbM definition used by Oxford Molecular's AbM antibody modeling software). See generally, for example, Protein Sequence and Structure Analysis of Antibody Variable Domains. In: Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). In some embodiments, CDRs may comprise amino acid sequences in the antibody variable region that confer antigen specificity and binding affinity. In some embodiments, an antibody may have three CDRs (VH-CDR1, VH-CDR2, and VH-CDR3) in each heavy chain variable region and three CDRs (VL-CDR1, VL-CDR2, and VL-CDR3) in each light chain variable region.In some embodiments, the amino acid sequence boundaries of a given CDR can be determined using any of several known schemes, including those described in Kabat et al. (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD (the "Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273,927-948 (the "Chothia" numbering scheme).

[0178] The antibodies described herein can be produced recombinantly, for example, using phage display or by using combinatorial methods. Phage display and combinatorial methods for generating antibodies are known in the art (see, e.g., U.S. Pat. No. 5,223,409 to Ladner et al.; International Publication No. WO 92 / 18619 to Kang et al.; International Publication No. WO 91 / 17271 to Dower et al.; International Publication No. WO 92 / 20791 to Winter et al.; International Publication No. WO 92 / 15679 to Markland et al.; International Publication No. WO 93 / 01288 to Breitling et al.; International Publication No. WO 92 / 01047 to McCafferty et al.; International Publication No. WO 92 / 09690 to Garrard et al.; International Publication No. WO 90 / 02809 to Ladner et al.; Fuchs et al. (1991) Bio / Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas 3:81-85;Huse et al. (1989) Science 246:1275-1281;Griffths et al. (1993) EMBO J 12:725-734;Hawkins et al. (1992) J Mol Biol 226:889-896;Clackson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS 89:3576-3580; Garrad et al. (1991) Bio / Technology 9:1373-1377; Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; and Barbas et al. (1991) PNAS 88:7978-7982, the contents of all of which are incorporated herein by reference.

[0179] In some embodiments, the antibodies described herein may be fully human antibodies (e.g., antibodies made in mice genetically engineered to produce antibodies from human immunoglobulin sequences), or non-human antibodies, such as rodent (mouse or rat), goat, primate (e.g., monkey), or camel antibodies. In some embodiments, the non-human antibodies may be rodent antibodies (mouse or rat antibodies). Methods of generating rodent antibodies are known in the art.

[0180] In some embodiments, the antibodies, antigen-binding fragments thereof, or functional fragments thereof described herein may be humanized antibodies or humanized antigen-binding fragments. As used herein, a "humanized" antibody refers to a form of a non-human (e.g., murine) antibody that is specific for a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof that contains minimal sequence derived from a non-human immunoglobulin. In some embodiments, a humanized antibody may be a human immunoglobulin (recipient antibody) in which residues from the recipient's complementarity determining regions (CDRs) have been replaced by residues from a CDR (donor antibody) of a non-human species, such as mouse, rat, or rabbit, that has the desired specificity, affinity, and biological activity. In some embodiments, a humanized antibody may have at least one or two, but typically all three recipient CDRs (of the heavy and / or light immunoglobulin chains) replaced with donor CDRs. In some embodiments, an antibody may have at least a portion of a non-human CDR replaced, or only a portion of the CDRs replaced with non-human CDRs. In some embodiments, the minimum number of CDRs required for binding to the antigen may be replaced. In some embodiments, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine or optimize antibody performance. In general, a humanized antibody will comprise at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are of a human immunoglobulin consensus sequence. A humanized antibody will optimally also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. The antibody may have an Fc region modified, for example, as described in WO99 / 58572.Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, or six) that have been altered with respect to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody. Humanized antibodies can be generated, for example, by modeling an antibody variable domain and generating an antibody using genetic engineering techniques such as CDR grafting or CDR replacement, where one, two, or all CDRs of an immunoglobulin chain can be replaced. Descriptions of various techniques for the production of humanized antibodies can be found, for example, in U.S. Patent 5,225,539; Morrison et al., (1984) Proc. Nat'l Acad. Sci. USA 81:6851-55; Whittle et al., (1987) Prot. Eng. 1:499-505; Co et al., (1990) J. Immunol. 148:1149-1154; Co et al., (1992) Proc. Nat'l Acad. Sci. USA 88:2869-2873; Carter et al., (1992) Proc. Nat'l Acad. Sci. USA 89:4285-4289; Routledge et al., (1991) Eur. J. Immunol. 21:2717-2725 and PCT Patent Publication Nos. WO 91 / 09967, WO 91 / 09968 and WO 92 / 113831. For example, human monoclonal antibodies can be made using transgenic mice carrying the human immunoglobulin genes rather than the mouse system.Spleen cells from these transgenic mice immunized with an antigen of interest can be used to generate hybridomas secreting human mAbs with specific affinity for epitopes derived from human proteins (see, e.g., Wood et al., International Application WO 91 / 00906; Kucherlapati et al., PCT Publication WO 91 / 10741; Lonberg et al., International Application WO 92 / 03918; Kay et al., International Application 92 / 03917; Lonberg, N. et al. 1994 Nature 368:856-859; Green, LL et al. 1994 Nature Genet. 7:13-21; Morrison, SL et al. 1994 Proc. Natl. Acad. Sci. USA 81:6851-6855; Bruggeman et al. 1993 Year Immunol 7:33-40; Tuaillon et al. 1993 PNAS 90:3720-3724; Bruggeman et al. 1991 Eur J Immunol 21:1323-1326). In some embodiments, an immunocompetent transgenic mouse can be used. In some embodiments, the immunocompetent transgenic mouse can include a human antibody heavy chain, a human antibody light chain, or a combination thereof. In some embodiments, the immunocompetent transgenic mouse can include a human antibody heavy chain, a human antibody lambda light chain, a human antibody kappa light chain, or a combination thereof. In some embodiments, one or more specific amino acids can be substituted, deleted, or added in the humanized antibody. Criteria for selecting donor-derived amino acids are described in US 5,585,089, e.g., columns 12-16 of US 5,585,089, e.g., columns 12-16 of US 5,585,089, the contents of which are incorporated herein by reference. Other techniques for humanizing antibodies are described in EP 519596 A1, published December 23, 1992, by Padlan et al.

[0181] In some embodiments, the antibody, its antigen-binding fragment, or its functional fragment described herein may comprise a CDR-grafted scaffold domain. In some embodiments, the scaffold domain may be based on a fibronectin domain, for example, a fibronectin type III domain. In some embodiments, the overall fold of the fibronectin type III (Fn3) domain may be closely related to that of the variable domain of the smallest functional antibody fragment of an antibody heavy chain. There are three loops at the end of Fn3, and the positions of the BC, DE, and FG loops roughly correspond to those of CDR1, 2, and 3 of the VH domain of an antibody. In some embodiments, Fn3 may not have disulfide bonds, and therefore, Fn3 may be stable under reducing conditions, unlike antibodies and their fragments (see, for example, WO98 / 56915; WO01 / 64942; WO00 / 34784). Fn3 domains can be modified (e.g., using CDRs or hypervariable loops as described herein) or altered to select domains that bind to, for example, antigens / markers / cells as described herein. In some embodiments, scaffold domains, e.g., folded domains, can be based on "minibody" scaffolds created by deleting three beta chains from the heavy chain variable domain of an antibody, e.g., a monoclonal antibody (see, e.g., Tramontano et al., 1994, J Mol. Recognit. 7:9; and Martin et al., 1994, EMBO J. 13:5303-5309). In some embodiments, minibodies can be used to present two hypervariable loops. In some embodiments, the scaffold domain can be a V-like domain (see, e.g., Coia et al., WO 99 / 45110) or a domain derived from tendamistatin, a 74-residue six-stranded beta-sheet sandwich held together by two disulfide bonds (see, e.g., McConnell and Hoess, 1995, J Mol. Biol. 250:460).For example, the loops of tendamistatin can be modified (e.g., using CDRs or hypervariable loops) or altered to select domains that bind to, for example, markers / antigens / cells as described herein. Another exemplary scaffold domain is a beta sandwich structure derived from the extracellular domain of CTLA-4 (see, for example, WO00 / 60070). Other exemplary scaffold domains can include, but are not limited to, T cell receptors, MHC proteins, extracellular domains (e.g., fibronectin type III repeats, EGF repeats), protease inhibitors (e.g., Kunitz domains, ecotin, BPTI, etc.), TPR repeats; trifoil structures, zinc finger domains, DNA binding proteins, particularly monomeric DNA binding proteins, RNA binding proteins, enzymes, such as proteases (particularly inactivating proteases), RNases, chaperones, such as thioredoxin, and heat shock proteins; and intracellular signaling domains (e.g., SH2 and SH3 domains). See, for example, US20040009530 and US7,501,121, which are incorporated herein by reference. In some embodiments, scaffold domains can be evaluated and selected, for example, by one or more of the following criteria: (1) amino acid sequence, (2) sequence of several homologous domains, (3) three-dimensional structure, and / or (4) stability data over a range of pH, temperature, salt, organic solvent, and oxidant concentration. In some embodiments, the scaffold domain can be a small, stable protein domain, for example, a protein of less than 100, 70, 50, 40, or 30 amino acids. The domain can include one or more disulfide bonds or can chelate a metal, for example, zinc.

[0182] In some embodiments, the antibodies, antigen-binding fragments, or functional fragments described herein may contain variable regions, or portions thereof, such as CDRs, made in non-human organisms (e.g., rats or mice). In some embodiments, the antibodies, antigen-binding fragments, or functional fragments described herein may be chimeric antibodies, CDR-grafted antibodies, or humanized antibodies. In some embodiments, the antibodies, antigen-binding fragments, or functional fragments described herein may be made and modified in non-human organisms. For example, antibodies, antigen-binding fragments, or functional fragments made in non-human organisms (e.g., rats or mice) may be modified in the variable framework regions or constant regions to reduce antigenicity and / or immunogenicity in humans. In some embodiments, chimeric antibodies can be produced by recombinant DNA techniques known in the art (Robinson et al., International Patent Publication No. PCT / US86 / 02269; Akira et al., European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al., European Patent Application 173,494; Neuberger et al., International Application WO86 / 01533; Cabilly et al., U.S. Pat. No. 4,816,567; Cabilly et al., European Patent Application 125,023; Better et al. (1988 Science 240:1041-1043); Liu et al. (1987) PNAS 84:3439-3443; Liu et al., 1987, J. Immunol. 139:3521-3526; Sun et al. (1987) PNAS 84:214-218; Nishimura et al., 1987, Canc. Res. 47:999-1005; Wood et al. (1985) Nature 314:446-449; and Shaw et al., 1988, J. Natl Cancer Inst. 80:1553-1559).

[0183] In some embodiments, the antibodies, antigen-binding fragments thereof, or functional fragments thereof described herein can selectively bind to an antigen of a target protein or an epitope on an antigen of a target protein. In some embodiments, the target can include erythropoietin (EPO) protein, an EPO receptor subunit of a homo-EPOR or hetero-EPOR, a CD131 subunit of a hetero-EPOR, or a combination thereof. In some embodiments, the target can include a hetero-EPOR. For example, the antibodies, antigen-binding fragments thereof, or functional fragments thereof described herein can selectively bind to a hetero-EPOR comprising an EPO receptor subunit and a CD131 subunit. In this embodiment, the antibodies, antigen-binding fragments thereof, or functional fragments thereof described herein can bind to both the EPO receptor subunit and the CD131 subunit of a hetero-EPOR. In some embodiments, the antibodies, antigen-binding fragments thereof, or functional fragments thereof described herein can be non-naturally occurring. In some embodiments, the antibodies, antigen-binding fragments thereof, or functional fragments thereof described herein can be isolated and / or purified. In some embodiments, the antibodies, antigen-binding fragments thereof, or functional fragments thereof described herein can be used in in vitro assays (eg, binding assays, functional assays, etc.).

[0184] In some embodiments, the antibodies or functional fragments thereof described herein can bind to targets and act as antagonists. In one example, an anti-EPO antibody can bind to an EPO protein and prevent the formation of a complex between an EPO protein and a homo-EPOR. In another example, an anti-EPO antibody can bind to an EPO protein and prevent the formation of a complex between an EPO protein and a hetero-EPOR. In yet another example, an anti-EPOR antibody can bind to an EPO receptor subunit and prevent the formation of a homo-EPOR complex, the formation of a hetero-EPOR complex, the formation of a complex between an EPO protein and a homo-EPOR, or the formation of a complex between an EPO protein and a hetero-EPOR. In yet another example, an anti-CDC131 antibody can bind to a CDC131 subunit of a hetero-EPOR and prevent the formation of a hetero-EPOR complex or the formation of a complex between an EPO protein and a hetero-EPOR. In some embodiments, preventing the formation of a homo-EPOR complex or the formation of a complex between an EPO protein and a homo-EPOR can result in the prevention of the activation or function of homo-EPOR. In some embodiments, preventing the formation of a hetero-EPOR complex or a complex between an EPO protein and a hetero-EPOR can result in the prevention of the activation or function of the hetero-EPOR. In some embodiments, an anti-EPO antibody can bind to an EPO protein and inhibit the activity or reduce the level of a homo-EPOR or a hetero-EPOR without affecting the binding of the EPO protein to the homo-EPOR or the hetero-EPOR. In some embodiments, an anti-EPOR antibody can bind to the EPO receptor subunit of a homo-EPOR or a hetero-EPOR and inhibit the activity or reduce the level of a homo-EPOR or a hetero-EPOR without affecting the formation of a homo-EPOR or a hetero-EPOR complex or the formation of a complex between an EPO protein and a homo-EPOR or between an EPO protein and a hetero-EPOR.In some embodiments, the anti-CD131 antibody binds to the CD131 subunit of hetero-EPOR and can inhibit the activity or reduce the level of hetero-EPOR without affecting hetero-EPOR complex formation or the binding of EPO protein to the hetero-EPOR.

[0185] In some embodiments, the antibodies or functional fragments thereof described herein can bind to targets and act as agonists. In one example, an anti-EPOR antibody can bind to the EPO receptor subunit of a homo-EPOR in a manner that mimics the binding of EPO to the homo-EPOR. In another example, an anti-EPOR antibody can bind to the EPO receptor subunit of a hetero-EPOR in a manner that mimics the binding of EPO to the hetero-EPOR. In yet another example, an anti-CD131 antibody can bind to the CD131 subunit of a hetero-EPOR in a manner that mimics the binding of EPO to the hetero-EPOR. In some embodiments, mimicking the binding of EPO to a homo-EPOR can result in the activation of the homo-EPOR. In some embodiments, mimicking the binding of EPO to a hetero-EPOR can result in the activation of the hetero-EPOR. In some embodiments, an anti-EPO antibody can promote or increase the activity of a homo-EPOR or a hetero-EPOR without affecting the binding affinity of the EPO protein to the homo-EPOR or the hetero-EPOR. In some embodiments, an anti-EPOR antibody can promote or increase the activity of a homo-EPOR or a hetero-EPOR without affecting the binding affinity of an EPO protein to the homo-EPOR or the hetero-EPOR, or the binding affinity of the homo-EPOR (e.g., between the two EPO receptor subunits of the homo-EPOR) or the hetero-EPOR (e.g., between an EPO receptor subunit and the CD131 subunit of the hetero-EPOR). In some embodiments, an anti-CD131 antibody can promote or increase the activity of a hetero-EPOR without affecting the binding affinity of an EPO protein to the hetero-EPOR, or the binding affinity of the hetero-EPOR (e.g., between an EPO receptor subunit and the CD131 subunit of the hetero-EPOR).

[0186] In some embodiments, homo-EPOR activity or hetero-EPOR activity may include, but is not limited to, phosphorylation of the intracellular domain of homo-EPOR, the intracellular domain of hetero-EPOR, Janus tyrosine kinase 2 (Jak2), or signal transduction and activator of transcription 5 (Stat5). In some embodiments, homo-EPOR activity or hetero-EPOR activity may include, but is not limited to, activation of Jak2, Jak2 pathway, Stat5 pathway, mitogen-activated protein kinase (MAPK), MAPK pathway, extracellular signal-regulated kinase (ERK), ERK pathway, phosphatidylinositol 3-kinase (PI3K), PI3K pathway, v-Akt murine thymoma viral oncogene / protein kinase-B (Akt / PKB), Akt / PKB pathway, mammalian target of rapamycin (mTOR), or mTOR pathway. In some embodiments, the antibodies or functional fragments thereof described herein can inhibit activation or phosphorylation of homo-EPOR, hetero-EPOR, Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB, or mTOR. In some embodiments, the antibodies or functional fragments thereof described herein can inhibit activation of Jak2, Jak2 pathway, Stat5, Stat5 pathway, MAPK, MAPK pathway, ERK, ERK pathway, PI3K, PIK3 pathway, Akt / PKB, Akt / PKB pathway, mTOR, or mTOR pathway. In some embodiments, the antibodies or functional fragments thereof described herein can promote activation or phosphorylation of homo-EPOR, hetero-EPOR, Jak2, Stat5, or mTOR. In some embodiments, the antibodies or functional fragments thereof described herein can promote activation of Jak2, Jak2 pathway, Stat5, Stat5 pathway, MAPK, MAPK pathway, ERK, ERK pathway, PI3K, PIK3 pathway, Akt / PKB, Akt / PKB pathway, mTOR, or mTOR pathway. In some embodiments, the antibodies or functional fragments thereof described herein may not affect activation or phosphorylation of homo-EPOR, hetero-EPOR, Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB, or mTOR.In some embodiments, the antibody or functional fragment thereof described herein may not affect the activation of Jak2, Jak2 pathway, Stat5, Stat5 pathway, MAPK, MAPK pathway, ERK, ERK pathway, PI3K, PIK3 pathway, Akt / PKB, Akt / PKB pathway, mTOR, or mTOR pathway. In some embodiments, the activation or phosphorylation of homo-EPOR, hetero-EPOR, Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB, or mTOR can be measured using any method known in the art. Examples of methods for measuring the activation level of Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB, or mTOR include, but are not limited to, Western blot, flow cytometry assay, cell proliferation assay, apoptosis assay, or enzyme-linked immunosorbent assay (ELISA).

[0187] In some embodiments, the anti-EPO antibody, anti-EPOR antibody, or functional fragment thereof described herein can bind to a target and act as an agonist for hetero-EPOR. In some embodiments, the anti-EPO antibody, anti-EPOR antibody, or functional fragment thereof described herein can be an agonist for hetero-EPOR and can selectively bind to hetero-EPOR. In some embodiments, the anti-EPO antibody, anti-EPOR antibody, or functional fragment thereof can be an agonist for hetero-EPOR and can have a higher binding affinity for hetero-EPOR than for homo-EPOR. In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof described herein may be agonists for hetero-EPOR and have a binding affinity that is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, or at least about 28% higher than the homo-EPOR binding affinity. or at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% higher hetero-EPOR binding affinity.

[0188] In some embodiments, an anti-EPO antibody, an anti-EPOR antibody, or a functional fragment thereof may be an agonist for a hetero-EPOR and may have binding specificity or selectivity for a hetero-EPOR. In some embodiments, an anti-EPO antibody, an anti-EPOR antibody, or a functional fragment thereof may be an agonist for a hetero-EPOR and may have higher specificity or selectivity for a hetero-EPOR than for a homo-EPOR. For example, an anti-EPO antibody, an anti-EPOR antibody, or a functional fragment thereof may be an agonist for a hetero-EPOR and may have higher hetero-EPOR binding specificity or selectivity than homo-EPOR binding specificity or selectivity.In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof described herein may be agonists for hetero-EPOR and have a homo-EPOR binding specificity or selectivity that is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, or at least about 28%. or at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% higher hetero-EPOR binding specificity or selectivity.

[0189] In some embodiments, the anti-EPO antibody, anti-EPOR antibody, or functional fragment thereof described herein can bind to a target and act as an antagonist to hetero-EPOR. In some embodiments, the anti-EPO antibody, anti-EPOR antibody, or functional fragment thereof described herein can be an antagonist to hetero-EPOR and can selectively bind to hetero-EPOR. In some embodiments, the anti-EPO antibody, anti-EPOR antibody, or functional fragment thereof can be an antagonist to hetero-EPOR and can have a higher binding affinity to hetero-EPOR than to homo-EPOR. In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof described herein may be antagonists for hetero-EPOR and have binding affinity that is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27% or less than the homo-EPOR binding affinity. and / or at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% higher hetero-EPOR binding affinity.

[0190] In some embodiments, an anti-EPO antibody, an anti-EPOR antibody, or a functional fragment thereof may be an antagonist for hetero-EPOR and may have binding specificity or selectivity for hetero-EPOR. In some embodiments, an anti-EPO antibody, an anti-EPOR antibody, or a functional fragment thereof may be an antagonist for hetero-EPOR and may have higher specificity or selectivity for hetero-EPOR than for homo-EPOR. For example, an anti-EPO antibody, an anti-EPOR antibody, or a functional fragment thereof may be an antagonist for hetero-EPOR and may have higher hetero-EPOR binding specificity or selectivity than homo-EPOR binding specificity or selectivity.In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof described herein may be antagonists for hetero-EPOR and have a binding specificity or selectivity that is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27% or less than the homo-EPOR binding specificity or selectivity. and / or at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% higher hetero-EPOR binding specificity or selectivity.

[0191] In some embodiments, the anti-EPO antibody, anti-EPOR antibody, or functional fragment thereof described herein can bind to a target and act as an agonist for homo-EPOR. In some embodiments, the anti-EPO antibody, anti-EPOR antibody, or functional fragment thereof described herein can be an agonist for homo-EPOR and can selectively bind to homo-EPOR. In some embodiments, the anti-EPO antibody, anti-EPOR antibody, or functional fragment thereof can be an agonist for homo-EPOR and can have a higher binding affinity for homo-EPOR than for hetero-EPOR. In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof described herein may be agonists for homo-EPOR and have binding affinity that is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27% or less than the hetero-EPOR binding affinity. and / or at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% higher homo-EPOR binding affinity.

[0192] In some embodiments, an anti-EPO antibody, an anti-EPOR antibody, or a functional fragment thereof may be an agonist for homo-EPOR and may have binding specificity or selectivity for homo-EPOR. In some embodiments, an anti-EPO antibody, an anti-EPOR antibody, or a functional fragment thereof may be an agonist for homo-EPOR and may have higher specificity or selectivity for homo-EPOR than for hetero-EPOR. For example, an anti-EPO antibody, an anti-EPOR antibody, or a functional fragment thereof may be an agonist for homo-EPOR and may have higher homo-EPOR binding specificity or selectivity than hetero-EPOR binding specificity or selectivity. In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof described herein may be agonists for homo-EPOR and have a hetero-EPOR binding specificity or selectivity that is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27% or less. or at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% higher homo-EPOR binding specificity or selectivity.

[0193] In some embodiments, the anti-EPO antibody, anti-EPOR antibody, or functional fragment thereof described herein can bind to a target and act as an antagonist to homo-EPOR. In some embodiments, the anti-EPO antibody, anti-EPOR antibody, or functional fragment thereof described herein can be an antagonist to homo-EPOR and can selectively bind to homo-EPOR. In some embodiments, the anti-EPO antibody, anti-EPOR antibody, or functional fragment thereof can be an antagonist to homo-EPOR and can have a higher binding affinity to homo-EPOR than to hetero-EPOR. In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof described herein may be antagonists for homo-EPOR and have binding affinity that is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27% or less than the hetero-EPOR binding affinity. In some embodiments, the homo-EPOR binding affinity may be at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% higher.

[0194] In some embodiments, an anti-EPO antibody, an anti-EPOR antibody, or a functional fragment thereof may be an antagonist for homo-EPOR and may have binding specificity or selectivity for homo-EPOR. In some embodiments, an anti-EPO antibody, an anti-EPOR antibody, or a functional fragment thereof may be an antagonist for homo-EPOR and may have higher specificity or selectivity for homo-EPOR than for hetero-EPOR. For example, an anti-EPO antibody, an anti-EPOR antibody, or a functional fragment thereof may be an antagonist for homo-EPOR and may have higher homo-EPOR binding specificity or selectivity than hetero-EPOR binding specificity or selectivity.In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof described herein may be antagonists for homo-EPOR and have a hetero-EPOR binding specificity or selectivity that is at least about 1%, at least about 2%, at least about 3%, at least about 4%, at least about 5%, at least about 6%, at least about 7%, at least about 8%, at least about 9%, at least about 10%, at least about 11%, at least about 12%, at least about 13%, at least about 14%, at least about 15%, at least about 16%, at least about 17%, at least about 18%, at least about 19%, at least about 20%, at least about 21%, at least about 22%, at least about 23%, at least about 24%, at least about 25%, at least about 26%, at least about 27%, or less. In some embodiments, the antibody may have a homo-EPOR binding specificity or selectivity that is at least about 28%, at least about 29%, at least about 30%, at least about 31%, at least about 32%, at least about 33%, at least about 34%, at least about 35%, at least about 36%, at least about 37%, at least about 38%, at least about 39%, at least about 40%, at least about 41%, at least about 42%, at least about 43%, at least about 44%, at least about 45%, at least about 46%, at least about 47%, at least about 48%, at least about 49%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% higher.

[0195] In some embodiments, the antibody described herein has specificity for EPO, hetero-EPOR, or homo-EPOR, including all of the forms described above.Antibody can be engineered for use in a particular organism.The organism can be human, canine, or commercially valuable livestock, such as pig, horse, dog, cat, chicken, or other bird.Engineering such antibodies can include, for example, CDR splicing, humanization, humaneering, chimerization, or isolating human (or other organism) antibodies using any of the repertoire or monoclonal techniques known in the art.

[0196] Certain examples of antibodies with alternative scaffolds may include, but are not limited to, nanobodies, affibodies, microbodies, evibodies, and domain antibodies. Certain examples of alternative scaffolds useful for generating antibodies include, but are not limited to, single domain antibodies from camelids; protease inhibitors; human serum transferrin; CTLA-4; fibronectin, including but not limited to, fibronectin type III domain; C-type lectin-like domains; lipocalin family proteins; ankyrin repeat proteins; Z-domain of protein A; gamma-crystallin; tendamistat; neocarzinostatin; CBM4-2; T cell receptor; Im9; designed AR protein; designed TPR protein;zinc finger domain;pVIII;avian pancreatic polypeptide;GCN4;WW domain;Src homology 3 (SH3) domain;Src homology 2 (SH2) domain;PDZ domain;TEM-1 beta-lactamase;GFP;thioredoxin;Staphylcoccal nuclease;PHD-finger domain;CI-2;BPTI;APPI;HPSTI;ecotin;LACI-D1;LDTI;MTI-II;scorpion toxin;insect defensin A peptide;EETI-II;Min-23;CBD;PBP;cytochrome b 562transferrin; LDL receptor domain A; and ubiquitin. Certain examples of alternative scaffolds are discussed in Hey et al., "Artificial, non-antibody binding proteins for pharmaceutical and industrial applications" Trends in Biotechnology, 23:514-22 (2005) and Binz et al., "Engineering novel binding proteins from nonimmunoglobulin domains" Nature Biotechnology, 23:1257-68 (2005), both of which are incorporated by reference in their entirety for all purposes.

[0197] A bispecific or bifunctional antibody may comprise two different heavy / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including, but not limited to, fusion of hybridomas or linking of Fab' fragments. See, for example, Songsivilai & Lachmann Clin. Exp. Immunol 79: 315-321 (1990); Kostelny et al. J. Immunol. 148:1547-1553 (1992), which are incorporated herein by reference in their entirety for all purposes.

[0198] Bispecific antibody molecules can be classified into five different structural groups: (i) bispecific immunoglobulin G (BsIgG); (ii) IgG with additional antigen-binding moieties added; (iii) bispecific antibody fragments; (iv) bispecific fusion proteins; and (v) bispecific antibody conjugates. BsIgG is a format that is monovalent for each antigen. Exemplary BsIgG formats include, but are not limited to, crossMab, DAF (two-in-one), DAF (four-in-one), DutaMab, DT-IgG, knobs-in-holes common LC, knobs-in-hole assembly, charge pair, Fab arm exchange, SEEDbody, triomab, LUZ-Y, Fcab, κλ-body, orthogonal Fab. See Spiess et al. Mol. Immunol. 67(2015):95-106. Exemplary BsIgGs include catumaxomab (Fresenius Biotech, Trion Pharma, Neopharm), which contains an anti-CD3 arm and an anti-EpCAM arm, and ertumaxomab (Neovii Biotech, Fresenius Biotech), which targets CD3 and HER2. In some embodiments, the BsIgG comprises a heavy chain that is engineered for heterodimerization. For example, the heavy chain can be engineered for heterodimerization using a "knobs-into-holes" strategy, the SEED platform, a common heavy chain (e.g., in a kappa lambda body), and the use of a heterodimeric Fc region. See Spiess et al. Mol. Immunol. 67(2015):95-106. Strategies that have been used to avoid homodimeric heavy chain pairing in BsIgG include knobs-in-holes, duobodies, azymetric, charge-pair, HA-TF, SEED bodies, and differential Protein A affinity. See id.BsIgG can be produced by separate expression of the component antibodies in different host cells and subsequent purification / assembly into BsIgG. BsIgG can also be produced by expression of the component antibodies in a single host cell. BsIgG can be purified using affinity chromatography, for example, using protein A and sequential pH elution. IgG with additional antigen-binding moieties appended is another format of bispecific antibody molecule. For example, monospecific IgG can be engineered to have bispecificity by appending additional antigen-binding units onto the monospecific IgG, for example, at the N- or C-terminus of either the heavy or light chain. Exemplary additional antigen-binding units include single domain antibodies (e.g., variable heavy or variable light chains), engineered protein scaffolds, and paired antibody variable domains (e.g., single chain variable fragments or variable fragments). See ibid. Examples of added IgG formats include dual variable domain IgG (DVD-Ig), IgG(H)-scFv, scFv-(H)IgG, IgG(L)-scFv, scFv-(L)IgG, IgG(L,H)-Fv, IgG(H)-V, V(H)-IgG, IgG(L)-V, V(L)-IgG, KIH IgG-scFab, 2scFv-IgG, IgG-2scFv, scFv4-Ig, zybody, and DVI-IgG (four-in-one). See Spiess et al. Mol. Immunol. 67(2015):95-106. An example of an IgG-scFv is MM-141 (Merrimack Pharmaceuticals), which binds IGF-1R and HER3. Examples of DVD-Igs include ABT-981 (AbbVie), which binds to IL-1α and IL-1β, and ABT-122 (AbbVie), which binds to TNF and IL-17A.

[0199] Bispecific antibody fragments (BsAbs) are a format of bispecific antibody molecules that lack some or all of the antibody constant domains. For example, some BsAbs lack the Fc region. In some embodiments, bispecific antibody fragments comprise heavy and light chain regions connected by a peptide linker that allows efficient expression of the BsAb in a single host cell. Exemplary bispecific antibody fragments include, but are not limited to, nanobodies, nanobody-HAS, BiTEs, diabodies, DARTs, TandAbs, scDiabodies, scDiabody-CH3, diabody-CH3, triplebodies, miniantibodies, minibodies, TriBi minibodies, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F(ab')2, F(ab')2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, scDiabody-Fc, diabody-Fc, tandem scFv-Fc, and intrabodies. For example, the BiTE format includes tandem scFvs, where the constituent scFvs bind to CD3 on T cells and to a surface antigen on a cancer cell. Bispecific fusion proteins include, for example, antibody fragments linked to other proteins to add additional specificity and / or functionality. An example of a bispecific fusion protein is immTAC, which comprises an anti-CD3 scFv linked to an affinity matured T cell receptor that recognizes an HLA-presented peptide. In some embodiments, the dock-and-lock (DNL) method can be used to create bispecific antibody molecules with higher valency. Fusion to albumin binding proteins or human serum albumin can also extend the serum half-life of antibody fragments. In some embodiments, chemical conjugation, e.g., chemical conjugation of antibodies and / or antibody fragments, can be used to create BsAb molecules. Exemplary bispecific antibody conjugates include the CovX body format, in which a low molecular weight drug is site-specifically conjugated to each Fab arm or a single reactive lysine in an antibody or fragment thereof. In some embodiments, the conjugation improves the serum half-life of the low molecular weight drug.An exemplary CovX body is CVX-241 (NCT01004822), which comprises an antibody conjugated to two short peptides that inhibit either VEGF or Ang2. In some instances, the bispecific antibody may further comprise a linker. In some instances, the bispecific antibody may further comprise an Fc domain. The Fc domain may be, for example, a human IgG1 Fc domain. The Fc domain may comprise knobs-in-holes. In some instances, the bispecific antibody may further comprise a linker and an Fc domain. In some embodiments, the linker may be a peptide linker. Non-limiting examples of peptide linkers include (GS). n , (GGS) n , (GGGS) n , (GGSG) n , (GGSGG) n , or (GGGGS) n where n can be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10. For example, the linking peptide can be (GGGGS) 3 or (GGGGS) 4 The linkers described herein can be used for multispecific antibodies. In this embodiment, the multispecific antibody can have two or more linkers. In this embodiment, the linkers can be the same. Alternatively, the linkers can be different.

[0200] Antibody molecules can be produced by recombinant expression, e.g., recombinant expression of at least one or more components, in a host system. Exemplary host systems include eukaryotic cells (e.g., mammalian cells, e.g., CHO cells, or insect cells, e.g., SF9 or S2 cells) and prokaryotic cells (e.g., E. coli). Bispecific antibody molecules can be produced by separate expression of the components in different host cells and subsequent purification / assembly. Alternatively, antibody molecules can be produced by expression of the components in a single host cell. Purification of bispecific antibody molecules can be performed by various methods, such as, for example, affinity chromatography using protein A and sequential pH elution. In other embodiments, affinity tags, e.g., histidine-containing tags, myc tags, or streptavidin tags, can be used for purification.

[0201] In some embodiments, the antibody may be part of a conjugate molecule that includes an antibody and all or a portion of a prodrug. The term "prodrug" refers to a precursor or derivative form of a pharma- ceutically active substance. Prodrugs may be less cytotoxic to cells compared to the parent drug and can be enzymatically activated or converted to a more active cytotoxic parent form. Exemplary prodrugs may include, but are not limited to, phosphate-containing prodrugs, thiophosphate-containing prodrugs, sulfate-containing prodrugs, peptide-containing prodrugs, D-amino acid modified prodrugs, glycosylated prodrugs, beta-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs and optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs, which may be converted to a more active cytotoxic free drug. Examples of cytotoxic drugs that may be derivatized into a prodrug form may include, but are not limited to, the cytotoxic agents described above. See, for example, U.S. Patent No. 6,702,705.

[0202] In some aspects, an anti-EPOR, anti-CD131, or anti-EPO antibody may comprise an antigen-binding domain or antigen-binding fragment. In some embodiments, the antigen-binding domain or antigen-binding fragment may comprise a heavy chain variable region (VH), a light chain variable region (VL), or a combination thereof. In some embodiments, the heavy chain variable region (VH) may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH sequences listed in Table 5. In some embodiments, the VH may comprise any one of the VH sequences listed in Table 5. In some embodiments, the VH may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH sequences listed in Table 7. In some embodiments, the VH may comprise any one of the VH sequences listed in Table 7. In some embodiments, the VH may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH sequences listed in Table 9. In some embodiments, the VH may comprise any one of the VH sequences listed in Table 9.

[0203] In some embodiments, the light chain variable region (VL) may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VL sequences listed in Table 5. In some embodiments, the VL may comprise any one of the VL sequences listed in Table 5. In some embodiments, the VL may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VL sequences listed in Table 7. In some embodiments, the VL may comprise any one of the VL sequences listed in Table 7. In some embodiments, the VL may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VL sequences listed in Table 9. In some embodiments, the VL may comprise any one of the VL sequences listed in Table 9.

[0204] In some embodiments, the VH may comprise VH complementarity determining region 1 (VH-CDR1), VH-CDR2, or VH-CDR3. In some embodiments, the VH may comprise VH complementarity determining region 1 (VH-CDR1), VH-CDR2, and VH-CDR3.

[0205] In some embodiments, the VH-CDR1 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH-CDR1 sequences listed in Table 14. In some embodiments, VH-CDR1 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of SEQ ID NOs: 2068-2255. In some embodiments, VH-CDR1 may comprise a sequence of any one of SEQ ID NOs: 2068-2255. In some embodiments, an anti-EPOR antibody that binds to an EPO receptor subunit may comprise a VH-CDR1 sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH-CDR1 sequences listed in Table 14.

[0206] In some embodiments, the VH-CDR1 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH-CDR1 sequences listed in Table 15. In some embodiments, VH-CDR1 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of SEQ ID NOs: 2820-2948. In some embodiments, VH-CDR1 may comprise a sequence of any one of SEQ ID NOs: 2820-2948. In some embodiments, an anti-CD131 antibody that binds to an EPO receptor subunit may comprise a VH-CDR1 sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH-CDR1 sequences listed in Table 15.

[0207] In some embodiments, the VH-CDR1 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH-CDR1 sequences listed in Table 16. In some embodiments, VH-CDR1 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of SEQ ID NOs: 3336-3471. In some embodiments, VH-CDR1 may comprise a sequence of any one of SEQ ID NOs: 3336-3471. In some embodiments, an anti-EPOR antibody that binds to both the EPO receptor subunit and the CD131 subunit of a hetero-EPOR may comprise a VH-CDR1 sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH-CDR1 sequences listed in Table 16.

[0208] In some embodiments, the VH-CDR2 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH-CDR2 sequences in Table 14. In some embodiments, VH-CDR2 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of SEQ ID NOs: 2256-2443. In some embodiments, VH-CDR2 may comprise a sequence of any one of SEQ ID NOs: 2256-2443. In some embodiments, an anti-EPOR antibody that binds to an EPO receptor subunit may comprise a VH-CDR2 sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH-CDR2 sequences listed in Table 14.

[0209] In some embodiments, the VH-CDR2 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH-CDR2 sequences listed in Table 15. In some embodiments, VH-CDR2 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of SEQ ID NOs: 2949-3077. In some embodiments, VH-CDR2 may comprise a sequence of any one of SEQ ID NOs: 2949-3077. In some embodiments, an anti-CD131 antibody that binds to an EPO receptor subunit may comprise a VH-CDR2 sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH-CDR2 sequences listed in Table 15.

[0210] In some embodiments, the VH-CDR2 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH-CDR2 sequences listed in Table 16. In some embodiments, VH-CDR2 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of SEQ ID NOs: 3472-3607. In some embodiments, VH-CDR2 may comprise a sequence of any one of SEQ ID NOs: 3472-3607. In some embodiments, an anti-EPOR antibody that binds to both the EPO receptor subunit and the CD131 subunit of a hetero-EPOR may comprise a VH-CDR2 sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH-CDR2 sequences listed in Table 16.

[0211] In some embodiments, the VH-CDR3 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH-CDR3 sequences listed in Table 4. In some embodiments, the VH-CDR3 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of SEQ ID NOs: 63-250. In some embodiments, the VH-CDR3 may comprise a sequence of any one of SEQ ID NOs: 63-250. In some embodiments, an anti-EPOR antibody that binds to an EPO receptor subunit may comprise a VH-CDR3 sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH-CDR3 sequences listed in Table 4.

[0212] In some embodiments, the VH-CDR3 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH-CDR3 sequences listed in Table 6. In some embodiments, the VH-CDR3 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of SEQ ID NOs: 815-943. In some embodiments, the VH-CDR3 may comprise a sequence of any one of SEQ ID NOs: 815-943. In some embodiments, an anti-CD131 antibody that binds to the CD131 subunit of a hetero-EPOR may comprise a VH-CDR3 sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH-CDR3 sequences listed in Table 6.

[0213] In some embodiments, the VH-CDR3 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH-CDR3 sequences listed in Table 8. In some embodiments, the VH-CDR3 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of SEQ ID NOs: 1331-1466. In some embodiments, the VH-CDR3 may comprise a sequence of any one of SEQ ID NOs: 1331-1466. In some embodiments, an anti-EPOR antibody that binds to both the EPO receptor subunit and the CD131 subunit of a hetero-EPOR may comprise a VH-CDR3 sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH-CDR3 sequences listed in Table 8.

[0214] In some embodiments, the VL may comprise VL complementarity determining region 1 (VL-CDR1), VL-CDR2, or VL-CDR3. In some embodiments, the VL may comprise VL complementarity determining region 1 (VL-CDR1), VL-CDR2, and VL-CDR3.

[0215] In some embodiments, the VL-CDR1 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VL-CDR1 sequences listed in Table 14. In some embodiments, VL-CDR1 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of SEQ ID NOs: 2444-2631. In some embodiments, VL-CDR1 may comprise a sequence of any one of SEQ ID NOs: 2444-2631. In some embodiments, an anti-EPOR antibody that binds to an EPO receptor subunit may comprise a VL-CDR1 sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VL-CDR1 sequences listed in Table 14.

[0216] In some embodiments, the VL-CDR1 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VL-CDR1 sequences listed in Table 15. In some embodiments, VL-CDR1 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of SEQ ID NOs: 3078-3206. In some embodiments, VL-CDR1 may comprise a sequence of any one of SEQ ID NOs: 3078-3206. In some embodiments, an anti-CD131 antibody that binds to an EPO receptor subunit may comprise a VL-CDR1 sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VL-CDR1 sequences listed in Table 15.

[0217] In some embodiments, the VL-CDR1 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VL-CDR1 sequences listed in Table 16. In some embodiments, VL-CDR1 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of SEQ ID NOs: 3608-3743. In some embodiments, VL-CDR1 may comprise a sequence of any one of SEQ ID NOs: 3608-3743. In some embodiments, an anti-EPOR antibody that binds to both the EPO receptor subunit and the CD131 subunit of a hetero-EPOR may comprise a VL-CDR1 sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VL-CDR1 sequences listed in Table 16.

[0218] In some embodiments, the VL-CDR2 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VL-CDR2 sequences in Table 14. In some embodiments, the VL-CDR2 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of SEQ ID NOs: 2632-2819. In some embodiments, the VL-CDR2 may comprise a sequence of any one of SEQ ID NOs: 2632-2819. In some embodiments, an anti-EPOR antibody that binds to an EPO receptor subunit may comprise a VL-CDR2 sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VL-CDR2 sequences listed in Table 14.

[0219] In some embodiments, the VL-CDR2 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VL-CDR2 sequences listed in Table 15. In some embodiments, the VL-CDR2 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of SEQ ID NOs: 3207-3335. In some embodiments, the VL-CDR2 may comprise a sequence of any one of SEQ ID NOs: 3207-3335. In some embodiments, an anti-CD131 antibody that binds to an EPO receptor subunit may comprise a VL-CDR2 sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VL-CDR2 sequences listed in Table 15.

[0220] In some embodiments, the VL-CDR2 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VL-CDR2 sequences listed in Table 16. In some embodiments, the VL-CDR2 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of SEQ ID NOs: 3744-3879. In some embodiments, the VL-CDR2 may comprise a sequence of any one of SEQ ID NOs: 3744-3879. In some embodiments, an anti-EPOR antibody that binds to both the EPO receptor subunit and the CD131 subunit of a hetero-EPOR may comprise a VL-CDR2 sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VL-CDR2 sequences listed in Table 16.

[0221] In some embodiments, the VL-CDR3 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH-CDR3 sequences listed in Table 4. In some embodiments, the VL-CDR3 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of SEQ ID NOs: 251-438. In some embodiments, the VL-CDR3 may comprise a sequence of any one of SEQ ID NOs: 251-438. In some embodiments, an anti-EPOR antibody that binds to an EPO receptor subunit may comprise a VL-CDR3 sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VL-CDR3 sequences listed in Table 4.

[0222] In some embodiments, the VL-CDR3 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VL-CDR3 sequences listed in Table 6. In some embodiments, the VL-CDR3 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of SEQ ID NOs: 944-1072. In some embodiments, the VL-CDR3 may comprise a sequence of any one of SEQ ID NOs: 944-1072. In some embodiments, an anti-CD131 antibody that binds to a CD131 subunit of a hetero-EPOR may comprise a VL-CDR3 sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VL-CDR3 sequences listed in Table 6.

[0223] In some embodiments, the VL-CDR3 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VL-CDR3 sequences listed in Table 8. In some embodiments, the VL-CDR3 may comprise a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of SEQ ID NOs: 1467-1602. In some embodiments, the VL-CDR3 may comprise a sequence of any one of SEQ ID NOs: 1467-1602. In some embodiments, an anti-EPOR antibody that binds to both the EPO receptor subunit and the CD131 subunit of a hetero-EPOR may comprise a VL-CDR3 sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VL-CDR3 sequences listed in Table 8.

[0224] In some embodiments, an anti-EPOR antibody that binds to an EPO receptor subunit of a hetero-EPOR has at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 91% or 92% affinity to any one of SEQ ID NOs: 2068-2255. , 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of SEQ ID NOs: 2256 to 2443; %, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of SEQ ID NOs: 63 to 250. , 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to the VH-CDR3.In some embodiments, an anti-EPOR antibody that binds to an EPO receptor subunit of a hetero-EPOR has at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 91% or 92% affinity to any one of SEQ ID NOs: 2444-2631. , 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of SEQ ID NOs: 2632 to 2819, and VL-CDR1 comprising a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99. VL-CDR2 comprising a sequence having 1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to the sequences of SEQ ID NOs: 251 to 438, and at least 100%, 99.9%, 99.8%, The VL-CDR3 may comprise a sequence having 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity.

[0225] In some embodiments, the anti-CD131 antibody has at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 60%, 85%, 8 ... VH-CDR1 comprising a sequence having 5%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of SEQ ID NOs: 2949 to 3077, or at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 99.8%, 99.9 ... VH-CDR2 comprising a sequence having 7%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to the sequences of SEQ ID NOs: 815 to 943, and at least 100%, 99.9%, 99.8%, 99.7%, The VH-CDR3 may comprise a VH-CDR3 comprising a sequence having 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity.In some embodiments, the anti-CD131 antibody has at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 70%, 85%, 90%, 95 ... %, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of SEQ ID NOs: 3207 to 3335; %, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to the sequences of SEQ ID NOs: 944 to 1072, and The VL-CDR3 may comprise a sequence having 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity.

[0226] In some embodiments, an anti-EPOR antibody that binds to both the EPO receptor subunit and the CD131 subunit of a hetero-EPOR has a binding affinity of at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 95% or 96% to any one of SEQ ID NOs: 3336-3471. , 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of SEQ ID NOs: 3472 to 3607, and at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99. 2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of SEQ ID NOs: 1331 to 1466, and at least 100%, 99.9%, The VH-CDR3 may comprise a VH-CDR3 comprising a sequence having 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity.In some embodiments, an anti-EPOR antibody that binds to both the EPO receptor subunit and the CD131 subunit has at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 99% or 99% specific for any one of SEQ ID NOs: 3608-3743. VL-CDR1 comprising a sequence having 1%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of SEQ ID NOs: 3744 to 3879; and VL-CDR2 comprising a sequence having 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to the sequences of SEQ ID NOs: 1467-1602, and at least 100%, 99.9%, 99. The VL-CDR3 may comprise a sequence having 8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity.

[0227] In some embodiments, an anti-EPOR antibody that binds to a hetero-EPOR EPO receptor subunit may comprise a VH comprising an amino acid sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH sequences listed in Table 5. In some embodiments, an anti-EPOR antibody that binds to a hetero-EPOR EPO receptor subunit may comprise a VL comprising an amino acid sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VL sequences listed in Table 5. In some embodiments, an anti-EPOR antibody that binds to a hetero-EPOR EPO receptor subunit may comprise a VH comprising any one of the amino acid sequences of SEQ ID NOs: 439-626, and a VL comprising any one of the amino acid sequences of SEQ ID NOs: 627-814.

[0228] In some embodiments, an anti-CD131 antibody may comprise a VH comprising an amino acid sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH sequences listed in Table 7. In some embodiments, an anti-CD131 antibody may comprise a VL comprising an amino acid sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VL sequences listed in Table 7. In some embodiments, the anti-CD131 antibody may comprise a VH comprising the amino acid sequence of any one of SEQ ID NOs: 1073-1201, and a VL comprising the amino acid sequence of any one of SEQ ID NOs: 1202-1330.

[0229] In some embodiments, an anti-EPOR antibody that binds to both the EPO receptor subunit and the CD131 subunit of a hetero-EPOR may comprise a VH comprising an amino acid sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH sequences listed in Table 9. In some embodiments, an anti-EPOR antibody that binds to both the EPO receptor subunit and the CD131 subunit of a hetero-EPOR may comprise a VL comprising an amino acid sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VL sequences listed in Table 9. In some embodiments, an anti-EPOR antibody that binds to both a hetero-EPOR EPO receptor subunit and a CD131 subunit of a hetero-EPOR EPO receptor subunit may comprise a VH comprising any one of the amino acid sequences of SEQ ID NOs: 1603-1738, and a VL comprising any one of the amino acid sequences of SEQ ID NOs: 1739-1874.

[0230] In some embodiments, the anti-EPOR, anti-CD131, or anti-EPO antibody may comprise a VH sequence and a kappa chain variable region (VK) sequence. In some embodiments, the anti-EPOR, anti-CD131, or anti-EPO antibody may comprise a VH sequence and a lambda chain variable region. In some embodiments, an anti-EPOR antibody, anti-CD131 antibody, or anti-EPO antibody may comprise a VH sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH sequences listed in Table 10. In some embodiments, an anti-EPOR antibody, anti-CD131 antibody, or anti-EPO antibody may comprise a VH sequence of any one of SEQ ID NOs: 1739-1955. For example, an anti-EPOR antibody, anti-CD131 antibody, or anti-EPO antibody can include a VH sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VH sequences in any one of SEQ ID NOs: 1739-1955. In some embodiments, an anti-EPOR antibody, anti-CD131 antibody, or anti-EPO antibody may comprise a VK sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VK sequences listed in Table 10.In some embodiments, the anti-EPOR antibody, anti-CD131 antibody, or anti-EPO antibody may comprise a VK sequence of any one of SEQ ID NOs: 1956-1972. For example, an anti-EPOR antibody, anti-CD131 antibody, or anti-EPO antibody can include a VK sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any one of the VK sequences in any one of SEQ ID NOs: 1956-1972.

[0231] In some aspects, an anti-EPOR antibody, anti-CD131 antibody, or anti-EPO antibody may bind to hetero-EPOR or homo-EPOR or EPO (respectively) with an affinity of about 1 pM to about 100 nM, about 2.0 to about 5.1 nM, about 45 nM to about 300 nM, or about 2.0 to about 300 nM. In some embodiments, an anti-EPOR antibody, anti-CD131 antibody, or anti-EPO antibody may bind with an affinity of at least about 300 nM, at least about 140 nM, at least about 100 nM, at least about 5.1 nM, at least about 3.8 nM, or at least about 2.4 nM. In some aspects, binding affinity may be measured using any method known in the art. For example, binding affinity can be determined using surface plasmon resonance (SPR; Biacore), Kinexa biosensor, scintillation proximity assay, enzyme-linked immunosorbent assay (ELISA), ORIGEN immunoassay (IGEN), fluorescence quenching, fluorescence transfer, yeast display, or any combination thereof. In some embodiments, binding affinity can be screened using a suitable bioassay.

[0232] In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, anti-CD131 antibodies described herein have a mAb of less than about 600 nM, less than about 590 nM, less than about 580 nM, less than about 570 nM, less than about 560 nM, less than about 550 nM, less than about 540 nM, less than about 530 nM, less than about 520 nM, less than about 510 nM, less than about 500 nM, less than about 490 nM, less than about 480 nM, less than about 470 nM, less than about 460 nM, less than about 450 nM, less than about 440 nM, less than about 430 nM, less than about 420 nM, less than about 410 nM, less than about 400 nM, less than about 390 nM, less than about 380 nM, less than about 370 nM, less than about 360 nM, less than about 350 nM, less than about 340 nM, less than about 330 nM, less than about 320 nM, less than about 310 nM, less than about 300 nM, less than about 290 nM, less than about 280 nM, less than about 270 nM, less than about 260 nM, less than about 250 nM, less than about 240 nM, less than about 230 nM, less than about 220 nM, less than about 210 nM, less than about 200 nM, less than about 190 nM, less than about 180 nM, less than about 170 nM, less than about 160 nM, less than about 150 nM, less than about 140 nM, less than about 130 nM, less than about 120 nM, less than about 110 nM, less than about 100 nM, less than about 90 nM, less than about 80 nM, less than about 70 nM, less than about 50 nM, less than about 50 nM, less than about 49 nM, less than about 48 nM, less than about 47 nM, less than about 46 nM, less than about 45 nM, less than about 44 nM, less than about 43 nM, less than about 42 nM, less than about 41 nM, less than about 40 nM, less than about 39 nM, less than about 38 nM, less than about 37 nM, less than about 36 nM, less than about 35 nM, less than about 34 nM, less than about 33 nM, less than about 32 nM, less than about 31 nM, less than about 30 nM, less than about 29 nM, less than about 28 nM, less than about 27 nM, less than about 26 nM, less than about 25 nM, less than about 24 nM less than about 23 nM, less than about 22 nM, less than about 21 nM, less than about 20 nM, less than about 19 nM, less than about 18 nM, less than about 17 nM, less than about 16 nM, less than about 15 nM, less than about 14 nM, less than about 13 nM, less than about 12 nM, less than about 11 nM, less than about 10 nM, less than about 9 nM, less than about 8 nM, less than about 7 nM, less than about 6 nM, less than about 5 nM, less than about 4 nM, less than about 3 nM, less than about 2 nM, less than about 1 nM, less than about 990 pM, less than about 980 pM, less than about 970 pM, less than about 960 pM, less than about 950 pM, less than about 940 pM, less than about 930 pM, less than about 920 pM,Less than about 910 pM, less than about 900 pM, less than about 890 pM, less than about 880 pM, less than about 870 pM, less than about 860 pM, less than about 850 pM, less than about 840 pM, less than about 830 pM, less than about 820 pM, less than about 810 pM, less than about 800 pM, less than about 790 pM, less than about 780 pM, less than about 770 pM, less than about 760 pM, less than about 750 pM, less than about 740 pM, less than about 730 pM, less than about 720 pM less than about 710 pM, less than about 700 pM, less than about 690 pM, less than about 680 pM, less than about 670 pM, less than about 660 pM, less than about 650 pM, less than about 640 pM, less than about 630 pM, less than about 620 pM, less than about 610 pM, less than about 600 pM, less than about 590 pM, less than about 580 pM, less than about 570 pM, less than about 560 pM, less than about 550 pM, less than about 540 pM, less than about 530 pM, less than about 520 pM, less than about pM, less than about 510 pM, less than about 500 pM, less than about 490 pM, less than about 480 pM, less than about 470 pM, less than about 460 pM, less than about 450 pM, less than about 440 pM, less than about 430 pM, less than about 420 pM, less than about 410 pM, less than about 400 pM, less than about 390 pM, less than about 380 pM, less than about 370 pM, less than about 360 pM, less than about 350 pM, less than about 340 pM, less than about 330 pM, The antibody may have a binding affinity of less than 320 pM, less than about 310 pM, less than about 300 pM, less than about 290 pM, less than about 280 pM, less than about 270 pM, less than about 260 pM, less than about 250 pM, less than about 240 pM, less than about 230 pM, less than about 220 pM, less than about 210 pM, less than about 200 pM, less than about 190 pM, less than about 180 pM, less than about 170 pM, less than about 160 pM, or any integer therebetween. In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, anti-CD131 antibodies described herein have a concentration of less than 150 pM, less than about 140 pM, less than about 130 pM, less than about 120 pM, less than about 110 pM, less than about 100 pM, less than about 95 pM, less than about 90 pM, less than about 85 pM, less than about 80 pM, less than about 75 pM, less than about 70 pM, less than about 65 pM, less than about 60 pM, less than about 55 pM, less than about 50 pM, less than about 45 pM, less than about 40 pM, less than about 35 pM, less than about 30 pM, less than about 25 pM, less than about 20 pM, less than about 15 pM, less than about 10 pM, less than about 9 pM, less than about 8 pM, less than about 7 pM, less than about 6 pM, less than about 5 pM, less than about 4 pM,The binding affinity may be less than about 2 pM, less than about 1 pM, less than about 0.9 pM, less than about 0.8 pM, less than about 0.7 pM, less than about 0.6 pM, less than about 0.5 pM, less than about 0.4 pM, less than about 0.3 pM, less than about 0.2 pM, less than about 0.1 pM, less than about 0.09 pM, less than about 0.08 pM, less than about 0.07 pM, less than about 0.06 pM, less than about 0.05 pM, less than about 0.04 pM, less than about 0.03 pM, less than about 0.02 pM, less than about 0.01 pM, or any integer therebetween.

[0233] In some instances, the anti-EPO antibodies, anti-EPOR antibodies, or anti-CD131 antibodies described herein may have antagonistic effects. In some embodiments, the anti-EPO antibodies described herein can bind to EPO and inhibit or block EPO / EPOR interaction. For example, the anti-EPO antibodies can bind to EPO and inhibit EPO from binding to homo-EPOR or hetero-EPOR. In some embodiments, the level of inhibition is at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 109%, at least about 110%, at least about 111 at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%.

[0234] In some embodiments, the anti-EPOR antibody described herein can bind to the EPOR subunit of homo-EPOR or hetero-EPOR and inhibit or block homo-EPOR complex formation, hetero-EPOR complex formation, EPO / homo-EPOR interaction, or EPO / hetero-EPOR interaction. For example, the anti-EPOR antibody can bind to the EPOR subunit and inhibit the formation of homo-EPOR or hetero-EPOR. For example, the anti-EPOR antibody can bind to the EPOR subunit of homo-EPOR or hetero-EPOR and inhibit homo-EPOR or hetero-EPOR from binding to EPO. In some embodiments, the level of inhibition is at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114%, at least about 115%, at least about 116%, at least about 117%, at least about 118%, at least about 119%, at least about 120%, at least about 121%, at least about 122%, at least about 123%, at least about 124%, at least about at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%.

[0235] In some embodiments, the anti-CD131 antibodies described herein can bind to CD131 and inhibit or block hetero-EPOR complex formation or EPO / hetero-EPOR interaction. For example, the anti-CD131 antibodies can bind to CD131 and inhibit the formation of hetero-EPOR. For example, the anti-CD131 antibodies can bind to the CD131 subunit of hetero-EPOR and inhibit hetero-EPOR from binding to EPO. In some embodiments, the level of inhibition is at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 10 ... at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%.

[0236] In some instances, the anti-EPO, anti-EPOR, or anti-CD131 antibodies described herein may have an agonistic effect. In some embodiments, the anti-EPO antibodies described herein may bind to EPO and enhance or promote EPO / EPOR interaction. In some embodiments, the EPO / EPOR interaction is enhanced or promoted by the anti-EPO antibody at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least or at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%.

[0237] In some embodiments, the anti-EPOR antibodies described herein can bind to the EPOR subunit of homo-EPOR or hetero-EPOR and enhance or promote homo-EPOR complex formation, hetero-EPOR complex formation, EPO / homo-EPOR interaction, or EPO / hetero-EPOR interaction. In some embodiments, homo-EPOR complex formation, hetero-EPOR complex formation, EPO / homo-EPOR interaction, or EPO / hetero-EPOR interaction is enhanced or promoted by the anti-EPOR antibody by at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 71%, at least about 72%, or at least about 74%. %, at least about 73%, at least about 74%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100% enhanced.

[0238] In some embodiments, the anti-CD131 antibodies described herein can bind to CD131 and enhance or promote hetero-EPOR complex formation or EPO / hetero-EPOR interaction. In some embodiments, hetero-EPOR complex formation or EPO / hetero-EPOR interaction is enhanced or promoted by the anti-CD131 antibody by at least about 1%, at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 71%, at least about 72%, at least about 73%, at least about 74%, at least about 75%, at least about 76%, at least about 77%, at least about 78%, at least about 79%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, at least about 100%, at least about 101%, at least about 102%, at least about 103%, at least about 104%, at least about 105%, at least about 106%, at least about 107%, at least about 108%, at least about 109%, at least about 109%, at least about 110%, at least about 111%, at least about 112%, at least about 113%, at least about 114 In one embodiment, the method is enhanced by at least about 74%, at least about 75%, at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 89%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or at least about 100%.

[0239] In some embodiments, affinity maturation can be used with the antibodies disclosed herein to obtain anti-EPOR, anti-CD131, or anti-EPO antibodies of desired affinity. When anti-EPOR, anti-CD131, or anti-EPO antibodies are obtained from animals (e.g., transgenic animals with human antibody repertoires), the antibodies produced in the transgenic animals can be subjected to affinity maturation. Alternatively, antibodies from transgenic animals, or other techniques (e.g., display techniques), can be affinity matured using chain shuffling approaches and / or mutations of nucleic acids encoding VH and VL, followed by screening and / or selection for antibodies with higher affinity.

[0240] The most widely used method for minimizing the immunogenicity of non-human antibodies while maintaining specificity and affinity may include grafting the CDRs of non-human antibodies onto human frameworks, which are typically selected for their structural homology to the non-human frameworks (Jones et al., 1986, Nature 321:522-5; U.S. Patent No. 5,225,539, both of which are incorporated herein by reference in their entirety). The inclusion of some non-human residues at key positions in the framework may improve the affinity of CDR-grafted antibodies (Bajorath et al., 1995, J Biol Chem 270:22081-4; Martin et al., 1991, Methods Enzymol. 203:121-53; Al-Lazikani, 1997, J Mol Biol 273:927-48, all of which are incorporated herein by reference in their entirety). Exemplary methods for antibody humanization by CDR grafting are disclosed, for example, in US Pat. No. 6,180,370, which is incorporated herein by reference in its entirety.

[0241] An improvement over the traditional CDR grafting approach can use a variety of hybrid selection approaches, in which portions of a non-human antibody are combined with a library of complementary human antibody sequences in subsequent rounds of selection for antigen binding, during which most of the non-human sequences are gradually replaced with human sequences. For example, in the chain shuffling technique (Marks, et al., 1992, Biotechnology 10:779-83, which is incorporated herein by reference in its entirety for all purposes), one chain of a non-human antibody can be combined with a naive human repertoire of other chains, on the rationale that the affinity of the non-human chain is sufficient to constrain the selection of the human partner for the same epitope on the antigen. The selected human partner can then be used to guide the selection of human counterparts for the remaining non-human chains.

[0242] Other methodologies may include chain replacement techniques in which the non-human CDR3 is retained and only the framework and remainder of the V region, including CDR1 and 2, are replaced individually in sequential steps (e.g., U.S. Patent Application No. 20030166871; Rader, et al., Proc Natl Acad Sci USA 95:8910-15, 1998; Steinberger, et al., J. Biol. Chem. 275:36073-36078, 2000; Rader, et al., J. Biol. Chem. 275:13668-13676, 2000, all of which are incorporated by reference in their entirety for all purposes).

[0243] These techniques can be used to generate antibodies suitable for use in non-human subjects by engineering CDRs into framework regions of the subject species using approaches similar to the CDR grafting methods used to generate antibodies for use in humans.

[0244] The present disclosure encompasses, as compounds, pharma- ceutically acceptable salts of anti-EPOR, anti-CD131, or anti-EPO antibodies, including salts of anti-EPOR, anti-CD131, or anti-EPO antibodies, including fragments thereof, including those with a positive net charge, those with a negative net charge, and those with no charge, in pharmaceutical compositions, in their therapeutic and diagnostic uses, and in their production.

[0245] In some embodiments, the anti-EPO, anti-EPOR, and anti-CD131 antibodies described herein may have a half-life in human plasma of 1 minute to 1 hour. In some embodiments, the anti-EPO, anti-EPOR, and anti-CD131 antibodies described herein may have a half-life of about 1 minute to 2 minutes, about 1 minute to about 4 minutes, about 1 minute to about 5 minutes, about 1 minute to about 10 minutes, about 1 minute to about 15 minutes, about 1 minute to about 20 minutes, about 1 minute to about 25 minutes, about 1 minute to about 30 minutes, about 1 minute to about 35 minutes, about 1 minute to about 40 minutes, about 1 minute to about 45 minutes, about 1 minute to about 50 minutes, about 1 minute to about 55 minutes, or about 1 minute to about 1 hour. In some embodiments, the anti-EPO, anti-EPOR, and anti-CD131 antibodies described herein may have a half-life of about 1 minute, about 2 minutes, about 3 minutes, about 4 minutes, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, or about 1 hour. In some embodiments, the anti-EPO, anti-EPOR, and anti-CD131 antibodies described herein may have a half-life in human plasma of 1 hour to 5 days. In some embodiments, the anti-EPO, anti-EPOR, and anti-CD131 antibodies described herein may have a half-life of about 1 hour to about 120 hours.In some embodiments, the anti-EPO antibody, anti-EPOR antibody, and anti-CD131 antibody described herein are administered for about 1 hour to about 5 hours, about 1 hour to about 10 hours, about 1 hour to about 12 hours, about 1 hour to about 24 hours, about 1 hour to about 36 hours, about 1 hour to about 48 hours, about 1 hour to about 60 hours, about 1 hour to about 72 hours, about 1 hour to about 84 hours, about 1 hour to about 96 hours, about 1 hour to about 120 hours, about 5 hours to about 10 hours, about 5 hours to about 12 hours, about 5 hours to about 24 hours, or about 5 hours to about 36 hours. , about 5 hours to about 48 hours, about 5 hours to about 60 hours, about 5 hours to about 72 hours, about 5 hours to about 84 hours, about 5 hours to about 96 hours, about 5 hours to about 120 hours, about 10 hours to about 12 hours, about 10 hours to about 24 hours, about 10 hours to about 36 hours, about 10 hours to about 48 hours, about 10 hours to about 60 hours, about 10 hours to about 72 hours, about 10 hours to about 84 hours, about 10 hours to about 96 hours, about 10 hours to about 120 hours, about 12 hours to about 24 hours, about 12 hours to about 36 hours, about 12 hours to about 48 hours, about 12 hours to about 60 hours, about 12 hours to about 72 hours, about 12 hours to about 84 hours, about 12 hours to about 96 hours, about 12 hours to about 120 hours, about 24 hours to about 36 hours, about 24 hours to about 48 hours, about 24 hours to about 60 hours, about 24 hours to about 72 hours, about 24 hours to about 84 hours, about 24 hours to about 96 hours, about 24 hours to about 120 hours, about 36 hours to about 48 hours, about 36 hours to about 60 hours, about 36 hours to about 72 hours, about 36 hours to about 84 hours, about 36 hours to about 96 hours, It may have a half-life of about 36 hours to about 120 hours, about 48 hours to about 60 hours, about 48 hours to about 72 hours, about 48 hours to about 84 hours, about 48 hours to about 96 hours, about 48 hours to about 120 hours, about 60 hours to about 72 hours, about 60 hours to about 84 hours, about 60 hours to about 96 hours, about 60 hours to about 120 hours, about 72 hours to about 84 hours, about 72 hours to about 96 hours, about 72 hours to about 120 hours, about 84 hours to about 96 hours, about 84 hours to about 120 hours, or about 96 hours to about 120 hours. In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, anti-CD131 antibodies described herein may have a half-life of about 1 hour, about 5 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, or about 120 hours.In some embodiments, the anti-EPO, anti-EPOR, and anti-CD131 antibodies described herein may have a half-life of at least about 1 hour, about 5 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, or about 96 hours. In some embodiments, the anti-EPO, anti-EPOR, and anti-CD131 antibodies described herein may have a half-life of up to about 5 hours, about 10 hours, about 12 hours, about 24 hours, about 36 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, about 96 hours, or about 120 hours. In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, and anti-CD131 antibodies described herein may have a half-life of at least about 10 days, about 11 days, about 11 days, 12 days, 13 days, 14 days, 15 days, 16 days, 17 days, 18 days, 19 days, or 20 days. In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, and anti-CD131 antibodies described herein may have a half-life of about 10 to about 11 days, about 10 to about 12 days, about 10 to about 13 days, 10 to about 14 days, about 10 to about 15 days, about 10 to about 16 days, about 10 to about 17 days, about 10 to about 18 days, about 10 to about 19 days, or about 10 to about 20 days. In some embodiments, the anti-EPO, anti-EPOR, and anti-CD131 antibodies described herein may have a half-life of about 14 to about 17 days.

[0246] The present disclosure also encompasses bispecific or multispecific antibodies that may have specificity for at least two antigens. For example, the anti-EPO, anti-EPOR, and anti-CD131 antibodies described herein may be made as bispecific antibodies that can also bind to another target. In some embodiments, the anti-EPO, anti-EPOR, and anti-CD131 antibodies described herein may be made as bispecific antibodies that can also bind to cell surface markers associated with immune cells, signaling molecules associated with immune cells, or antigens associated with tumors. In some embodiments, the bispecific antibodies described herein may enhance the specificity and / or selectivity of the anti-EPO, anti-EPOR, and anti-CD131 antibodies described herein. For example, a bispecific antibody that can bind to cell surface markers of immune cells and either EPO, homo-EPOR, or hetero-EPOR may be used to target EPO, homo-EPOR, or hetero-EPOR in immune cells. For example, bispecific antibodies that can bind to immune cell signaling molecules and EPO, homo-EPOR, or hetero-EPOR can be used to target EPO, homo-EPOR, or hetero-EPOR in immune cells.For example, bispecific antibodies that can bind to tumor-associated antigens and EPO, homo-EPOR, or hetero-EPOR can be used to target EPO, homo-EPOR, or hetero-EPOR in tumor or cancer cells.

[0247] In some embodiments, the bispecific antibody can bind to (i) EPO receptor subunit of EPO, homo-EPOR or hetero-EPOR, CD131 subunit of hetero-EPOR, homo-EPOR, hetero-EPOR; and (ii) cell surface marker associated with immune cells. Examples of cell surface marker associated with immune cells may include, but are not limited to, lymphocyte antigen 75 (DEC205), XC motif chemokine receptor 1 (XCR1), or XC motif chemokine ligand 1 (XCL1). In some embodiments, the bispecific antibody described herein can enhance the specificity and / or selectivity of the anti-EPO, anti-EPOR, anti-CD131 antibody described herein to target immune cells. For example, a bispecific antibody that can bind to cell surface marker associated with immune cells and either EPO, homo-EPOR, or hetero-EPOR can be used to target EPO, homo-EPOR, or hetero-EPOR in immune cells. In some embodiments, the bispecific antibodies described herein can specifically and / or selectively target EPO, homo-EPOR, or hetero-EPOR in immune cells, and specifically and / or selectively increase or decrease the homo-EPOR or hetero-EPOR activity described herein in immune cells. In some embodiments, the bispecific antibodies described herein can be used to enhance the specificity and / or selectivity of the agonistic anti-EPO, anti-EPOR, anti-CD131 binding described herein in immune cells to promote immune tolerance before / after organ transplantation (e.g., bone marrow, kidney, heart, lung, liver, etc.). In some embodiments, the immune cells can include macrophages, dendritic cells, T cells, natural killer cells, or B cells.

[0248] In some embodiments, the bispecific antibody can bind to (i) EPO receptor subunit of EPO, homo-EPOR or hetero-EPOR, CD131 subunit of hetero-EPOR, homo-EPOR, hetero-EPOR; and (ii) signaling molecule associated with immune cells. Examples of signaling molecules associated with immune cells may include, but are not limited to, programmed cell death ligand 1 (PD-L1), T cell immunoglobulin and mucin domain containing 3 (Tim3), or triggering receptor expressed in myeloid ce...

Claims

1. A composition comprising an antibody or a functional fragment thereof, (i) the antibody or functional fragment thereof selectively binds to a target comprising erythropoietin (EPO) protein, an EPO receptor subunit, a CD131 subunit, or a combination thereof; (ii) binding of said antibody or said functional fragment thereof to said target (a) prevents the formation of an EPO protein-hetero-EPO receptor complex, said hetero-EPO receptor comprising said EPO receptor subunit and said CD131 subunit, (b) prevents the formation of a hetero-EPO receptor between said EPO receptor subunit and said CD131 subunit, or (c) prevents the activation of a hetero-EPO receptor comprising said EPO receptor subunit and said CD131 subunit; (iii) A composition, wherein the antibody or the functional fragment thereof comprises an antigen-binding domain.

2. The antigen-binding domain comprises: a heavy chain variable region (VH) comprising a VH complementarity determining region 1 (VH-CDR1) sequence, a VH-CDR2 sequence, and a VH-CDR3 sequence; and a light chain variable region (VL) comprising a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence; VH and kappa chain variable regions (VK); or VH and Lambda Chain Variable Regions The composition of claim 1 , comprising:

3. Formation of the EPO protein-hetero-EPO receptor complex; the formation of the hetero-EPO receptor between the EPO receptor subunit and the CD131 subunit; or Activation of a hetero-EPO receptor comprising the EPO receptor subunit and the CD131 subunit The composition of claim 1 or 2, wherein said prevention inhibits immune tolerance.

4. Formation of the EPO protein-hetero-EPO receptor complex; the formation of the hetero-EPO receptor between the EPO receptor subunit and the CD131 subunit; or Activation of a hetero-EPO receptor comprising the EPO receptor subunit and the CD131 subunit 4. The composition of claim 3, wherein said prevention promotes differentiation of a plurality of naive T cells into a plurality of effector T cells.

5. 5. The composition of claim 4, wherein the plurality of effector T cells express cluster of differentiation 45 (CD45), CD3, CD8, perforin, interferon gamma (IFNγ), granzyme B, or tumor necrosis factor alpha (TNFα).

6. Formation of the EPO protein-hetero-EPO receptor complex; the formation of the hetero-EPO receptor between the EPO receptor subunit and the CD131 subunit; or Activation of a hetero-EPO receptor comprising the EPO receptor subunit and the CD131 subunit 4. The composition of claim 3, wherein said prevention inhibits differentiation of a plurality of naive T cells into a plurality of regulatory T cells.

7. 7. The composition of claim 6, wherein the plurality of regulatory T cells express cluster of differentiation 4 (CD4), CD25, CD127, forkhead box P3 (FoxP3), CD39, protein tyrosine phosphatase receptor type C (CD45RA), interleukin-2 (IL-2), or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

8. Formation of the EPO protein-hetero-EPO receptor complex; the formation of the hetero-EPO receptor between the EPO receptor subunit and the CD131 subunit; or Activation of a hetero-EPO receptor comprising the EPO receptor subunit and the CD131 subunit The composition of claim 1 or 2, wherein said prevention increases a plurality of precursor exhaustion T cells.

9. 9. The composition of claim 8, wherein the plurality of precursor exhausted T cells express cluster of differentiation 44 (CD44), signaling lymphocyte activation molecule family member 6 (SLAMF6), or T cell factor 1 (TCF1).

10. Formation of the EPO protein-hetero-EPO receptor complex; the formation of the hetero-EPO receptor between the EPO receptor subunit and the CD131 subunit; or Activation of a hetero-EPO receptor comprising the EPO receptor subunit and the CD131 subunit The composition of claim 1 or 2, wherein said prevention stimulates an immune response in cancer.

11. Formation of the EPO protein-hetero-EPO receptor complex; the formation of the hetero-EPO receptor between the EPO receptor subunit and the CD131 subunit; or Activation of a hetero-EPO receptor comprising the EPO receptor subunit and the CD131 subunit 3. The composition of claim 1 or 2, wherein said prevention sensitizes the cancer cell to an immune checkpoint inhibitor.

12. 12. The composition of claim 11, wherein the immune checkpoint inhibitor comprises a cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitor, a programmed cell death 1 (PD-1) inhibitor, or a programmed cell death ligand 1 (PD-L1) inhibitor.

13. The composition of claim 12, wherein the CTLA-4 inhibitor comprises an anti-CTLA-4 antibody.

14. The composition of claim 12, wherein the PD-1 inhibitor comprises an anti-PD-1 antibody.

15. The composition of claim 12, wherein the PD-L1 inhibitor comprises an anti-PD-L1 antibody.

16. Formation of the EPO protein-hetero-EPO receptor complex; the formation of the hetero-EPO receptor between the EPO receptor subunit and the CD131 subunit; or Activation of a hetero-EPO receptor comprising the EPO receptor subunit and the CD131 subunit The composition of claim 1 or 2, wherein said prevention attenuates tumor growth.

17. The composition of any one of claims 1 to 16, wherein the antibody or functional fragment thereof is, is derived from, or is a combination of IgG, IgM, IgE, IgA, IgD.

18. The composition of any one of claims 1 to 17, wherein the antibody or the functional fragment thereof comprises a monoclonal antibody, a grafted antibody, a chimeric antibody, a human antibody, a humanized antibody, or a combination thereof.

19. The antigen-binding domain is Fab, Fab', (Fab') ２ 19. The composition of any one of claims 1 to 18, comprising a variable fragment (Fv), a single chain variable fragment (scFv), a scFv-Fc, a Fab-Fc, a VHH, a non-antibody scaffold, or a combination thereof.

20. The composition of any one of claims 1 to 19, wherein the antigen binding domain is isolated, recombinant, synthetic, or a combination thereof.

21. The composition of any one of claims 2 to 20, wherein the VH-CDR3 comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 63 to 250.

22. The composition of any one of claims 2 to 20, wherein the VH-CDR3 comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 815 to 943.

23. The composition of any one of claims 2 to 20, wherein the VH-CDR3 comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1331 to 1466.

24. The composition of any one of claims 2 to 20, wherein the VL-CDR3 comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 251 to 438.

25. The composition of any one of claims 2 to 20, wherein the VL-CDR3 comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 944 to 1072.

26. The composition of any one of claims 2 to 20, wherein the VL-CDR3 comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1467-1602.

27. The composition of any one of claims 2 to 20, wherein the VH comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 439-626.

28. The composition of any one of claims 2 to 20, wherein the VH comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1073-1201.

29. The composition of any one of claims 2 to 20, wherein the VH comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1603 to 1738.

30. The composition of any one of claims 2 to 20, wherein the VL comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 627-814.

31. The composition of any one of claims 2 to 20, wherein the VL comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1202 to 1330.

32. The composition of any one of claims 2 to 20, wherein the VL comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1739-1874.

33. The composition of any one of claims 2 to 20, wherein the VH comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1739-1955.

34. The composition of any one of claims 2 to 20, wherein the VK comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1956-1972.

35. A composition comprising a nucleic acid sequence encoding an antibody or a functional fragment thereof of the composition according to any one of claims 1 to 34.

36. A cell comprising the composition of any one of claims 1 to 35.

37. 36. A method of treating a disease or condition in a subject in need thereof, comprising administering to said subject a composition according to any one of claims 1 to 35.

38. 38. The method of claim 37, further comprising inhibiting immune tolerance in the subject.

39. 40. The method of claim 38, wherein inhibiting immune tolerance comprises increasing an immune response to the vaccine when the vaccine is administered to the subject.

40. 40. The method of claim 38, wherein the step of inhibiting immune tolerance comprises increasing an immune response to a viral or bacterial infection in the subject.

41. 40. The method of claim 38, wherein the step of inhibiting immune tolerance comprises increasing an immune response to an antigen produced by the cancer.

42. 38. The method of claim 37, wherein the disease or condition comprises cancer or an infectious disease.

43. 43. The method of claim 42, wherein the cancer comprises lung cancer, breast cancer, colon cancer, brain cancer, melanoma, hepatocellular carcinoma, or liver cancer.

44. 43. The method of claim 42, wherein the cancer is melanoma.

45. 43. The method of claim 42, wherein the cancer is liver cancer.

46. 43. The method of claim 42, wherein the cancer is colon cancer.

47. 43. The method of claim 42, wherein the cancer is breast cancer.

48. 1. A method for treating cancer, comprising administering a composition or a derivative thereof to a subject having or at risk of having cancer, wherein the composition or derivative thereof inhibits hetero-erythropoietin (EPO) receptor activity in the subject.

49. The method of claim 48, wherein the hetero-EPO receptor is expressed in a myeloid cell.

50. A composition comprising an antibody or a functional fragment thereof, (i) the antibody or functional fragment thereof selectively binds to a target comprising erythropoietin (EPO) protein, an EPO receptor subunit, a CD131 subunit, or a combination thereof; (ii) binding of said antibody or said functional fragment thereof to said target (a) promotes the formation of an EPO protein-hetero-EPO receptor complex, said hetero-EPO receptor comprising said EPO receptor subunit and said CD131 subunit, (b) promotes the formation of a hetero-EPO receptor between said EPO receptor subunit and said CD131 subunit, or (c) promotes the activation of a hetero-EPO receptor comprising said EPO receptor subunit and said CD131 subunit; (iii) A composition, wherein the antibody or the functional fragment thereof comprises an antigen-binding domain.

51. The antigen-binding domain comprises: a heavy chain variable region (VH) comprising a VH complementarity determining region 1 (VH-CDR1) sequence, a VH-CDR2 sequence, and a VH-CDR3 sequence; and a light chain variable region (VL) comprising a VL-CDR1 sequence, a VL-CDR2 sequence, and a VL-CDR3 sequence; VH and kappa chain variable regions (VK); or VH and Lambda Chain Variable Regions 51. The composition of claim 50, comprising:

52. The composition of claim 50 or claim 51, wherein the hetero-EPO receptor is expressed in a myeloid cell.

53. Formation of the EPO protein-hetero-EPO receptor complex; the formation of the hetero-EPO receptor between the EPO receptor subunit and the CD131 subunit; or Activation of the hetero-EPO receptor between the EPO receptor subunit and the CD131 subunit The composition of any one of claims 50 to 52, wherein said promotion induces antigen-specific immune tolerance.

54. Formation of the EPO protein-hetero-EPO receptor complex; the formation of the hetero-EPO receptor between the EPO receptor subunit and the CD131 subunit; or Activation of the hetero-EPO receptor between the EPO receptor subunit and the CD131 subunit 53. The composition of claim 52, wherein said promotion inhibits differentiation of a plurality of naive T cells into a plurality of effector T cells.

55. 55. The composition of claim 54, wherein the plurality of effector T cells express cluster of differentiation 45 (CD45), CD3, CD8, perforin, interferon gamma (IFNγ), granzyme B, or tumor necrosis factor alpha (TNFα).

56. Formation of the EPO protein-hetero-EPO receptor complex; the formation of the hetero-EPO receptor between the EPO receptor subunit and the CD131 subunit; or Activation of the hetero-EPO receptor between the EPO receptor subunit and the CD131 subunit 53. The composition of claim 52, wherein said promoting promotes differentiation of a plurality of naive T cells into a plurality of regulatory T cells.

57. 57. The composition of claim 56, wherein the plurality of regulatory T cells express cluster of differentiation 4 (CD4), CD25, CD127, forkhead box P3 (FoxP3), CD39, protein tyrosine phosphatase receptor type C (CD45RA), interleukin-2 (IL-2), or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

58. The composition of any one of claims 50 to 57, wherein the antibody or functional fragment thereof does not affect homo-EPO receptor activity.

59. The composition of any one of claims 50 to 57, wherein the antibody or functional fragment thereof does not bind to a homo-EPO receptor comprising at least two EPO receptor subunits.

60. Formation of the EPO protein-hetero-EPO receptor complex; the formation of the hetero-EPO receptor between the EPO receptor subunit and the CD131 subunit; or Activation of the hetero-EPO receptor between the EPO receptor subunit and the CD131 subunit 60. The composition of any one of claims 50 to 59, wherein said promotion reduces an immune response when administered to a subject with an autoimmune disease or a subject with a transplanted organ.

61. 61. The composition of claim 60, wherein the transplanted organ comprises bone marrow, kidney, liver, lung, or heart.

62. 61. The composition of claim 60, wherein the autoimmune disease comprises rheumatoid arthritis, systemic lupus erythematosus, or multiple sclerosis.

63. Formation of the EPO protein-hetero-EPO receptor complex; the formation of the hetero-EPO receptor between the EPO receptor subunit and the CD131 subunit; or Activation of the hetero-EPO receptor between the EPO receptor subunit and the CD131 subunit 60. The composition of any one of claims 50-59, wherein said promotion reduces systemic chronic inflammation when administered to a subject suffering from systemic chronic inflammation.

64. The composition of any one of claims 50 to 63, wherein the antibody or functional fragment thereof is, is derived from, or is a combination of IgG, IgM, IgE, IgA, IgD.

65. The composition of any one of claims 50 to 64, wherein the antibody or the functional fragment thereof comprises a monoclonal antibody, a grafted antibody, a chimeric antibody, a human antibody, a humanized antibody, or a combination thereof.

66. The antigen-binding domain is Fab, Fab', (Fab') ２ , a variable fragment (Fv), a single chain variable fragment (scFv), a scFv-Fc, a Fab-Fc, a VHH, a non-antibody scaffold, or a combination thereof.

67. 67. The composition of any one of claims 50 to 66, wherein the antigen binding domain is isolated, recombinant, synthetic, or a combination thereof.

68. The composition of any one of claims 51 to 67, wherein the VH-CDR3 comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 63 to 250.

69. The composition of any one of claims 51 to 67, wherein the VH-CDR3 comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 815 to 943.

70. The composition of any one of claims 51 to 67, wherein the VH-CDR3 comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1331 to 1466.

71. The composition of any one of claims 51 to 67, wherein the VL-CDR3 comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 251 to 438.

72. The composition of any one of claims 51 to 67, wherein the VL-CDR3 comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 944 to 1072.

73. The composition of any one of claims 51 to 67, wherein the VL-CDR3 comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1467 to 1602.

74. The composition of any one of claims 51 to 67, wherein the VH comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 439 to 626.

75. The composition of any one of claims 50-67, wherein the VH comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1073-1201.

76. The composition of any one of claims 50-67, wherein the VH comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1603-1738.

77. The composition of any one of claims 50 to 67, wherein the VL comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 627 to 814.

78. The composition of any one of claims 50 to 67, wherein the VL comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1202 to 1330.

79. The composition of any one of claims 50 to 67, wherein the VL comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1739 to 1874.

80. The composition of any one of claims 50-67, wherein the VH comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1739-1955.

81. 68. The composition of any one of claims 50-67, wherein the VK comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1956-1972.

82. 2. The composition of any one of the preceding claims, wherein the antibody further comprises a binding domain that selectively binds to an antigen associated with a tumor, a cell surface marker associated with an immune cell, or a signaling molecule associated with an immune cell.

83. 83. The composition of claim 82, wherein the tumor-associated antigen is selected from the group consisting of PD1, HER2, EpCAM, CEA, CEACAM5, EGFR, CD33, CD19, CD20, CD22, and any combination thereof.

84. The composition of claim 82, wherein the cell surface marker is DEC205, XCR1, or XCL1.

85. The composition of claim 82, wherein the signaling molecule is PD-L1, Tim3, or TREM2.

86. A composition comprising a nucleic acid sequence encoding an antibody or a functional fragment thereof of the composition according to any one of claims 50 to 85.

87. A cell comprising the composition of any one of claims 50 to 86.

88. 90. A method of treating a disease or condition in a subject in need thereof, comprising administering to said subject a composition according to any one of claims 50 to 86.

89. 89. The method of claim 88, wherein the disease or condition comprises an autoimmune disease.

90. 90. The method of claim 88, wherein the subject has received or will receive an organ transplant or an exogenous therapeutic protein.

91. 1. A composition for administration to a subject having cancer or a chronic infectious disease condition, wherein said composition or a derivative thereof inhibits erythropoietin (EPO) receptor activity in myeloid cells of said subject.

92. The composition of claim 91, wherein the composition is an antibody or a functional fragment thereof.

93. 92. The composition of claim 91, wherein the myeloid cells are selected from the group consisting of macrophages, monocytes, dendritic cells, basophils, neutrophils, and eosinophils.

94. The composition of claim 91, wherein the EPO receptor comprises a homo-EPO receptor comprising at least two EPO receptor subunits, or a hetero-EPO receptor comprising an EPO receptor subunit and a CD131 subunit.

95. The composition of claim 91, wherein the EPO receptor is a hetero-EPO receptor comprising an EPO receptor subunit and a CD131 subunit.

96. The composition of claim 91, wherein the composition is an antibody or a functional fragment thereof.

97. The composition according to any one of claims 91 to 96, wherein the composition is a soluble fragment of the EPO receptor.

98. The composition of claim 97, wherein the soluble fragment is capable of binding to EPO to form a complex.

99. 99. The composition of claim 98, wherein the complex is capable of preventing EPO receptor activity.

100. 92. The composition of claim 91, wherein the composition or derivative thereof comprises an engineered erythropoietin (EPO) protein, wherein the engineered EPO protein inhibits hetero-erythropoietin (EPO) receptor activity in myeloid cells.

101. A composition comprising an engineered erythropoietin (EPO) protein, wherein the engineered EPO protein inhibits hetero-erythropoietin (EPO) receptor activity in myeloid cells.

102. The engineered EPO protein is K20A, N24Q, N24A, N24S, N38Q, N38A, N38S, K45A, K52A, Q58A, E62R, E62A, Q65A, L69A, E72A, R76E, R76A, L80A, N83Q, N83A, N83S, S84A, S85A, K97A, K116A, G151A, R103A, K45D, N147K, R150E, Q65A, E72R, N83A, K140A, K152A, or K154A 102. The composition of claim 100 or 101, comprising at least one amino acid substitution comprising:

103. The composition of claim 101 or 102, wherein the composition or a derivative thereof inhibits immune tolerance.

104. The composition of claim 101 or 102, wherein the composition or a derivative thereof promotes an immune response.

105. 103. The composition of claim 101 or 102, wherein the composition or a derivative thereof promotes differentiation of a plurality of naive T cells into a plurality of effector T cells.

106. The composition of claim 105, wherein the plurality of effector T cells express cluster of differentiation 45 (CD45), CD3, CD8, perforin, interferon gamma (IFNγ), granzyme B, or tumor necrosis factor alpha (TNFα).

107. The composition of claim 101 or 102, wherein the composition or a derivative thereof inhibits differentiation of a plurality of naive T cells into a plurality of regulatory T cells.

108. 108. The composition of claim 107, wherein the plurality of regulatory T cells express cluster of differentiation 4 (CD4), CD25, CD127, forkhead box P3 (FoxP3), CD39, protein tyrosine phosphatase receptor type C (CD45RA), interleukin-2 (IL-2), or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

109. The composition of claim 101 or 102, wherein the composition or a derivative thereof increases a plurality of precursor exhaustion T cells.

110. The composition of claim 109, wherein the plurality of precursor exhausted T cells express cluster of differentiation 44 (CD44), signaling lymphocyte activation molecule family member 6 (SLAMF6) or T cell factor 1 (TCF1).

111. The composition of claim 101 or 102, wherein the composition or a derivative thereof stimulates an immune response in cancer.

112. The composition of claim 101 or 102, wherein the composition or a derivative thereof sensitizes cancer cells to an immune checkpoint inhibitor.

113. 113. The composition of claim 112, wherein the immune checkpoint inhibitor comprises a cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) inhibitor, a programmed cell death 1 (PD-1) inhibitor, or a programmed cell death ligand 1 (PD-L1) inhibitor.

114. The composition of claim 113, wherein the CTLA-4 inhibitor comprises an anti-CTLA-4 antibody.

115. The composition of claim 113, wherein the PD-1 inhibitor comprises an anti-PD-1 antibody.

116. The composition of claim 113, wherein the PD-L1 inhibitor comprises an anti-PD-L1 antibody.

117. 103. The composition of claim 101 or 102, wherein the composition or a derivative thereof reduces the size of the cancer or attenuates the growth of the cancer.

118. The composition of any one of claims 102-117, wherein the at least one amino acid substitution comprises R103A.

119. The composition of any one of claims 102-117, wherein the at least one amino acid substitution comprises E72A.

120. The composition of any one of claims 102-117, wherein the at least one amino acid substitution comprises Q58A.

121. The composition of any one of claims 102-117, wherein the at least one amino acid substitution comprises L69A.

122. The composition of any one of claims 102 to 117, wherein the at least one amino acid substitution comprises L80A.

123. The composition of any one of claims 102-117, wherein the at least one amino acid substitution comprises N147K or R103A.

124. The composition of any one of claims 102-117, wherein the at least one amino acid substitution comprises R150E or R103A.

125. The composition of any one of claims 102-117, wherein the at least one amino acid substitution comprises Q65A or E72R.

126. The composition of any one of claims 102-117, wherein the at least one amino acid substitution comprises Q65A, E72R, or N83A.

127. The composition of any one of claims 102-117, wherein the at least one amino acid substitution comprises K20A, K45A, or K52A.

128. The composition of any one of claims 102-117, wherein the at least one amino acid substitution comprises K140A or K152A.

129. The composition of any one of claims 102-117, wherein the at least one amino acid substitution comprises K140A, K152A, or K154A.

130. The composition of any one of claims 102-117, wherein the at least one amino acid substitution comprises K20A, K45A, K52A, K140A, K152A, or K154A.

131. The composition of any one of claims 102 to 130, wherein the position is determined by alignment with SEQ ID NO:

1.

132. The composition of any one of claims 101-131, wherein the engineered EPO further comprises amino acid modifications including carbamylation or PEGylation.

133. The composition of claim 132, wherein the amino acid modification comprises carbamylation of one or more lysine residues.

134. The composition of any one of claims 101 to 133, wherein the engineered EPO protein has a lower binding affinity to a hetero-EPO receptor compared to a corresponding wild-type EPO protein without the at least one amino acid substitution.

135. The composition of any one of claims 101 to 134, wherein the hetero-EPO receptor activity comprises phosphorylation of the intracellular domain of the hetero-EPO receptor or activation of Janus tyrosine kinase 2 (Jak2), signal transducer and activator of transcription 5 (Stat5), mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K), v-Akt murine thymoma viral oncogene / protein kinase-B (Akt / PKB), or mammalian target of rapamycin (mTOR).

136. The composition of any one of claims 101 to 135, wherein the hetero-EPO receptor activity is measured by Western blot, enzyme-linked immunosorbent assay (ELISA), flow cytometry assay, cell proliferation assay, apoptosis assay, or a combination thereof.

137. The composition of any one of claims 101 to 136, wherein the engineered EPO protein has a higher binding affinity to the homo-EPO receptor compared to the corresponding wild-type EPO protein without the at least one amino acid substitution.

138. The composition of any one of claims 101 to 136, wherein the engineered EPO protein has the same level of binding affinity to the homo-EPO receptor as compared to the corresponding wild-type EPO protein without the at least one amino acid substitution.

139. The composition of any one of claims 101 to 136, wherein the engineered EPO protein binds to a homo-EPO receptor with a lower binding affinity than to a hetero-EPO receptor.

140. 137. The composition of any one of claims 101 to 136, wherein the engineered EPO protein does not affect or inhibit homo-EPO receptor activity.

141. 141. The composition of any one of claims 101-140, wherein the engineered EPO has a half-life of at least 5 hours.

142. The composition of any one of claims 101 to 141, wherein the engineered EPO protein comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1973 to 2019.

143. The composition of any one of claims 101-142, wherein the myeloid cells comprise granulocytes, monocytes, macrophages, or dendritic cells.

144. A composition comprising a nucleic acid sequence encoding an engineered EPO protein of any one of claims 101 to 143.

145. A cell comprising the composition of any one of claims 101 to 144.

146. 146. A method of treating a disease or condition in a subject in need thereof, comprising administering to the subject a composition according to any one of claims 101 to 144 or a cell according to claim 145.

147. 146. A method of treating anemia in a subject in need thereof, comprising administering to the subject a composition of any one of claims 101-144 or a cell of claim 145, wherein the subject has cancer.

148. 148. The method of any one of claims 101-147, wherein the cancer comprises lung cancer, breast cancer, colon cancer, brain cancer, melanoma, hepatocellular carcinoma, or liver cancer.

149. 148. The method of any one of claims 101 to 147, wherein the cancer is melanoma.

150. The method of any one of claims 101 to 147, wherein the cancer is liver cancer.

151. 148. The method of any one of claims 101 to 147, wherein the cancer is colon cancer.

152. The method of any one of claims 101 to 147, wherein the cancer is breast cancer.

153. A composition comprising an engineered erythropoietin (EPO) protein, wherein the engineered EPO protein promotes hetero-erythropoietin (EPO) receptor activity to reduce an immune response, the hetero-EPO receptor comprising an EPO receptor subunit and a CD131 subunit.

154. The engineered EPO protein is At least one amino acid modification, and / or at least one amino acid substitution including K20A, N24Q, N24A, N24S, N38Q, N38A, N38S, K45A, K52A, Q58A, E62R, E62A, Q65A, L69A, E72A, R76E, R76A, L80A, N83Q, N83A, N83S, S84A, S85A, K97A, K116A, G151A, R103A, K45D, N147K, R150E, Q65A, E72R, N83A, K140A, K152A, or K154A; The composition of claim 153, comprising:

155. The composition of claim 153 or 154, wherein the promotion of hetero-EPO receptor activity reduces an immune response when administered to a subject with an autoimmune disease or a subject with a transplanted organ.

156. 156. The composition of claim 155, wherein the transplanted organ comprises bone marrow, kidney, liver, lung, or heart.

157. The composition of claim 155, wherein the autoimmune disease comprises rheumatoid arthritis, systemic lupus erythematosus, or multiple sclerosis.

158. The composition of claim 153 or 154, wherein the promotion of hetero-EPO receptor activity reduces systemic chronic inflammation when administered to a subject suffering from systemic chronic inflammation.

159. The composition of any one of claims 153 to 158, wherein said promotion of said hetero-EPO receptor activity induces antigen-specific immune tolerance.

160. The composition of any one of claims 153 to 158, wherein said promotion of said hetero-EPO receptor activity inhibits differentiation of naive T cells into effector T cells.

161. The composition of claim 160, wherein the plurality of effector T cells express cluster of differentiation 45 (CD45), CD3, CD8, perforin, interferon gamma (IFNγ), granzyme B, or tumor necrosis factor alpha (TNFα).

162. The composition of any one of claims 153 to 158, wherein said promotion of said hetero-EPO receptor activity promotes differentiation of a plurality of naive T cells into a plurality of regulatory T cells.

163. 163. The composition of claim 162, wherein said plurality of regulatory T cells express cluster of differentiation 4 (CD4), CD25, CD127, forkhead box P3 (FoxP3), CD39, protein tyrosine phosphatase receptor type C (CD45RA), interleukin-2 (IL-2), or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

164. The composition of any one of claims 153 to 163, wherein the at least one amino acid substitution comprises Q65A.

165. The composition of any one of claims 153 to 163, wherein the at least one amino acid substitution comprises N83A.

166. The composition of any one of claims 153 to 166, wherein the amino acid residue positions are determined by alignment with SEQ ID NO:

1.

167. The composition of any one of claims 153 to 166, wherein the at least one amino acid modification comprises a chemical modification comprising carbamylation or PEGylation.

168. The composition of claim 167, wherein the at least one amino acid modification comprises carbamylation of one or more lysine residues.

169. The composition of claim 168, wherein the at least one amino acid modification comprises carbamylation of all lysine residues.

170. The composition of any one of claims 153 to 169, wherein the engineered EPO protein has a higher binding affinity to the hetero-EPO receptor compared to a corresponding wild-type EPO protein without the at least one amino acid substitution.

171. The composition of any one of claims 153 to 170, wherein the hetero-EPO receptor activity comprises phosphorylation of the intracellular domain of the hetero-EPO receptor or activation of Janus tyrosine kinase 2 (Jak2), signal transducer and activator of transcription 5 (Stat5), mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K), v-Akt murine thymoma viral oncogene / protein kinase-B (Akt / PKB), or mammalian target of rapamycin (mTOR).

172. The composition of any one of claims 153 to 171, wherein the hetero-EPO receptor activity is measured by Western blot, enzyme-linked immunosorbent assay (ELISA), flow cytometry assay, cell proliferation assay, apoptosis assay, or a combination thereof.

173. The composition of any one of claims 153 to 172, wherein the engineered EPO protein has a lower binding affinity to a homo-EPO receptor comprising at least two EPO receptor subunits compared to a corresponding wild-type EPO protein without the at least one amino acid substitution.

174. The composition of any one of claims 153 to 172, wherein the engineered EPO protein has the same level of binding affinity to the homo-EPO receptor compared to the corresponding wild-type EPO protein without the at least one amino acid substitution.

175. The composition of any one of claims 153 to 174, wherein the engineered EPO protein does not affect or inhibit the homo-EPO receptor activity.

176. The composition of any one of claims 153 to 175, wherein the homo-EPO receptor activity comprises phosphorylation of the intracellular domain of the homo-EPO receptor or activation of Janus tyrosine kinase 2 (Jak2), signal transducer and activator of transcription 5 (Stat5), mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K), v-Akt murine thymoma viral oncogene / protein kinase-B (Akt / PKB), or mammalian target of rapamycin (mTOR).

177. The composition of claim 176, wherein the homo-EPO receptor activity is measured by Western blot, enzyme-linked immunosorbent assay (ELISA), flow cytometry assay, cell proliferation assay, apoptosis assay, or a combination thereof.

178. 178. The composition of any one of claims 153 to 177, wherein the engineered EPO has a half-life of at least 5 hours.

179. The composition of any one of claims 153 to 178, wherein the engineered EPO protein comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1973 to 2019.

180. The composition of any one of claims 153 to 179, wherein the hetero-EPOR is on an immune cell.

181. The composition of claim 180, wherein the immune cells comprise macrophages, dendritic cells, T cells, natural killer cells, or B cells.

182. The composition of claim 181 , wherein the T cells comprise cytotoxic T cells.

183. The composition of any one of claims 153 to 179, wherein the hetero-EPOR is on an endothelial cell.

184. A composition comprising a nucleic acid sequence encoding an EPO protein of the composition of any one of claims 153 to 183.

185. A cell comprising the composition of any one of claims 153 to 184.

186. 185. A method of treating a disease or condition in a subject in need thereof, comprising administering to the subject a composition according to any one of claims 153 to 184 or a cell according to claim 185.

187. 187. The method of claim 186, wherein the disease or condition comprises an autoimmune disease.

188. The method of claim 186, wherein the subject has received or is scheduled to receive an organ transplant or an exogenous therapeutic protein.

189. A composition comprising an engineered erythropoietin (EPO) protein, said engineered EPO protein promoting homo-erythropoietin (EPO) receptor activity and having a reduced effect on hetero-EPO receptor activity, said homo-EPO receptor comprising at least two EPO receptor subunits, said hetero-EPO receptor comprising an EPO receptor subunit and a CD131 subunit.

190. The engineered EPO protein is K20A, N24Q, N24A, N24S, N38Q, N38A, N38S, K45A, K52A, Q58A, E62R, E62A, Q65A, L69A, E72A, R76E, R76A, L80A, N83Q, N83A, N83S, S84A, S85A, K97A, K116A, G151A, R103A, K45D, N147K, R150E, Q65A, E72R, N83A, K140A, K152A, or K154A The composition of claim 189, comprising at least one amino acid substitution comprising:

191. The composition of claim 189 or 190, wherein the engineered EPO has no substantial effect on the hetero-EPO receptor activity.

192. The composition of claim 189 or 190, wherein the engineered EPO inhibits the hetero-EPO receptor activity.

193. The composition of claim 190, wherein the engineered EPO protein comprises at least one amino acid substitution including E72A, Q58A, L69A, or L80A.

194. The composition of claim 190, wherein the engineered EPO protein comprises Q65A, E72R, and N83A amino acid substitutions.

195. The composition of claim 190, wherein the engineered EPO protein comprises K20A, K45A, and K52A amino acid substitutions.

196. The composition of any one of claims 189 to 192, wherein the position is determined by alignment with SEQ ID NO:

1.

197. The composition of any one of claims 189 to 193, wherein the engineered EPO further comprises amino acid modifications including carbamylation or PEGylation.

198. The composition of claim 197, wherein the amino acid modification comprises carbamylation of one or more lysine residues.

199. The composition of any one of claims 189 to 198, wherein the engineered EPO protein has a higher binding affinity to the homo-EPO receptor compared to a corresponding wild-type EPO protein without the at least one amino acid substitution.

200. The composition of any one of claims 189 to 199, wherein the homo-EPO receptor activity comprises phosphorylation of the intracellular domain of the homo-EPO receptor or activation of Janus tyrosine kinase 2 (Jak2), signal transducer and activator of transcription 5 (Stat5), mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K), v-Akt murine thymoma viral oncogene / protein kinase-B (Akt / PKB), or mammalian target of rapamycin (mTOR).

201. The composition of any one of claims 189 to 200, wherein the homo-EPO receptor activity is measured by Western blot, enzyme-linked immunosorbent assay (ELISA), flow cytometry assay, cell proliferation assay, apoptosis assay, or a combination thereof.

202. The composition of any one of claims 189 to 201, wherein the engineered EPO protein has the same level of binding affinity to the hetero-EPO receptor compared to the corresponding wild-type EPO protein without the at least one amino acid substitution.

203. The composition of any one of claims 189 to 202, wherein the hetero-EPOR activity comprises phosphorylation of the intracellular domain of the homo-EPO receptor or activation of Janus tyrosine kinase 2 (Jak2), signal transducer and activator of transcription 5 (Stat5), mitogen-activated protein kinase (MAPK), extracellular signal-regulated kinase (ERK), phosphatidylinositol 3-kinase (PI3K), v-Akt murine thymoma viral oncogene / protein kinase-B (Akt / PKB), or mammalian target of rapamycin (mTOR).

204. The composition of any one of claims 189 to 203, wherein the hetero-EPO receptor activity is measured by Western blot, enzyme-linked immunosorbent assay (ELISA), flow cytometry assay, cell proliferation assay, apoptosis assay, or a combination thereof.

205. The composition of any one of claims 202 to 204, wherein the engineered EPO protein does not affect immune tolerance.

206. The composition of claim 205, wherein the engineered EPO protein does not affect the differentiation of a plurality of naive T cells into a plurality of effector T cells.

207. The composition of claim 206, wherein the plurality of effector T cells express cluster of differentiation 45 (CD45), CD3, CD8, perforin, interferon gamma (IFNγ), granzyme B, or tumor necrosis factor alpha (TNFα).

208. The composition of claim 205, wherein the engineered EPO protein does not affect the differentiation of a plurality of naive T cells into a plurality of regulatory T cells.

209. The composition of claim 208, wherein said plurality of regulatory T cells express cluster of differentiation 4 (CD4), CD25, CD127, forkhead box P3 (FoxP3), CD39, protein tyrosine phosphatase receptor type C (CD45RA), interleukin-2 (IL-2), or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).

210. The composition of any one of claims 202 to 204, wherein the engineered EPO protein does not affect the immune response.

211. The composition of any one of claims 189 to 210, wherein the engineered EPO has a half-life of at least 5 hours.

212. The composition of any one of claims 189 to 211, wherein the engineered EPO protein comprises an amino acid sequence having at least 80% sequence identity to any one of SEQ ID NOs: 1973 to 2019.

213. The composition of any one of claims 189-211, wherein the homo-EPOR is located on an erythroid progenitor cell.

214. A composition comprising a nucleic acid sequence encoding an EPO protein of the composition of any one of claims 189 to 213.

215. A cell comprising the composition of any one of claims 189 to 214.

216. 216. A method of treating a disease or condition in a subject in need thereof, comprising administering to the subject a composition according to any one of claims 189 to 214 or a cell according to claim 215.

217. 217. The method of claim 216, wherein the disease or condition comprises cancer.

218. 216. A method of treating anemia in a subject in need thereof, comprising administering to the subject a composition of any one of claims 189-214 or a cell of claim 215, wherein the subject has cancer.

219. 219. The method of claim 217 or 218, wherein the cancer comprises lung cancer, breast cancer, colon cancer, brain cancer, melanoma, hepatocellular carcinoma, or liver cancer.

220. 219. The method of claim 217 or 218, wherein the cancer is melanoma.

221. The method of claim 217 or 218, wherein the cancer is liver cancer.

222. 219. The method of claim 217 or 218, wherein the cancer is colon cancer.

223. The method of claim 217 or 218, wherein the cancer is breast cancer.

224. A composition for administration to a subject having cancer or a chronic infectious disease condition, comprising a compound, or a pharma- ceutically acceptable salt, solvate, or stereoisomer thereof, that inhibits erythropoietin (EPO) receptor activity in myeloid cells of the subject.

225. The composition of claim 224, wherein the EPO receptor is a hetero-EPO receptor.

226. The composition of claim 224 or 225, wherein the hetero-EPO receptor comprises an EPO subunit and a CD131 subunit.

227. The composition of any one of claims 224 to 226, wherein the hetero-EPO receptor is on a macrophage, monocyte, dendritic cell, basophil, neutrophil, or eosinophil.

228. The composition of any one of claims 224 to 227, wherein the compound is an inhibitor of hypoxia inducible factor (HIF), IL-1α, IL-1β, TNF-α, IL-6, estrogen receptor, phospholipase C-γ1, or stimulation of the Cbl / p85 / Episin-1 pathway.

229. The composition of any one of claims 224 to 228, wherein the compound is an inhibitor of hypoxia inducible factor (HIF), IL-1α, IL-1β, TNF-α, IL-6, or estrogen receptor.

230. The composition of any one of claims 224 to 229, wherein the compound is an inhibitor of hypoxia inducible factor (HIF).

231. The compound is selected from the group consisting of CAY10585 (LW6), chetomin, chrysin, dimethyl-bisphenol A, ethinomycin, 2-methoxyestradiol (2ME2), SYP-5, PX-478 2HCl, KC7F2, GN44028, verucopeptin, FM19G11, PT2399, PT2385, belzutifan, HIF-2a-IN-1, HIF-2a-IN-2, HIF-2a-IN-3, HIF-2a-IN-4, TC-S700, IDF-11774, paeoniflorin, emetine hydrochloride, glucosamine, PX12, vitexin, BAY87-2243, lificiguat (YC-1), vorinostat, tanespimycin, silibinin, diaryl. The composition of any one of claims 224 to 230, which is selected from the group consisting of dihydrotrisulfide (DATS), herboxidiene (GEX1A), celastrol, phenethyl isothiocyanate (PEITC), gliotoxin, sulforaphane, acriflavine, emodin, cardenolide, 3,3'-diindolylmethane (DIM), pseudoralic acid-B (PAB), babachinin, andrographolide, isoliquiritigenin, wondonin, thymoquinone, and curcumin.

232. The compound is CAY10585 (LW6), chetomin, chrysin, dimethyl-bisphenol A, echinomycin, 2-methoxyestradiol (2ME2), SYP-5, PX-478 2HCl, KC7F2, GN44028, berucopeptin, FM19G11, PT2399, PT2385, belzutifan, HIF-2a-IN-1, HIF-2a-IN-2, HIF-2a-IN-3, HIF-2a-IN-4, TC-S700, IDF-11774, paeoniflorin, emetine hydrochloride, glucosamine, PX12, vitexin, BAY87-2243, lificiguat (YC-1), vorinostat, or tanespimycin.

233. The composition of any one of claims 224 to 232, wherein the compound is chetomin, echinomycin, PT2399, velzutifan, vorinostat, or tanespimycin.

234. The composition of any one of claims 224 to 231, wherein the compound is silibinin, diallyl trisulfide (DATS), herboxidiene (GEX1A), celastrol, phenethyl isothiocyanate (PEITC), gliotoxin, sulforaphane, acriflavine, emodin, cardenolide, 3,3'-diindolylmethane (DIM), pseudoralic acid-B (PAB), babachinin, andrographolide, isoliquiritigenin, wondonin, thymoquinone, or curcumin.

235. A composition for administration to a subject having cancer or a chronic infectious disease condition, comprising a compound, or a pharma- ceutically acceptable salt, solvate, or stereoisomer thereof, that inhibits erythropoietin (EPO) receptor activity such that immune checkpoint blockade resistance is reversed in the subject.

236. The composition of claim 235, wherein the EPO receptor is a hetero-EPO receptor.

237. The composition of claim 235 or 236, wherein the hetero-EPO receptor comprises an EPO subunit and a CD131 subunit.

238. The composition of any one of claims 235-237, wherein the immune checkpoint blockade is an inhibitor of CTLA-4, PD-1, or PD-L1.

239. The composition of claim 238, wherein the CTLA-4, PD-1, or PD-L1 inhibitor is nivolumab, pembrolizumab, cemiplimab, atezolizumab, avelumab, durvalumab, ipilimumab, lirilumab, and BMS-986016.

240. The composition of any one of claims 235 to 239, wherein the hetero-EPO receptor is on a macrophage, monocyte, dendritic cell, basophil, neutrophil, or eosinophil.

241. 241. The composition of any one of claims 235-240, wherein the compound is an inhibitor of hypoxia inducible factor (HIF), IL-1α, IL-1β, TNF-α, IL-6, estrogen receptor, phospholipase C-γ1, or the Cbl / p85 / Episin-1 pathway.

242. The composition of any one of claims 235-241, wherein the compound is an inhibitor of hypoxia inducible factor (HIF), IL-1α, IL-1β, TNF-α, or IL-6.

243. The composition of any one of claims 235-242, wherein the compound is an inhibitor of hypoxia inducible factor (HIF).

244. The compound is selected from the group consisting of CAY10585 (LW6), chetomin, chrysin, dimethyl-bisphenol A, ethinomycin, 2-methoxyestradiol (2ME2), SYP-5, PX-478 2HCl, KC7F2, GN44028, verucopeptin, FM19G11, PT2399, PT2385, belzutifan, HIF-2a-IN-1, HIF-2a-IN-2, HIF-2a-IN-3, HIF-2a-IN-4, TC-S700, IDF-11774, paeoniflorin, emetine hydrochloride, glucosamine, PX12, vitexin, BAY87-2243, lificiguat (YC-1), vorinostat, tanespimycin, silibinin, diaryl. The composition of any one of claims 235 to 243, which is selected from the group consisting of dihydrotrisulfide (DATS), herboxidiene (GEX1A), celastrol, phenethyl isothiocyanate (PEITC), gliotoxin, sulforaphane, acriflavine, emodin, cardenolide, 3,3'-diindolylmethane (DIM), pseudoralic acid-B (PAB), babachinin, andrographolide, isoliquiritigenin, wondonin, thymoquinone, and curcumin.

245. The compound is CAY10585 (LW6), chetomin, chrysin, dimethyl-bisphenol A, echinomycin, 2-methoxyestradiol (2ME2), SYP-5, PX-478 2HCl, KC7F2, GN44028, berucopeptin, FM19G11, PT2399, PT2385, belzutifan, HIF-2a-IN-1, HIF-2a-IN-2, HIF-2a-IN-3, HIF-2a-IN-4, TC-S700, IDF-11774, paeoniflorin, emetine hydrochloride, glucosamine, PX12, vitexin, BAY87-2243, lificiguat (YC-1), vorinostat, or tanespimycin.

246. The composition of any one of claims 235 to 245, wherein the compound is chetomin, echinomycin, PT2399, velzutifan, vorinostat, or tanespimycin.

247. The composition of any one of claims 235 to 244, wherein the compound is silibinin, diallyl trisulfide (DATS), herboxidiene (GEX1A), celastrol, phenethyl isothiocyanate (PEITC), gliotoxin, sulforaphane, acriflavine, emodin, cardenolide, 3,3'-diindolylmethane (DIM), pseudoralic acid-B (PAB), babachinin, andrographolide, isoliquiritigenin, wondonin, thymoquinone, or curcumin.

248. A composition for administration to a subject, comprising a compound or a pharma- ceutically acceptable salt, solvate, or stereoisomer thereof that promotes hetero-erythropoietin (EPO) receptor activity, said hetero-EPO receptor comprising an EpoR subunit and a CD131 subunit such that immune tolerance to an antigen is increased in said subject, said compound having no substantial effect on homo-EPO receptor activity, said homo-EPO receptor comprising at least two EPO receptor subunits.

249. The composition of claim 248, wherein the hetero-EPO receptor is on a macrophage, monocyte, dendritic cell, basophil, neutrophil, or eosinophil.

250. The composition of claim 248 or 249, wherein the compound is an inhibitor of HIF-prolyl hydroxylase (PHD), NHF-4, GATA factors, IL-17, AKT / NFkB / HIF1 pathway, estrogen receptor, epithelial membrane protein 1 (EMP-1).

251. The composition of any one of claims 248 to 250, wherein the compound is an inhibitor of HIF-prolyl hydroxylase (PHD), NHF-4, GATA factors, or IL-17.

252. The composition of any one of claims 248 to 251, wherein the compound is an inhibitor of HIF-prolyl hydroxylase (PHD).

253. The composition of any one of claims 248 to 252, wherein the compound is roxadustat, vadadustat, enarodustat, decidustat, molidustat, dimethyloxaloylglycine, daprodustat, prolyl hydroxylase inhibitor 1, TM6089, TRC160334, PHD-1-IN-1, MK-8617, JNJ-42041935, TP0463518, IOX (JICL38), IOX4, IOX3 (FG-2216), densitin, HIF-PHD-IN-1, AKB-6899, VH298, M1001, ML228, dimethyloxalylglycine (DMOG), mitoxantrone, angiotensin II (Ang II), or 17β-estradiol.

254. The composition of any one of claims 248 to 253, wherein the compound is roxadustat, vadadustat, enarodustat, decidustat, molidustat, dimethyloxaloylglycine, daprodustat, prolyl hydroxylase inhibitor 1, TM6089, TRC160334, PHD-1-IN-1, MK-8617, JNJ-42041935, TP0463518, IOX (JICL38), IOX4, IOX3 (FG-2216), densitin, HIF-PHD-IN-1, AKB-6899, VH298, M1001, ML228, or dimethyloxalylglycine (DMOG).

255. The composition of any one of claims 248-253, wherein the compound is mitoxantrone, angiotensin II (Ang II), or 17β-estradiol.

256. The composition of claim 248 or 249, wherein the compound is an EPOR agonist.

257. The composition of claim 256, wherein the compound is LG5640.

258. The composition of any one of claims 248 to 257, wherein the immune tolerance is to a transplanted organ or to a self-antigen.

259. The composition of any one of claims 248 to 258, wherein the immune tolerance is to a transplanted organ.

260. The composition of any one of claims 248 to 259, wherein the immune tolerance is to an immunosuppressive condition.

261. The composition of any one of claims 248 to 258, wherein the immune tolerance is to a self-antigen.

262. The composition of any one of claims 248 to 258, wherein the immune tolerance is to a self-antigen.

263. A composition for administration to a subject having cancer, comprising an RNA interference (RNAi) molecule, wherein the RNAi binds to an RNA molecule selected from the group consisting of an mRNA molecule encoding an erythropoietin (EPO) protein, an mRNA molecule encoding an EPO receptor subunit, an mRNA molecule encoding a CD131 subunit, and any combination thereof, and wherein, upon administration of the RNAi to the subject, the tumor burden in the subject is reduced.

264. The tumor volume is 0.5 cm ３ The composition of claim 263, wherein the concentration is reduced to less than 100%.

265. The tumor volume is 0.2 cm ３ The composition of claim 263 or 264, wherein the concentration is reduced to less than 100%.

266. The tumor volume is about 0.2 cm ３ The composition of claim 263 or 264, wherein the

267. A composition for administration to a subject having cancer, comprising an RNA interference (RNAi) molecule, wherein the RNAi binds to an RNA molecule selected from the group consisting of an mRNA molecule encoding an erythropoietin (EPO) protein, an mRNA molecule encoding an EPO receptor subunit, an mRNA molecule encoding a CD131 subunit, and any combination thereof, and wherein upon administration of the RNAi to the subject, the immune response of the subject is enhanced by increasing the expression of more effector T (T ｅｆｆ ) A composition that increases by inducing cells.

268. The composition of any one of claims 263 to 267, wherein the cancer is hepatocellular carcinoma.

269. The composition of any one of claims 263-268, wherein the RNAi reduces EPO half-life in a subject.

270. The composition of any one of claims 263-268, wherein the RNAi reduces EPO levels in a subject.

271. The composition of claim 269, wherein the reduced EPO half-life increases survival rate.

272. The composition of claim 271, wherein the survival rate is increased by 2-fold.

273. The composition of any one of claims 263-272, wherein the RNAi is in a nanoparticle.

274. The composition of claim 273, wherein the nanoparticle is a lipid nanoparticle.

275. The composition of any one of claims 263-274, wherein the RNAi molecule is an siRNA molecule, an miRNA molecule, an antisense RNA molecule, or an lncRNA molecule.

276. The composition of any one of claims 263 to 275, wherein the RNAi is a siRNA molecule.

277. The composition of claim 276, wherein the siRNA molecule has a sequence length of about 15 to about 30 nucleotides.

278. The composition of claim 276 or 277, wherein the siRNA molecule has a sequence length of about 21 to about 30 nucleotides.

279. The composition of any one of claims 276 to 278, wherein the siRNA molecule is double-stranded or single-stranded.

280. The single stranded siRNA molecule is selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35, SEQ ID NO:36, The composition of claim 279, comprising a nucleic acid sequence that is at least 80%, 85%, 90%, or 95% identical to at least one of SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, and SEQ ID NO:

62.

281. The single stranded siRNA molecule is SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, SEQ ID NO:28, SEQ ID NO:29, SEQ ID NO:30, SEQ ID NO:31, SEQ ID NO:32, SEQ ID NO:33, SEQ ID NO:34, SEQ ID NO:35 , SEQ ID NO:36, SEQ ID NO:37, SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49, SEQ ID NO:50, SEQ ID NO:51, SEQ ID NO:52, SEQ ID NO:53, SEQ ID NO:54, SEQ ID NO:55, SEQ ID NO:56, SEQ ID NO:57, SEQ ID NO:58, SEQ ID NO:59, SEQ ID NO:60, SEQ ID NO:61, and SEQ ID NO:

62.

282. A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising a single-stranded siRNA from claim 280 or claim 281 at a dose and schedule sufficient to reduce expression levels of erythropoietin (EPO) protein, EPO receptor subunit, or CD131 subunit.

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