EPO receptor agonists and antagonists

Abstract

The present disclosure provides EPO analogs, anti-EPOR antibodies, anti-EPO antibodies, and fragments thereof, which specifically bind to heterologous EPOR, or homologous EPOR, or EPO with high affinity. Also provided herein are engineered EPOs. These EPO analogs, anti-EPOR antibodies, anti-EPO antibodies, and / or engineered EPO may be used to treat a patient.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 317,943, filed on March 8, 2022, which is incorporated herein by reference in its entirety.

[0003] Background Art Erythropoietin (EPO) induces hematopoiesis by dimerizing EPO receptor (EPOR) molecules, leading to 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 expressed on erythroid progenitor cells, inhibiting apoptosis and promoting cell survival, proliferation, and differentiation in the production of mature erythrocytes ( Figure 1 However, EPOR expression is not limited to erythroid tissues. EPOR is also expressed in many non-hematopoietic tissues and triggers tissue protection in ischemic injury and promotes wound healing, cardiovascular protection, angiogenesis, neuroprotection, regulation of metabolic homeostasis, and bone remodeling. Summary of the Invention

[0004] There are two main tyrosine kinase receptors for EPO: homodimeric EPOR / EPOR ("homo-EPOR") and heterodimeric EPOR / CD131 receptor ("hetero-EPOR"). Homologous EPOR signaling is crucial for erythropoiesis, however, heterologous EPOR signaling is known to have tissue protective activity and may participate in EPO-mediated immunomodulatory functions of immune cells (e.g., myeloid cells, T cells, and B cells). Regulating EPO signaling through heterologous EPOR can provide benefits in various pathological conditions, including but not limited to suppressing or stimulating immune responses, inducing or destroying antigen-specific tolerance, stimulating erythropoiesis without immune tolerogenicity or inhibitory effects, providing neuroprotection and tissue protection without stimulating erythropoiesis, and inducing preventive or therapeutic immunity.

[0005] 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. EPO analogs can bind to heterologous EPORs and not to homologous EPORs, and can be agonists or antagonists of heterologous EPORs. Other EPO analogs can bind to homologous EPORs and not to heterologous EPORs, and can be agonists or antagonists of homologous EPORs. EPO analogs can bind to homologous EPORs and heterologous EPORs, and can be agonists of both, antagonists of both, or agonists of one and antagonists of the other. At least four types of anti-EPO receptor (anti-EPOR) antibodies can be obtained. Anti-EPOR antibodies can be agonists or antagonists of heterologous EPORs, and anti-homologous EPOR antibodies can be agonists or antagonists of homologous EPORs. At least two types of anti-CD131 antibodies can be obtained. Anti-CD131 antibodies can be agonists or antagonists of heterologous EPORs. At least three types of anti-EPO antibodies can be obtained. Anti-EPO antibodies can inhibit the binding of EPO to a homologous EPOR, inhibit the binding of EPO to a heterologous EPOR, or inhibit the binding of EPO to both a homologous EPOR and a heterologous EPOR.

[0006] The antibodies disclosed herein may include fragments thereof that specifically bind to homologous EPOR, heterologous EPOR, EPO, CD131, or a combination thereof with high binding affinity (heterologous EPOR and homologous EPOR are collectively referred to as "EPOR"). The antibody may be a monoclonal antibody and may be a human antibody, a chimeric antibody, or a humanized antibody. Chimeric anti-EPOR antibodies and / or anti-EPO antibodies (including fragments thereof) may have non-human (e.g., mouse) complementary determining regions (CDRs) and / or non-human framework regions, and optionally one or more human constant domains. Humanized anti-EPOR antibodies 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 transplanted antibodies.

[0007] The humanized antibodies disclosed herein may refer to anti-EPOR and / or anti-EPO antibodies obtained by transplanting CDRs into a human framework of the heavy chain and / or a human framework of the light chain, along with a selected number of framework residues from a mouse antibody, as well as anti-EPOR and / or anti-EPO antibodies from murine antibodies. The anti-EPOR and / or anti-EPO antibodies disclosed herein also include those obtained from affinity maturation libraries made from anti-EPOR antibodies or anti-EPO antibodies. The anti-EPOR and / or anti-EPO antibodies may also comprise a heavy chain variable region having 99%, 95%, 90%, 80% or 70% sequence identity to one of the heavy chains and a light chain variable region having 99%, 95%, 90%, 80% or 70% sequence identity to one of the light chains. The anti-EPOR antibodies may bind to homologous EPOR or heterologous 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 nM to about 100 nM. Anti-EPOR antibodies can bind to homologous EPOR or heterologous 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. Anti-EPO antibodies 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 nM to about 100 nM. Anti-EPO antibodies 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.

[0008] The anti-EPOR antibodies and / or anti-EPO antibodies described herein may include modifications that provide desired properties to the antibodies. For example, the modifications may increase the serum half-life of the antibody, or the modifications may decrease the serum half-life. The modifications may also increase or decrease the effector function of the antibody. The modifications may reduce immunogenicity or reduce other unwanted side effects or adverse events caused by the antibody.

[0009] EPO analogs as antagonists of heterologous EPOR, anti-heterologous EPOR antibodies as antagonists of heterologous EPOR, anti-CD131 antibodies as antagonists of heterologous EPOR and / or anti-EPO antibodies that inhibit the binding of EPO to heterologous EPOR can be used to overcome the immunosuppressive or tolerogenic state of the subject. For example, these EPO analogs, anti-heterologous EPOR antibodies, anti-CD131 antibodies and / or anti-EPO antibodies can be used to overcome the tumor immunosuppressive microenvironment, enhance the immune response to vaccines and / or enhance the immune response during the acute inflammatory response to a disease (e.g., infection from a microorganism or virus).

[0010] EPO analogs as agonists of heterologous EPOR, anti-CD131 antibodies as agonists of heterologous EPOR and / or anti-EPOR antibodies as agonists of heterologous EPOR can be used to induce negative immune regulation (e.g., immunosuppression or tolerogenic state) of the subject. For example, these EPO analogs, anti-CD131 antibodies as agonists of heterologous EPOR and / or anti-heterologous EPOR antibodies can be used to inhibit transplant rejection, induce antigen-specific immune tolerance, reduce the immune response of autoimmune diseases, reduce systemic chronic inflammation, and reduce damage to neural tissue and other tissues during injury or other stress. These EPO analogs, anti-CD131 antibodies as agonists of heterologous EPOR and / or anti-heterologous EPOR antibodies can also be administered together with an antigen to induce an immune tolerogenic state against the antigen.

[0011] For cancer patients requiring ESA treatment, an EPO analog that is an agonist of the homologous EPOR and does not bind to the heterologous EPOR or acts as an antagonist thereof, and / or an anti-EPO antibody that inhibits the binding of EPO to the heterologous EPOR, and / or an anti-CD131 antibody that inhibits the binding of EPO to the heterologous EPOR, and / or an anti-heterologous EPOR antibody that acts as an antagonist of the heterologous EPOR can be used with or without an erythropoietin-stimulating agent (ESA). A combination of ESA and these EPO analogs, and / or anti-EPOR antibodies, and / or anti-EPO antibodies can be provided to any cancer patient requiring ESA.

[0012] RNA or small molecules can be utilized to regulate the signal transduction of homologous EPOR or heterologous EPOR. Stimulation of the signal transduction from homologous EPOR or heterologous EPOR can be achieved by delivering the mRNA of a positive regulator, the siRNA of a negative regulator, the small molecule that raises the positive regulator, or the small molecule that lowers the negative regulator. Inhibition of the signal transduction from homologous EPOR or heterologous EPOR can be achieved by delivering the mRNA of a negative regulator, the siRNA of a positive regulator, the small molecule that raises the negative regulator, or the small molecule that lowers the positive regulator.

[0013] In some aspects, provided herein is a composition comprising an antibody or a functional fragment thereof, wherein: (i) the antibody or the functional fragment thereof selectively binds to a target comprising an erythropoietin (EPO) protein, an EPO receptor subunit, a CD131 subunit, or a combination thereof; (ii) binding of the antibody or the functional fragment thereof to the target prevents (a) formation of an EPO protein-heterologous EPO receptor complex, wherein the heterologous EPO receptor comprises the EPO receptor subunit and the CD131 subunit, (b) formation of a heterologous EPO receptor between the EPO receptor subunit and the CD131 subunit, or (c) activation of a heterologous EPO receptor, wherein the heterologous EPO receptor comprises the EPO receptor subunit and the CD131 subunit; and (iii) the antibody or the functional fragment thereof comprises an antigen binding domain.

[0014] In some aspects, provided herein is a method for treating cancer, wherein the method comprises administering a composition or a derivative thereof to a subject having or at risk of having cancer, wherein the composition or the derivative thereof inhibits the activity of the subject's allogeneic erythropoietin (EPO) receptor.

[0015] In some aspects, provided herein is a composition comprising an antibody or a functional fragment thereof, wherein: (i) the antibody or the functional fragment thereof selectively binds to a target comprising an erythropoietin (EPO) protein, an EPO receptor subunit, a CD131 subunit, or a combination thereof; (ii) binding of the antibody or the functional fragment thereof to the target promotes (a) formation of an EPO protein-heterologous EPO receptor complex, wherein the heterologous EPO receptor comprises the EPO receptor subunit and the CD131 subunit, (b) formation of a heterologous EPO receptor between the EPO receptor subunit and the CD131 subunit, or (c) activation of a heterologous EPO receptor, wherein the heterologous EPO receptor comprises the EPO receptor subunit and the CD131 subunit; and (iii) the antibody or the functional fragment thereof comprises an antigen binding domain.

[0016] In some aspects, provided herein is a composition for administration to a subject having cancer or a chronic infectious condition, wherein the composition or a derivative thereof inhibits erythropoietin (EPO) receptor activity in myeloid cells of the subject.

[0017] In some aspects, provided herein is a composition comprising an engineered erythropoietin (EPO) protein, wherein the engineered EPO protein inhibits heterologous erythropoietin (EPO) receptor activity in myeloid cells. In some embodiments, the engineered EPO protein comprises 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.

[0018] In some aspects, provided herein is a composition comprising an engineered erythropoietin (EPO) protein, wherein: the engineered EPO protein comprises at least one amino acid substitution, the 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; and the engineered EPO protein inhibits heterologous erythropoietin (EPO) receptor activity in myeloid cells.

[0019] In some aspects, provided herein is a composition comprising an engineered erythropoietin (EPO) protein, wherein the engineered EPO protein promotes the activity of a heterologous erythropoietin (EPO) receptor to reduce an immune response, wherein the heterologous 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.

[0020] In some aspects, provided herein is a composition comprising an engineered erythropoietin (EPO) protein, wherein: the engineered EPO protein comprises at least one amino acid modification and / or at least one amino acid substitution, wherein the at least one amino acid modification and / or at least one amino acid substitution comprises: 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; and the engineered EPO protein promotes the activity of a heterologous erythropoietin (EPO) receptor, wherein the heterologous EPO receptor comprises an EPO receptor subunit and a CD131 subunit.

[0021] In some aspects, provided herein is a composition comprising an engineered erythropoietin (EPO) protein that promotes homologous erythropoietin (EPO) receptor activity and has a reduced effect on heterologous EPO receptor activity, wherein the homologous EPO receptor comprises at least two EPO receptor subunits and the heterologous 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 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.

[0022] In some aspects, provided herein is a composition comprising an engineered erythropoietin (EPO) protein, wherein: the engineered EPO protein comprises 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, S84 A, S85A, K97A, K116A, G151A, R103A, K45D, N147K, R150E, Q65A, E72R, N83A, K140A, K152A or K154A; and the engineered EPO protein promotes homologous erythropoietin (EPO) receptor activity and has no substantial effect on heterologous EPO receptor activity, wherein the homologous EPO receptor comprises at least two EPO receptor subunits and the heterologous EPO receptor comprises an EPO receptor subunit and a CD131 subunit.

[0023] In some aspects, provided herein is a composition comprising an engineered erythropoietin (EPO) protein, wherein: the engineered EPO protein comprises 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, S84 A, S85A, K97A, K116A, G151A, R103A, K45D, N147K, R150E, Q65A, E72R, N83A, K140A, K152A or K154A; and the engineered EPO protein promotes heterologous erythropoietin (EPO) receptor activity and has no substantial effect on homologous EPO receptor activity, wherein the homologous EPO receptor comprises at least two EPO receptor subunits and the heterologous EPO receptor comprises an EPO receptor subunit and a CD131 subunit.

[0024] In some aspects, provided herein is a composition for administration to a subject having cancer or a chronic infectious condition, the composition comprising a compound, a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, wherein the compound inhibits erythropoietin (EPO) receptor activity in myeloid cells of the subject.

[0025] In some aspects, provided herein is a composition for administration to a subject having cancer or a chronic infectious condition, the composition comprising a compound, a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, wherein the compound inhibits erythropoietin (EPO) receptor activity such that resistance to immune checkpoint blockade is reversed in the subject.

[0026] In some aspects, provided herein is a composition for administration to a subject, comprising a compound, a pharmaceutically acceptable salt, solvate, or stereoisomer thereof, wherein the compound promotes the activity of a heterologous erythropoietin (EPO) receptor, wherein the heterologous EPO receptor comprises an EpoR subunit and a CD131 subunit, such that the subject's immune tolerance to the antigen is increased; and wherein the compound has no substantial effect on the activity of a homologous EPO receptor, wherein the homologous EPO receptor comprises at least two EPO receptor subunits.

[0027] In some aspects, provided herein is a composition for administration to a subject having cancer, the composition comprising an RNA interference (RNAi) molecule, wherein the RNAi binds to an RNA molecule selected from 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; wherein after administration of the RNAi to the subject, the subject's tumor mass is reduced.

[0028] In some aspects, provided herein is a composition for administration to a subject having cancer, the composition comprising an RNA interference (RNAi) molecule, wherein the RNAi binds to an RNA molecule selected from 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; wherein after administration of the RNAi to the subject, the subject's immune response is increased by inducing more effector T (Teff) cells.

[0029] In some aspects, provided herein is a method for treating cancer in a subject, 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 for a schedule sufficient to reduce the expression level of erythropoietin (EPO) protein, EPO receptor subunit, or CD131 subunit.

[0030] Incorporation by reference

[0031] 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] A better understanding of the features and advantages of the present disclosure will be obtained by referring to the following detailed description and the accompanying drawings, which set forth illustrative embodiments of the present disclosure.

[0033] Figure 1 An overview of erythropoiesis mediated by EPO and the cognate EPOR in erythroid progenitor cells.

[0034] Figure 2 An overview of immune tolerance mediated by EPO and allogeneic EPOR in dendritic cells and macrophages.

[0035] Figures 3A-3B EpoR + (EpoR + ) for EpoR - (EpoR -) gene expression analysis of genes in . Figure 3A Display EpoR + XCR1 + CD8α + cDC1 on EpoR - XCR1 + CD8α + Volcano plot of upregulated and downregulated genes in cDC1. XCR1: XC-chemokine receptor 1. CD8α: cluster of differentiation 8α. cDC1: conventional type 1 dendritic cell. Figure 3B Displays EpoR + EpoR - Heatmap of RNA-seq gene expression for the most significantly up-regulated and down-regulated genes.

[0036] Figures 4A-4C Shown are the effects of heterologous EPOR knockout in dendritic cells (DCs). Figure 4A Heterologous EPOR expression in DCs was shown. Figure 4B Display mice (C57BL / 6J), Batf3 knockout mice (Batf3 - / - ), with CD11c Cre mice (CD11c Cre ), with EPOR flox / flox mice (EPOR flox / flox ) and mice with heterologous EPOR knockout in dendritic cells (EPOR ΔCD11c ) donor TCRβ + Number of T cells. TCRβ: T cell receptor β. Batf3: basic leucine zipper ATF-like transcription factor 3. CD11c: cluster of differentiation 11c. P values ​​are determined by two-tailed t-test of independent means. *P < .05; **P < .01; ***P < .001; ns, not significant (P > .05). Figure 4C It was shown that after cardiac transplantation (TX), C57BL / 6J, Batf3 - / - EPOR flox / flox and EPOR ΔCD11c Percent cardiac survival of mice. WT TX (C57BL / 6J) versus Batf3 - / - TX: P < .001; WTTX (C57BL / 6J) on EpoR ΔXCR1 TX: P < .001, log-rank; Mantel-Cox test.

[0037] Figures 5A-5B Shown is the induction of antigen (Ag)-specific regulatory T cells (Tregs) in dendritic cells (cDC1) via EPOR. Figure 5A(i) cDC1 expressing heterologous EPOR (EpoR + cDC) or (ii) not expressing heterologous EPOR (EpoR - FoxP3 in transgenic mice (OTII mice) expressing mouse α-chain and β-chain T cells + Percentage of Tregs. FoxP3: Forkhead box P3. P values ​​are determined by two-tailed t-test of independent means. *P < .05; **P < .01; ***P < .001; ns, not significant (P > .05). Figure 5B We show that measurements of C57BL / 6 mice or mice with EPOR knockout in dendritic cells (EPOR ΔCD11c ) in mice treated with whole lymphoid irradiation and antithymocyte serum (TLI / ATS) or untreated (UNT).

[0038] Figures 6A-6B Shown are tumor burdens in mice in which allogeneic EPOR was deleted from myeloid cells. Figure 6A Shown are (i) wild-type (WT) mice, (ii) wild-type mice with PD-L1 treatment (WT+αPD-L1), and (iii) mice with heterologous EPOR knockout in macrophages (EpoR ΔLysM ) of lung tumor size (Lewis lung cancer). PD-L1: programmed death ligand 1. Figure 6B Figure 3. (i) WT mice and (ii) mice with heterologous EPOR knockout in dendritic cells (EPOR ΔCD11c ) of the tumor size (breast adenocarcinoma).

[0039] Figures 7A-7C Shown are tumor burdens in mice with heterologous EPOR-deficient dendritic cells. Figure 7A Show Zbtb46 gfp / + EpoR tdTomato / + Expression of EPOR-tdT in various immune cells of mice. Figure 7B (i) Mice with heterologous EPOR deletion in dendritic cells (EpoR ΔXCR1 ) compared to (ii) mice without EPOR deletion (EPOR flox / flox ) of tumor size (colon cancer). Figure 7B showed that (i) mice with mTOR deletion in dendritic cells (mTOR ΔXCR1 ) compared to (ii) mice without mTOR deletion (mTOR flox / flox) tumor size. 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. Figure 7C Display EPOR for day 14 ΔXCR1 and EPOR flox / flox Picture of mice (left) and EPOR on day 14 ΔXCR1 EPOR flox / flox Tumor size in mice with mTOR deletion (right). Scale bars are as shown. Mean tumor sizes are shown. P values ​​were determined by two-tailed t-test of independent means. ***P < .001.

[0040] Figures 8A-8C Mice with EPOR-deficient macrophages (Epor ΔLysM Altered resistance to immune checkpoint blockade in cold tumors of WT and WT tumors. Figure 8A The experimental scheme of administering anti-programmed death 1 antibody (αPD-1) to mice bearing cold hepatocellular carcinoma (HCC) is shown. A spontaneous model of cold HCC was created by delivering plasmids pCMV-SB13, pT3-EF1a-C-Myc-IRES-luciferase, and pX330-sgRNA targeting Trp53 to the liver of mice using in vivo hydrodynamic tail vein injection (HDTV). After two weeks, C57BL / 6 wild type (WT) and Epor were treated with 2 mg / kg αPD-1 or IgG isotype control via intraperitoneal injection every three days. ΔLysM strain of mice, for a total of five doses. Trp53: cellular tumor antigen p53. C-myc: c-Myc oncoprotein. Luc: luciferase. Figure 8B Shown are wild-type mice treated with IgG isotype (WT IgG isotype), wild-type mice treated with αPD-1 (WTαPD-1), mice with macrophage-specific heterologous EPOR knockout (Epor ΔLysM IgG isotype) and mice with macrophage-specific heterologous EPOR knockout (Epor ΔLysM The tumor growth kinetics of αPD-1 (αPD-1) were analyzed by measuring luciferin-based bioluminescence. Figure 8C Shows WT IgG isotype, WT αPD-1, Epor ΔLysM IgG isotype and Epor ΔLysM Survival curve of αPD-1.

[0041] Figures 9A-9CShown are changes in immune checkpoint blockade-resistant cold tumors in mice with EPO knockout in dendritic cells. Figure 9A Shown is the experimental scheme of melanoma mice treated with αPD-1 (programmed death-1). Figure 9B Shown are (i) control mice, (ii) control mice treated with αPD-1) control + αPD-1), (iii) mice with heterologous EPOR deletion in dendritic cells (EpoR ΔXCR1 ) and (iv) mice with heterologous EPOR deletion in dendritic cells treated with αPD-1 (EpoR ΔXCR1 +αPD-1) tumor size (melanoma). *P<0.05, **P<0.01, ***P<0.001 and ****P<0.0001, two-way ANOVA. Figure 9C showed that measurements of mice without heterologous EPOR deletion (EPOR flox / flox ) and mice with heterologous EPOR deletion in dendritic cells (EpoR ΔXCR1 Flow cytometric data of perforin, granzyme B, interferon-γ (IFNγ), and tumor necrosis factor α (TNFα) in ).

[0042] Figure 10 Shown are percent survival data from The Cancer Genome Atlas of Hepatocellular Carcinoma (TCGA-LIHC) for hepatocellular carcinoma patients with (i) low levels of EPO compared to (ii) high levels of EPO.

[0043] Figures 11A-11C Shown is the effect of EPO on tumor progression in regressing hepatocellular carcinoma (HCC) mice. Figure 11A The experimental scheme for establishing a regression HCC model is shown. 3 x 10 6 Hepa1-6 cells were orthotopically implanted into C57BL / 6 mice. OE Two Hepa1-6 stable cell lines were generated by lentivirus containing either ) or empty vector (Hepa1-6_EV). Figure 11B Display with Hepa1-6_EV or Hepa1-6_Epo OE Tumors from treated HCC mice were harvested on days 14 and 21 after injection. Figure 11C Display with Hepa1-6_EV or Hepa1-6_Epo OE Quantification of tumor volume and complete regression (CR) rate measurements in treated HCC mice.

[0044] Figures 12A-12B Shown are colon tumor growth in mice with or without liver metastases. Figure 12A Shown are changes in colon tumor volume in wild-type mice with or without liver metastases. Figure 12B EPOR deficiency (EpoR ΔLysM ) and changes in colon tumor volume in mice with or without liver metastases.

[0045] Figures 13A-13C Hepatocellular carcinoma mice (Hepa1-6_Epo OE ) The effect of phage-targeted liposomes loaded with siRNA targeting EPOR (siEpor). Figure 13A The experimental scheme of liposome treatment in two HCC models is shown. OE : 3x 10 6 EPO-overexpressing Hepa1-6 cells were orthotopically implanted into C57BL / 6 mice. One week later, mice were treated with liposomes containing 50 μg of siEpor (siRNA targeting EPOR) or siNTC (non-target control) RNA via intravenous injection every four days for a total of three doses. HDTV: A spontaneous model of cold HCC was created by delivering plasmids pCMV-SB13, pT3-EF1a-C-Myc, and pX330-sgRNA targeting Trp53 to the liver of mice using in vivo hydrodynamic tail vein injection (HDTV). After two weeks, mice were treated with liposomes containing 50 μg of siEpor or siNTC RNA via intravenous injection every four days for a total of six doses. Figure 13B Shown are tumors harvested from hepatocellular carcinoma mice treated with liposomes containing siEpor or siNTC (left) and tumor volumes (right). Figure 13C Shown are livers harvested from cold-HCC mice treated with liposomes containing siEpor or siNTC (left) and liver weight (right).

[0046] Figures 14A-14C Shown are the effects of macrophage-targeted liposomes loaded with siRNA targeting heterologous EPOR. Figure 14A Physical properties of macrophage-targeted liposomes were demonstrated. Figure 14B Flow cytometric analysis indicated macrophages as the major cell type taking up liposomes. OE Liposomes loaded with 50 μg of fluorescein isothiocyanate (FITC)-conjugated siRNA were administered to C57BL / 6 mice. 24 hours later, tumors were harvested and isolated into single-cell suspensions. Flow cytometry analysis was performed to measure the expression of FITC in different myeloid cell types. + The percentage of cells. Figure 14C The knockdown efficiency of EPOR in tumor-infiltrating macrophages was shown.6 Epo-overexpressing Hepa1-6 cells were orthotopically implanted into C57BL / 6 mice. One week later, mice were treated with liposomes containing 50 μg of siRNA targeting EpoR (siEpor) or a non-target control siRNA (siNTC) via intravenous injection every four days for a total of three doses. Tumors were harvested three weeks after injection and isolated into single-cell suspensions. Macrophages were isolated using magnetic-activated cell sorting, and RNA was extracted for real-time PCR quantification.

[0047] Figures 15A-15B Shown from breast cancer ( Figure 15A ) and breast cancer left axillary lymph node metastasis site ( Figure 15B ) of human fresh cancer samples. Figure 15A Shown are the EPOR expression levels analyzed by flow cytometry. + Cancer infiltrating lymphocytes gated as live-dead aqua-CD45 + Histograms showing EPOR expression on individual myeloid cell subsets. Left: Breast cancer. Right: Surrounding healthy tissue. Bottom: Gated on dendritic cells for CD11c + HLA - DR + CD14 - CD16 - EPOR expression. Figure 15B EPOR expression on tumor-infiltrating lymphocytes in a breast cancer left axillary lymph node (LN) metastasis. Top: EPOR expression on dendritic cells. Bottom: EPOR expression on HLA-DR- cells.

[0048] Figures 16A-16B EPOR expression on myeloid cells in human fresh liver metastasis sites paired with peripheral blood samples is shown. Samples were collected from three separate patients with different original tumor types. Figure 16A CD45 expression in liver metastatic sites as analyzed by flow cytometry + EPOR expression levels in tumor-infiltrating lymphocytes. CD45 + Cancer infiltrating lymphocytes gated as live-dead aqua-CD45 + Right: Comparison of EPOR expression in peripheral blood samples from patients with liver metastasis and blood from healthy donors. + The percentage of cells is shown in the red rectangle. Figure 16B Shows EPOR in blood of patients with liver metastasis, blood of healthy donors, and blood of patients with liver cancer or cirrhosis +Percentage of cells. Statistical analysis was performed using an unpaired two-tailed t-test. *P < 0.05; **P < 0.01; ***P < 0.001 and ****P < 0.0001.

[0049] Figure 17 Examples of EPO blocking efficiencies of the hybridoma clones listed in Table 11 are shown.

[0050] Figures 18A-18B TLI / ATS-induced tolerance to allogeneic (allo) bone marrow (BM) and heart transplantation is shown. Figure 18A The experimental protocol for performing cardiac transplantation on mice, treating the mice with TLI / ATS, performing bone marrow transplantation, and examining allogeneic BM chimerism and cardiac survival is shown. Figure 18B Showing wild type and Baf3 - / - Heart transplant survival in mice (left) and wild-type and Baf3 - / - BM chimerism of mice on day 34 after BM transplantation (TX) (right).

[0051] Figures 19A-19E TLI / ATS induced local apoptosis and extramedullary erythropoiesis, as well as dendritic cell (DC) enrichment and systemic upregulation of EPO. Figure 19A Representative images of TUNEL staining of sections of untreated spleen (UNT) and spleen treated with TLI / ATS are shown. Figure 19B Shown is an analysis of the changes in the cell composition of different cell populations in untreated spleens (UNT), spleens treated with ATS, spleens treated with TLI, and spleens treated with TLI / ATS. The pie chart shows the mean frequency of the indicated populations from one representative experiment (n=4). T cells (TCRβ + CD19 - ), B cells (TCRβ - CD19 + ), erythroid progenitor cells (TER119 + CD71 + )、DC(CD11c 高 MHCII 高 ), CD11b + Myeloid cells are subdivided into LyG + 、Ly6C + and F4 / 80 + (RPM, red pulp macrophages). Figure 19C Shown is the gating strategy for erythroid progenitors using untreated and TLI-treated spleens. Figure 19D Extramedullary erythropoiesis is shown in the spleen treated with TLI and in the bone marrow treated with TLI. Figure 19ESystemic EPO was shown to be increased in peripheral serum as measured by enzyme-linked immunosorbent assay (ELISA).

[0052] Figures 20A-20H Shown are the effects of CD8α sorted from TLI / ATS-conditioned mice versus untreated (UNT) mice. + RNA-seq analysis of cDC1. Figure 20A Shown are the total splenocyte numbers of mice that were untreated or treated with TLI / ATS. Figure 20B Live cells (DAPI) of mice that were not treated or treated with TLI / ATS were shown. - ) CD11c 高 MHCII 高 The frequency of DC. Figure 20C CD8α + CD11b - and CD11b + CD8α - Gating strategy for cDCs (left) and CD11 高 MHCII 高 DC, UNT relative to CD8a in TLI / AT + CD11b - Frequency of DCs (right). Representative samples from mice conditioned with TLI / AT are shown. Figures 20A-20C The numbers in the figure indicate the percentage of positively stained cells within each gate. Data are mean ± sem, ***p < 0.001 and ****p < 0.001, determined by unpaired Student's t-test. The number of mice per group is as indicated. The results represent one of at least three similar experiments. Figure 20D To show CD8α + Principal component analysis (PCA) plots of different clustering of TLI / AT by DC and UNT. Figure 20E Shown is a heat map of RNA-seq gene expression for the top 30 upregulated (P≤0.01 and fold change≥log2) genes in the TLI / AT-treated group versus the UNT group. Biological replicates for each group (n=2, pooled from 3-5 mice per group) are shown separately. Heat maps were generated based on differential expression using DESeq2 in R studio software. Figure 20F Shown is a gene set enrichment analysis (GSEA) performed after TLI / AT using hallmark gene sets from the Molecular Signatures Database (MSigDB). NES: Normalized enrichment score. FDR: False discovery rate. Right half of the figure: Upregulated pathways. Left half of the figure: Downregulated pathways. Figure 20GShows spleen CD8α + cDC1 and CD11b + Real-time PCR of the indicated genes in cDC2 cells. CD8α cells were sorted by flow cytometry from (i) UNT, (ii) ATS, (iii) TLI, and (iv) TLI / ATS-conditioned mice on the day after the last TLI dose. + cDC1 (top) and CD11b + cDC2 (below). Gating strategy Figure 20C Data are mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.001, ns (not significantly different) determined by unpaired Student's T test. Figure 20H CD8α in EPOR-tdT mice treated with UNT, TLI, and TLI / ATS is shown. + EPOR expression in cDC1. In the TLI and TLI / ATS groups, spleens were harvested the day after the last TLI dose.

[0053] Figure 21 The results show that wild-type (WT), EPOR flox / flox mice, and DC-specific heterologous EPOR gene deletion (EPOR ΔCD11c TLI-ATS-induced chimerism in B cells, T cells, granulocytes, and macrophages (MΦ) of BM-transplanted mice and Baft3 knockout (KO) mice. 14 days after BM transplantation, T cells (TCRβ + ), B cells (B220 + ) and granulocytes (Ly6G + ) in the uterus. Bars indicate the mean percentage of donor cells. P values ​​were determined by two-tailed t-test of independent means. *P < 0.05; **P < 0.01; ***P < 0.001; ns, not significantly different.

[0054] Figures 22A-22B We show that the establishment and maintenance of bone marrow chimerism is abrogated by administering diphtheria toxin (DT) to FoxP3-DTR (forkhead box P3-diphtheria toxin receptor) recipient mice. Figure 22A Figure 2 shows the experimental scheme for two groups of FoxP3-DTR mice treated with different DTs. FoxP3-DTR mice were conditioned with TLI / ATS and DT was administered intravenously (iv) on day 3 after allogeneic BM infusion (Group A; top) or on day 15 after BM chimerism was established (Group B; bottom). DT was administered every 2 days. Both groups were examined for bone marrow chimerism on days 14 and 29. Figure 22BThe expression of donor (MHCI-H2kb)-derived T cells (TCRβ) in group A (left 3 bars in all 4 figures) and group B (right 3 bars in all 4 figures) is shown. + ), B cells (B220 + ), macrophages (MΦ; CD64 + ) and granulocytes (Ly6G + ) percentage.

[0055] Figures 23A-23D CD8α + cDC1 for antigen-specific CD4 + FoxP3 + Requirement for Treg induction and expansion. C57BL / 6 (wild type) or Batf3 - / - mouse, or EPOR ΔCD11c Recipient mice were either untreated (UNT) or conditioned with TLI. One day after the last TLI dose, macrophage-negatively selected OTII cells (cells expressing ovalbumin (Ova)-specific αβ TCR) were injected intravenously (iv), and on another day, bone marrow cells expressing Ova were injected iv. Five days later, adoptively transferred OTII cells (defined as TCR-vα2 + CD4 + ) on FoxP3 expression. Figure 23A and 23C Display of TCR-vα2 + CD4 + FoxP3 in OTII cells + Figure 1 shows the gating strategy for Tregs. Each figure shows C57BL / 6 cells that were untreated (UNT) or treated with TLI ( Figure 23A ), mice with Batf knockout (KO) ( Figure 23A ), mice with heterologous EPOR deleted in dendritic cells (EPOR ΔCD11c )( Figure 23C ) of the spleen TCRvα2 + FoxP3 in CD4+ living OTII cells + The percentage of Tregs. Figure 23B and 23D Histograms showing FoxP3 expression in adoptively transferred OTII cells. Each graph shows the expression of FoxP3 in UNT versus TLI-treated C57BL / 6 mice or mice with Batf3 KO ( Figure 23B ) or EPOR ΔCD11c Mouse ( Figure 23D) FoxP3 mean fluorescence intensity (MFI). Data are mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001 and ****p < 0.001, ns (not significantly different) determined by unpaired Student's T test, the number of mice in each group is as indicated.

[0056] Figures 24A-24D CD4 + FoxP3 - CD73 + Folate receptor 4+ (FR4 + ) induction of anergic T cells, and the induction is dependent on the presence of Tregs. Figure 24A BM chimerism is shown in B cells, T cells, granulocytes and macrophages (MΦ) in the absence (w / o) and presence of diphtheria toxin (DT). DT was injected on day -1 to day 1. Figure 24B Shown are analyses of intercellular IFNγ expression by Tregs and anergic T cells at day 5 after allogeneic BM loading with or without DT. Figure 24C show Figure 24B Statistical analysis. Figure 24D Display FoxP3 + Treg frequency (X axis) and CD4 + FoxP3 - CD73 + FR4 + Correlation between anergic T cell frequencies (Y axis). Linear regression was determined using Prism. Data are mean ± SEM, *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.001, ns (no significant difference) determined by unpaired Student's T test, with the number of mice in each group as indicated.

[0057] Figures 25A-25C Shown after TLI through CD8α + cDC1 5-chloromethylfluorescein diacetate + (CMFDA + ) Allogeneic bone marrow uptake. Figure 25A To show that live allogeneic BM cells are expressed in recipient CD11 高 MHCII 高 Engulfment in DCs (left) and CD11 12 hours after BM injection, respectively. 高 MHCII 高 CMFDA in recipient DCs + CD8α + and CD8α - Comparison of cDC frequencies (right). Figure 25B Gated CD11 showing untreated (UNT) or treated with TLI 高 MHCII 高 CD8α + CMFDA in cDC1 + The percentage of cells. Figure 25C CD8α + CMFDA + CD103 and DEC-205 expression in cDC1. Distribution of UNT (black) and TLI (pink) overlap.

[0058] Figures 26A-26B CD8α + cDC1 and CD11b + Expression of Epo-EPOR downstream signaling molecules in cDC2s and the percentage of donor cells from various mouse strains. Figure 26A Shown are CD8α from untreated (UNT) mice, mice treated with TLI, and mice treated with TLI and ATS, as measured by MFI. + cDC1 and CD11b + Expression of EPO-EPOR downstream signaling molecules in cDC2s Intracellular phospho-flow cytometry was performed one day after the last TLI dose. Figure 26B showed that C57 / 6J, Baftf3- / -, CD11c Cre EPOR flox / flox 、mTOR flox / flox EPOR ΔXCR1 and mTOR ΔXCR1 The percentage of donor cells containing B cells, T cells, granulocytes and macrophages.

[0059] Figures 27A-27C Shown are the effects of XCR1-specific EpoR or mTOR deletion on tumor Ag-specific CD8+ T cells in tumor-draining lymph nodes (tdLN). Figure 27A Analysis of control mice, mice with EpoR knockout in dendritic cells (EpoR ΔXCR1 ) and mice with mTOR knockout in dendritic cells (mTOR ΔXCR1 ) in OT-I (CD8+ T cells expressing T cell antigen receptor). Figure 27B Shown are the measurement of CD44, SLAMF6, PD-1, and Tim3 expressing cells and the measurement of proliferating cells (cell trace violet (CTV)) via flow cytometry. Figure 27C Display control, EpoR ΔXCR1 Mice and mTORΔXCR1 Percentage of proliferating OT-1 in mice. ***P<0.001; ns=not significant.

[0060] Figures 28A-28B Analysis of antibodies in supernatants of hybridoma clones is shown. Figure 28A Shown are the percentage of cell staining of 293T cells expressing EPOR, CD131, or both, binding kinetics data (EPOR-CD131-Fc, EPOR-Fc, and CD131-Fc), and data on the percentage of blocking EPO / EPOR interaction for 17 clones with unique antibody sequences. Figure 28B Flow cytometry showed that the anti-EPOR and Alexa Fluor 5000 ligands were labeled with phycoerythrin (PE) and Alexa Fluor 5000 ligands, respectively. The expression of human EPOR (hEPOR) and human CD131 (hCD131) was measured by anti-CD131 labeled with AF647 (AF647).

[0061] Figures 29A-29D Shown are 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 EPOR and CD131 (293T / EPOR / CD131) stained with purified antibodies. Figure 29A Shown are the MFIs of 293T / EPOR cells labeled with purified hybridoma clones M2 and M41 at different antibody concentrations. Figure 29B Shown are the MFIs of 293T / EPOR / CD131 cells labeled with purified hybridoma clones M2 and M41 at different antibody concentrations. Figure 29C Shown are the MFIs of 293T / CD131 cells labeled with purified hybridoma clones M2 and M41 at different antibody concentrations. Figure 29D Shown are the MFIs of UT-7 cells labeled with purified hybridoma clones M2 and M41 at different antibody concentrations.

[0062] Figure 30 Figure 2 shows phosphorylated STAT5 analyzed using a flow-based assay. UT-7 cells, 293T cells expressing EPOR (293T / EPOR), and 293T cells expressing EPOR and CD131 (293T / EPOR / CD131) were incubated with anti-EPOR antibodies (hybridoma clone M2, top panel; or hybridoma clone M41, bottom panel) after stimulation with (EPO+M2 or EPO+M41) or without recombinant human EPO (no EPO control). The same cells not incubated with anti-EPOR antibodies after EPO stimulation were used as controls (EPO, no Ab control).

[0063] Figures 31A-31B Shown are SDS-PAGE analyses of IME001, IME003, IME004, carbamylated EPO (CEPO), and recombinant human EPO (rhEPO). Figure 31A SDS-PAGE of expression vectors IME001 and IME003 in which EPO is fused to the N-terminus of human IgG4 or human serum albumin, and expression vector IME004 in which EPO is fused to the C-terminus of human albumin is shown. Figure 31B Shown are SDS-PAGEs of BSA control, rhEPO with or without Lyc-C digestion, and CEPO with or without Lyc-C digestion.

[0064] Figures 32A-32D Shown is a cell staining assay to measure the receptor binding activity of IME001, IME003, and IME004. Figure 32A Flow cytometric analysis of 293T cells (left) or 293T / EPOR cells (right) stained with anti-EPOR PE conjugate is shown. Figure 32B Shown are flow cytometric analyses of 293T / EPOR cells incubated with 1 μg / ml (left), 0.1 μg / ml (middle), or 0.01 μg / ml (right) IME001 and stained with anti-human Fc PE conjugate. Figure 32C Shown are flow cytometric analyses of 293T / EPOR cells incubated with 10 μg / ml (left), 1 μg / ml (middle), or 0.1 μg / ml (right) IME003 (upper panel) or IME004 (lower panel), biotinylated anti-HSA (human serum albumin), and streptavidin-PE conjugate. Figure 32D The binding of IME003 EPOR-Fc, IME003 IME 020, IME004 EPOR-Fc, and IME004 IME020 under various IME003 / IME004 viruses is shown.

[0065] Figures 33A-33B Shown is the analysis of STAT5 phosphorylation in 293T / EPOR cells stimulated with various EPO proteins. Figure 33A Shown are Western blot analyses of 293T / EPOR cells untreated (control) or stimulated with CEPO, IME001, IME003, or IME004 using human phospho-STAT5a / b (Y694 / Y699). Figure 33BShown are the results of phosphorylated STAT5 enzyme-linked immunosorbent assay (ELISA) using lysates of 293T / EPOR cells that were untreated (untreated control) or stimulated with IME001, IME003, IME004, IME005, IME008, or IME013.

[0066] Figure 34 The amino acid sequence and nucleic acid sequence of human EPO are shown, including the signal peptide sequence.

[0067] Figures 35A-35E EpoR expression on peripheral lymph node (pLN) migratory cDC1 is shown. Figure 35A EpoR tdt / + 、Zbtb46 gfp / + EpoR tdT / + 、CCR7 - / - EpoR tdT / + and Batf3 - / - EpoR tdT / + Flow cytometric analysis of EpoR expression in mouse pLN migratory and resident cDC1s. Figure 35B Histograms showing EpoR expression in migratory and resident cDC1s from individual mouse lines. Figure 35C Flow analysis of EPOR expression in individual inguinal, axillary, branchial, or superficial cervical lymph nodes is shown. Figure 35D Flow cytometric analysis showing EPOR and CD103 expression in pLN migratory cDCs. Figure 35E The experimental scheme showing the cross between EpoR-tdT-cre mice and Rosa26-lox-Stop-lox-EYFP mice and the expression of pLN migratory cDC1 (MHCII 高 CD11 inter XCR1 + ) Flow cytometric analysis of EYFP expression.

[0068] Figure 36 Display expression DEC205 + and CCR7 + Peripheral LN migratory EpoR + XCR1 + Flow cytometric analysis of cDC1. Figure 36 It also showed that EpoR 高 Expression of PD-L1, Tim3, Axl, and CD131 on migrating cDC1 and EpoR 低 Histogram comparing migratory cDCs.

[0069] Figures 37A-37C Showing peripheral LN (pLN) migratory EpoR + XCR1+ Effect of cDC1 on the induction of Ag-specific Tregs against DEC205-Ova and Ova-expressing cells. Figure 37A Display of pLN migratory and resident EpoR + cDC1 and EpoR - Flow cytometric analysis of cDC1. Figure 37B showed that with or without TGFβ treatment, the expression of CD45.2 + CellTrace culture of cDC1, purified macrophages, and DEC-205-Ova TM Violet (CTV) labeled naive CD45.1 + Flow cytometry and quantification of FoxP3 expression in OT-II cells. Figure 37C Shown in the presence or absence of TGFβ treatment or in the presence or absence of EPO treatment with CD45.2 + cDC1, purified macrophages and Gy-irradiated Act-mOVA thymocytes (CD45.2 + )CellTrace TM Violet (CTV) labeled naive CD45.1 + Flow cytometry and quantification of FoxP3 expression in OT-II cells.

[0070] Figure 38A -38C shows in vitro antigen (Ag)-specific regulatory T cell (Treg) induction with carbamylated EPO (CEPO) treatment. Figure 38A CD11c expression in the presence of EPO or CEPO treatment Int MHCII 高 XCR1 + FoxP3 expression and proliferation of cDC1 (CellTrace TM Flow cytometric analysis of CTV. Figure 38A Also shown are quantifications of the percentage of FoxP3+ Tregs in viable OTII untreated or in the presence of EPO or CEPO treatment. Figure 38B showed that using mice with mTOR knockout in dendritic cells (mTOR ΔXCR1 ), mice with EPOR knockout in dendritic cells (EPOR ΔXCR1 ) and littermate controls to study the effects of EPO or CEPO on antigen-specific tolerance.

[0071] Figures 39A-39C Shown is the expression of EPOR in migratory cDCs carrying apoptotic cells. Figure 39AShown is the experimental protocol in which mice were injected with cDC1 in the 3rd mammary fat pad. Figure 39B Flow cytometric analysis showing EPOR expression in cDC1s from the third mammary fat pad. Figure 39C Shows the injection of PKH67-labeled CD45.1 + Flow cytometric analysis of EPOR expression in draining lymph nodes (inguinal LN) of dexamethasone (DEX)-induced apoptotic thymocytes.

[0072] Figures 40A-40B Shown are the effects of EPO on peripheral Ag-specific tolerance to a cell-associated antigen (Ova) in draining lymph nodes. Figure 40A Shown is an IV injection of 5x10 on day -1 5 Purified macrophages and CellTrace TM Violet (CTV) labeled naive CD45.1 + Experimental protocol for OT-II cells. On day 0, dexamethasone (DEX)-induced apoptotic Act-mOVA thymocytes were injected subcutaneously into the third mammary fat pad. 50 IU EPO was administered ip for 4 consecutive days. Figure 40B The results showed that the expression of CD45.1 in draining lymph nodes (inguinal LN) was significantly correlated with the presence or absence of EPO. + Flow cytometric analysis and quantification of FoxP3 expression in OT-II.

[0073] Figures 41A-41C The binding activities of IME003 and IME004 are shown. Figure 41A The binding of IME003 and IME004 to IME083 or IME020 at various IME003 and IME004 concentrations is shown. Figure 41B The binding of IME061 / IME062, IME061 / IME063, IME061 / IME064, and IME063 / IME084 to IME003 at different IME003 concentrations is shown. Figure 41C The binding of IME061 / IME062, IME061 / IME063, IME061 / IME064, IME063 / IME084 to IME004 at different IME004 concentrations is shown.

[0074] Figures 42A-42D Shown are 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 The amino acid and nucleic acid sequences of human EPOR ECD in IME020 and IME061 are shown. Figure 42B The amino acid and nucleic acid sequences of human CD131 ECD in IME062 are shown. Figure 42C The amino acid and nucleic acid sequences of the human CD131 D3D4 domain in IME063 are shown. Figure 42D The amino acid and nucleic acid sequences of the human EPOR (F93A) domain in IME083 and IME034 are shown. DETAILED DESCRIPTION

[0075] Although various embodiments of the present disclosure are described herein, it will be apparent to those skilled in the art that such embodiments are provided by way of example only. Many modifications and variations, as well as modifications and substitutions to the embodiments described herein will be apparent to those skilled in the art without departing from the present disclosure. It should be understood that various alternatives to the embodiments described herein may be employed in practicing the present disclosure. It should also be understood that each embodiment of the present disclosure may be optionally combined with any one or more of the other embodiments described herein that are consistent with that embodiment.

[0076] When elements are presented in list form (eg, in the form of Markush groups), it is understood that every possible subgroup of the elements is also disclosed, and any one or more elements may be removed from the list or group.

[0077] It should also be understood that, unless explicitly indicated to the contrary, in any method described or claimed herein that includes more than one act or step, 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 encompasses embodiments where the order is so limited.

[0078] It should also be understood that, generally, where an embodiment in the specification or claims is referred to as comprising one or more features, the disclosure also encompasses embodiments consisting of or consisting essentially of such features.

[0079] It should also be understood that any embodiment of the present disclosure (eg, any embodiment found in the prior art) can be explicitly excluded from the claims, regardless of whether the specific exclusion is recited in the specification.

[0080] It should also be understood that, unless otherwise specifically stated or the context clearly indicates otherwise, references herein to peptides, polypeptides or proteins (such as antibodies or fragments thereof) include pharmaceutically acceptable salts thereof. Such salts may have a positive net charge, a negative net charge or no net charge.

[0081] Headings are included herein for reference and to help locate certain sections. The headings are not intended to limit the scope of the embodiments and concepts described in the sections under those headings, and those embodiments and concepts may be applicable in other sections throughout the disclosure.

[0082] All patent documents and all non-patent documents cited herein are hereby incorporated by reference in their entirety to the same extent as if each individual patent document or non-patent document was specifically and individually indicated to be incorporated by reference in its entirety.

[0083] In addition to erythroid progenitor cells, there is increasing evidence that EPOR is widely expressed in non-erythroid cells, such as hematopoietic stem cells (HSC), megakaryocytes, B cells, T cells, macrophages (MΦ), endothelial cells and neurons (Broxmeyer, J Exp Med 2013:210:205-208). It is worth noting that the immunomodulatory effect of EPO is increasingly recognized (Cantarelli et al., Am J Transplant 2019:19:2407-2414; Peng et al., Cell Death Dis 2020:11:79). The participation of EPO signaling suppresses inflammatory responses by inhibiting NFκB-induced immune pathways (Nairz et al., Immunity 2011:34:61-74). In addition, EPO contributes to the effective efferocytosis of MΦ, thereby preventing autoimmunity (Luo et al., Immunity 2016:44:287-302).

[0084] EPO has a cardioprotective effect in ischemia-reperfusion injury and myocardial infarction. EPO improves cardiac function associated with neovascularization, which is 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).

[0085] EPO stimulates new blood vessel formation and angiogenesis by activating endothelial cells (EC) and endothelial progenitor cells (EPC) in physiological and pathological conditions (e.g., ischemic cardiovascular disease and tumors). Activation of EPOR leads to mobilization, proliferation, migration and differentiation of EC and EPC (Annese et al., Experimental Cell Research, 2019: 374 (2): 266-273).

[0086] In the central nervous system, EPO and EPOR are expressed by neurons, glial cells and cerebral vascular endothelium. In vitro and in animal models of nerve damage associated with trauma, stroke, ischemia, inflammation and epileptic seizures, EPO shows neurotrophic and neuroprotective effects. The beneficial effects of EPO have also been demonstrated in clinical studies of stroke, schizophrenia and progressive multiple sclerosis. EPO directly protects neurons by preventing apoptosis, and indirectly protects neurons by regulating inflammatory processes and stimulating neurogenesis and angiogenesis (Wang et al., Stroke 2004:35:1732-7).

[0087] EPO is not only embodied in the regulation of metabolism by oxygen delivery, but also contributes to the maintenance of white adipose tissue and metabolic homeostasis. EPO is protective in terms of 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 regulates the pro-inflammatory response of macrophage infiltration in white adipose tissue, and promotes anti-inflammatory phenotype (Alnaeeli et al., Diabetes Metab. Res. Rev. 2014:63:2415-2431) by suppressing the expression of proinflammatory cytokines and reducing macrophage infiltration.

[0088] It has been shown that some of the cytoprotective effects of EPO are mediated by its binding to heterodimers containing the classical EPOR and a common β receptor (βcR or CD131) (Brines et al., Proc Natl Acad Sci USA 2004; 101: 14907-14912). Interestingly, carbamylated EPO binds to these heterologous receptors and exerts tissue protective effects, whereas it does not bind to the classical EPOR and does not stimulate erythropoiesis. βcR is not required for erythropoiesis. It is hypothesized that the combination of βcR and EPOR expressed by non-hematopoietic cells constitutes a tissue protective receptor, thus generating a tissue protective heterologous receptor.

[0089] 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. The binding of EPO to EPOR on erythroid progenitor cells increases the expression of transcription factors GATA1 and TAL1, thereby transactivating EPOR expression (Suresh et al., Front Physiol. 2020: 10: 1534). EPOR is regulated at the protein level. P85 promotes EPOR endocytosis and degradation. Prolyl hydroxylase D3 (PHD3) mediates proline hydroxylation of EPOR, leading to proteasomal degradation. TFR2 and Scribble promote the recycling of EPOR recycling (Bhoopalan et al., F1000 Res. 2020; 9: F1000 Faculty Rev-1153).

[0090] The inventors recently discovered that EPOR plays a key role in inducing tumor immune tolerance (including systemic antigen-specific immune tolerance induced by liver metastasis) in myeloid cells (including dendritic cells (DC) and macrophages (MΦ)) in a variety of primary and metastatic tumors ( Figure 2 In addition, EPOR is essential for myeloid cell-mediated tolerance in allogeneic organ transplantation (e.g., kidney, liver, lung, heart, etc.). Figure 2 ).

[0091] definition

[0092] Unless defined otherwise or explicitly indicated otherwise when used herein, 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.

[0093] As used in the specification and the appended claims, the indefinite articles "a" and "an" and the definite article "the" may include plural as well as singular referents unless specifically stated otherwise or the context clearly dictates otherwise.

[0094] The term "about" or "approximately" means an acceptable error of a specific value as determined by those of ordinary skill in the art, depending on the measurement or determination of the value. In certain embodiments, the term "about" or "approximately" means within a standard deviation. In some embodiments, when there is no description of a specific error limit (for example, the standard deviation of the mean value given in a graph or table of data), the term "about" or "approximately" means covering the range of the value and by rounding up to the range included in the value, taking into account significant figures. In certain embodiments, the term "about" or "approximately" means within ± 10%, 5%, 4%, 3%, 2% or 1% of a specified value. Whenever the term "about" or "approximately" is located before the first numerical value in a series of two or more numerical values ​​or a series of two or more numerical ranges, the term "about" or "approximately" is applicable to each numerical value in the series of numerical values ​​or the series of numerical ranges.

[0095] The term "antibody" refers to a protein that is functionally defined as conjugated proteins and structurally defined as comprising an amino acid sequence that is believed to be derived from a framework region of an immunoglobulin (Ig) encoding gene. Antibodies can comprise one or more polypeptides that are substantially encoded by immunoglobulin genes or fragments of immunoglobulin genes. Identified immunoglobulin genes include kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes and countless immunoglobulin variable region genes. Light chain can be classified as kappa or lambda. Heavy chain can be classified as gamma, mu, alpha, delta, or epsilon, which then define immunoglobulin classes IgG, IgM, IgA, IgD, and IgE, respectively. In some embodiments, these can be further subdivided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2.

[0096] It is known that a typical gamma immunoglobulin (antibody) structural unit comprises a tetramer. Each tetramer can be composed of two pairs of identical 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 can define a variable region of about 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. The term variable light chain (V L ) and variable heavy chain (V H ) may refer to these light and heavy chains, respectively.

[0097] Antibodies can exist as complete immunoglobulins or as many well-characterized fragments. Thus, for example, pepsin can digest the antibody below the disulfide bond in the hinge region to produce F(ab)'2, which is a dimer of Fab', which itself is naturally a light chain connected to the VH-CH1-hinge by a disulfide bond. F(ab)'2 can be reduced under mild conditions to destroy the disulfide bond in the hinge region, thereby converting the (Fab')2 dimer into a Fab' monomer. The Fab' monomer is essentially a Fab with a portion of the hinge region (for a more detailed description of other antibody fragments, see Fundamental Immunology, WE Paul, ed., Raven Press, NY (1993)). Although various antibody fragments are defined based on the digestion of intact antibodies, it will be understood by those skilled in the art that fragments can be synthesized chemically or by utilizing recombinant DNA methods to de novo. Therefore, the term "antibody" as used herein can also include antibody fragments produced by modifying whole antibodies or antibody fragments synthesized using recombinant DNA methods. Preferred antibodies can include V H -V L Dimers include single-chain antibodies (antibodies that exist as a single polypeptide chain), such as single-chain Fv antibodies (sFv or scFv) in which the variable heavy chain and variable light chain regions are linked together (directly or through a peptide linker) to form a continuous polypeptide. Single-chain Fv antibodies are covalently linked V H -V L Heterodimers can be derived from V H and V L The nucleic acid encoding the sequence is expressed (e.g., Huston et al., Proc. Nat. Acad. Sci. USA, 85:5879-5883, 1988, which is hereby incorporated by reference in its entirety). H and V L They are linked to each other as a single polypeptide chain, but V H and V LThe domains are non-covalently associated. Alternatively, the antibody can be another fragment. Other fragments can also be generated, including using recombinant technology. For example, if one chain (heavy chain or light chain) is fused to the g3 capsid protein and the complementary chain is exported to the periplasm as a soluble molecule, the Fab molecule can be displayed on the phage. The two chains can be encoded on the same or different replicons; the two antibody chains in each Fab molecule are assembled after translation, and the dimer is bound to the phage particle via a bond with one chain of g3p (see, for example, U.S. Patent No. 5,733,743, which is hereby incorporated by reference in its entirety). scFv antibodies and many other structures that convert naturally aggregated but chemically separated light and heavy chain polypeptide chains from antibody V regions into molecules folded into a three-dimensional structure are known to those skilled in the art, and this three-dimensional structure is substantially similar to the structure of the antigen binding site (see, for example, U.S. Patent Nos. 5,091,513, 5,132,405 and 4,956,778, all of which are hereby incorporated by reference in their entirety). Particularly preferred antibodies may include all antibodies that have been displayed on phage or produced by recombinant technology using vectors in which each chain is secreted as a soluble protein, such as scFv, Fv, Fab, (Fab') 2. Antibodies may also include diabodies and minibodies.

[0098] Antibodies may also include heavy chain dimers, such as antibodies from camelids. H The V region of the heavy chain does not have to make hydrophobic interactions with the light chain, so in camelids, the region of the heavy chain that normally contacts the light chain is changed to hydrophilic amino acid residues. H Domain V HH domain.

[0099] In camelids, the diversity of the antibody repertoire can be measured by V H or V HH The complementarity determining regions (CDRs) 1, 2, and 3 in the camel V HH The CDR3 in the V region can be characterized by its relatively long length, averaging 16 amino acids (Muyldermans et al., 1994, Protein Engineering 7(9):1129, which is hereby incorporated by reference in its entirety). This is in contrast to the CDR3 regions of antibodies from many other species. For example, the mouse V H The CDR3 may have an average of 9 amino acids.

[0100] Libraries of camelid-derived antibody variable regions that maintain the in vivo diversity of camelid variable regions can be prepared, for example, by the methods disclosed in U.S. Patent Application Publication No. US20050037421, published on February 17, 2005, which is hereby incorporated by reference in its entirety.

[0101] The terms "functional fragment", "antigen binding portion", "antigen binding fragment", "antigen binding domain" or "antibody fragment" are used interchangeably herein to refer to one or more fragments of an antibody that retain the ability to specifically bind to an antigen. Representative antigen binding fragments can include, but are not limited to, Fab, Fab', (Fab')2, Fv, scFv, dsFv, variable heavy chain domain, variable light chain domain, variable NAR domain, bispecific scFv, bispecific Fab2, trispecific Fab3, Minibodies, diabodies, maxibodies, camelid antibodies, VHHs, intrabodies, fusion proteins comprising an antibody portion (e.g., domain antibodies), single-chain binding polypeptides, scFv-Fc, or Fab-Fc.

[0102] In some cases, antibody or its functional fragment can comprise separated antibody or its functional fragment, purified antibody or its functional fragment, recombinant antibody or its functional fragment, modified antibody or its functional fragment or synthetic antibody or its functional fragment. It should be understood that antibodies as described herein can be modified as described herein or as known in the art. In some cases, antibodies as described herein and functional fragments thereof can be produced in part or in whole synthetically. Antibody or its functional fragment can be the binding domain that has, can be antigen-binding domain or can be with its homologous polypeptide or protein. In some cases, antibody or its functional fragment can be produced in appropriate in vivo animal model, then be separated and / or purified.

[0103] The term "Fc region" can be used to define the C-terminal region of an immunoglobulin heavy chain. An "Fc region" can be a native sequence Fc region or a variant Fc region. An immunoglobulin Fc region can typically include two constant domains, CH2 and CH3.

[0104] "Antibodies" may include, but are not limited to, monoclonal antibodies, polyclonal antibodies, chimeric antibodies, bispecific antibodies, multispecific antibodies, heteroconjugate antibodies, humanized antibodies, human antibodies, deimmunized antibodies, mutants thereof, fusions thereof, immunoconjugates thereof, antigen-binding fragments thereof, functional fragments thereof and / or any other modified configuration of an immunoglobulin molecule comprising an antigen recognition site of desired specificity, including glycosylation variants of the antibody, amino acid sequence variants of the antibody and / or covalently modified antibodies.

[0105] The antibody can be a human antibody. A human antibody can be an antibody having an amino acid sequence corresponding to an antibody produced by a human and / or has been prepared using any technique known in the art or disclosed herein for preparing a human antibody. This definition of a human antibody includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. One such example is an antibody comprising a mouse light chain and a human heavy chain polypeptide. Human antibodies can be produced using various techniques known in the art. In one embodiment, the human antibody is selected from a phage library, wherein the phage library expresses 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 made by introducing human immunoglobulin loci into transgenic animals (e.g., mice in which 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 the target antigen (such B lymphocytes can be recovered from the subject or can be 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.

[0106] As used herein, the term "binding specificity" or "antibody specificity" of an antibody may refer to the identity of the antigen to which the antibody binds, preferably the identity of the epitope to which the antibody binds.

[0107] As used herein, the term "chimeric polynucleotide" may refer to a polynucleotide comprising a wild-type region and a mutant region. It may also refer to a polynucleotide comprising a wild-type region from one polynucleotide and a wild-type region from another related polynucleotide.

[0108] As used herein, the term "complementarity determining region" or "CDR" may refer to art-recognized terms, as exemplified by Kabat and Chothia. CDRs are also often referred to as hypervariable regions or hypervariable loops (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 hereby incorporated by reference in their entirety). "Framework regions" or "FRs" may refer to the regions of the V domain that flank the CDRs. The positions of the CDRs and framework regions can be determined using various definitions well known in the art, such as Kabat, Chothia, the International ImMunoGeneTics database (IMGT), and AbM (see, e.g., Johnson et al., supra; Chothia and 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)).The definition of antigenic combining sites is also described by 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 hereby incorporated by reference in their entirety.

[0109] As used herein, the term "affinity" may refer to the equilibrium constant for the reversible binding of two agents and is expressed as binding affinity (K D ). In some cases, K D It can be expressed as k off With k on The ratio, k off Can refer to the rate constant for the dissociation of an antibody from an antibody or antigen-binding fragment / antigen complex, k on Can refer to the rate constant for the association of an antibody, antigen-binding domain or antigen-binding fragment with an antigen. Binding affinity can be determined using methods known in the art, 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 can also be screened using a suitable bioassay. The binding affinity (K) of an antibody, antigen-binding domain or antigen-binding fragment herein is D) can be less than 600nM, 590nM, 580nM, 570nM, 560nM, 550nM, 540nM, 530nM, 520nM, 510nM, 500nM, 490nM, 480nM, 470nM, 460nM, 450nM, 440nM, 430nM, 420nM, 410nM , 400nM, 390nM, 380nM, 370nM, 360nM, 350nM, 340nM, 330nM, 320nM, 310nM, 3 00nM, 290nM, 280nM, 270nM, 260nM, 250nM, 240nM, 230nM, 220nM, 210nM, 200n M, 190nM, 180nM, 170nM, 160nM, 150nM, 140nM, 130nM, 120nM, 110nM, 100nM, 90nM, 80nM, 70nM, 50nM, 50nM, 49nM, 48nM, 47nM, 46nM, 45nM, 44nM, 43nM, 42n M, 41nM, 40nM, 39nM, 38nM, 37nM, 36nM, 35nM, 34nM, 33nM, 32nM, 31nM, 30nM, 29nM, 28nM, 27nM, 26nM, 25nM, 24nM, 23nM, 22nM, 21nM, 20nM, 19nM, 18nM, 17n M, 16nM, 15nM, 14nM, 13nM, 12nM, 11nM, 10nM, 9nM, 8nM, 7nM, 6nM, 5nM, 4nM, 3 nM, 2nM, 1nM, 990pM, 980pM, 970pM, 960pM, 950pM, 940pM, 930pM, 920pM, 910 pM, 900pM, 890pM, 880pM, 870pM, 860pM, 850pM, 840pM, 830pM, 820pM, 810pM , 800pM, 790pM, 780pM, 770pM, 760pM, 750pM, 740pM, 730pM, 720pM, 710pM, 70 0pM, 690pM, 680pM, 670pM, 660pM, 650pM, 640pM, 630pM, 620pM, 610pM, 600p M, 590pM, 580pM, 570pM, 560pM, 550pM, 540pM, 530pM, 520pM, 510pM, 500pM, 4 90pM, 480pM, 470pM, 460pM, 450pM, 440pM, 430pM, 420pM, 410pM, 400pM, 390 pM, 380pM, 370pM, 360pM, 350pM, 340pM, 330pM, 320pM, 310pM, 300pM, 290pM,280 pM, 270 pM, 260 pM, 250 pM, 240 pM, 230 pM, 220 pM, 210 pM, 200 pM, 190 pM, 180 pM, 170 pM or any integer therebetween.

[0110] If an antibody can bind to a target with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other substances, it can selectively bind to a target. For example, an anti-EPO antibody or a functional fragment thereof that selectively binds to an EPO protein is an antibody or functional fragment that can bind to this target with greater affinity, avidity, more readily, and / or for a longer duration than it binds to a protein other than an EPO protein.

[0111] As used herein, the term "EPO analog" may refer to a polypeptide having a modification of its polypeptide structure or a polypeptide having a shorter, longer, and / or different amino acid sequence than wild-type human erythropoietin, all of which bind to a heterologous EPOR or a homologous EPOR with high affinity. An EPO analog may be an antagonist or agonist of a heterologous EPOR or a homologous EPOR. An EPO analog may block the activity of a heterologous EPOR or a homologous EPOR. An EPO analog may activate a heterologous EPOR without activating a homologous EPOR. An EPO analog may activate a homologous EPOR without activating a heterologous EPOR. An EPO analog may inhibit a heterologous EPOR without inhibiting a homologous EPOR. An EPO analog may inhibit a homologous EPOR without inhibiting a heterologous EPOR.

[0112] Whenever the term "at least" or "greater than" precedes the first value in a series of two or more values, the term "at least" or "greater than" applies to every value in the series.

[0113] The term "heterologous" may refer to an amino acid or nucleotide sequence with which it is not naturally associated.

[0114] As used herein, the term "immunotherapy" may refer to a specific therapy that is intended to modulate immune system components (such as antibodies or immune cells) or to stimulate, inhibit or otherwise modulate the immune system with drugs or other agents. For example, "immunotherapy" may refer to checkpoint inhibitor therapy, adoptive cell therapy and / or autologous or allogeneic CAR T cell therapy.

[0115] Whenever the term "or no more than" or "less than" precedes the first value in a series of two or more values, the term "or no more than" or "less than" applies to each of the values ​​in the series.

[0116] The term "polynucleotide" can refer to a polymer consisting of nucleotide units. Polynucleotides can include naturally occurring nucleic acids, such as deoxyribonucleic acid ("DNA") and ribonucleic acid ("RNA"), and nucleic acid analogs. Nucleic acid analogs can include those containing non-naturally occurring bases, nucleotides connected to other nucleotides except naturally occurring phosphodiester bonds, or / and bases connected by linkages except phosphodiester bonds. Non-limiting examples of nucleotide analogs can include phosphorothioates, phosphorodithioates, phosphotriesters, phosphoramidates, boranophosphates, methylphosphonates, chiral methylphosphonates, 2-O-methyl ribonucleotides, peptide-nucleic acids (PNA), etc. Such polynucleotides can, for example, be synthesized using an automated DNA synthesizer. The term "nucleic acid molecule" can refer to larger polynucleotides. The term "oligonucleotide" can refer to shorter polynucleotides. In certain embodiments, an oligonucleotide can comprise no more than about 50 nucleotides. It should be understood that when a nucleotide sequence is represented by a DNA sequence (ie, A, T, G, C), this also includes RNA sequences (ie, A, U, G, C) in which "U" replaces "T".

[0117] The term "polypeptide" may refer to a polymer composed of natural and / or non-natural amino acid residues, naturally occurring structural variants thereof, and / or 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 recombinantly produced in cells expressing a nucleic acid sequence encoding the polypeptide. The term "protein" may refer to a larger polypeptide. The term "peptide" may refer to a shorter polypeptide. In certain embodiments, a peptide may comprise no more than about 50, about 40, or about 30 amino acid residues. Polypeptides may include antibodies and fragments thereof. Conventional symbols are used herein to describe polypeptide sequences: the left end of the polypeptide sequence is the amino (N) terminus; the right end of the polypeptide sequence is the carboxyl (C) terminus.

[0118] A polypeptide may include one or more modifications made during the synthesis or cellular production of the polypeptide, such as one or more post-translational modifications, whether or not the modification(s) 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 intra- or intermolecular disulfide bonds, formation of lactams between two side chains, formation of pyroglutamate, carbamylation, and ubiquitination. As another example, a polypeptide may be linked to a natural polymer (e.g., a polysaccharide) or a synthetic polymer (e.g., polyethylene glycol [PEG]), lipidated (e.g., with a C8-C 20The polypeptide can be 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.

[0119] The term "conservative substitution" can refer to replacing an amino acid in a polypeptide with a functionally, structurally, or chemically similar natural or non-natural amino acid. In certain embodiments, each of the following groups contains natural amino acids that are conservative substitutions for each other:

[0120] 1) Glycine (Gly / G), Alanine (Ala / A);

[0121] 2) Isoleucine (leu; I), leucine (Leu / L), methionine (Met / M), valine (Val / V);

[0122] 3) Phenylalanine (Phe / F), tyrosine (Tyr / Y), tryptophan (Trp / W);

[0123] 4) Serine (Ser / S), threonine (Thr / T), and cysteine ​​(Cys / C);

[0124] 5) Asparagine (Asn / N), glutamine (Gln / Q);

[0125] 6) Aspartic acid (Asp / D), glutamic acid (Glu / E); and

[0126] 7) Arginine (Arg / R), Lysine (Lys / K), Histidine (His / H).

[0127] In further embodiments, each of the following groups contains natural amino acids that are conservative substitutions for one another:

[0128] 1) Non-polar: Ala, Val, Leu, Ile, Met, Pro (proline / P), Phe, Trp;

[0129] 2) Hydrophobicity: Val, Leu, Ile, Phe, Tyr, Trp;

[0130] 3) Aliphatic: Ala, Val, Leu, Ile;

[0131] 4) Aromatic: Phe, Tyr, Trp, His;

[0132] 5) Uncharged polar or hydrophilic: Gly, Ala, Pro, Ser, Thr, Cys, Asn, Gln, Tyr (tyrosine can be considered a hydrophobic amino acid with a polar side group);

[0133] 6) Containing aliphatic hydroxyl or sulfhydryl groups: Ser, Thr, Cys;

[0134] 7) Containing amides: Asn, Gln;

[0135] 8) Acidic: Asp, Glu;

[0136] 9) Basic: Lys, Arg, His; and

[0137] 10) Small: Gly, Ala, Ser, Cys.

[0138] In other embodiments, the amino acids may be grouped as follows:

[0139] 1) Hydrophobicity: Val, Leu, Ile, Met, Phe, Trp, Tyr;

[0140] 2) Aromatic: Phe, Tyr, Trp, His;

[0141] 3) Neutral hydrophilicity: Gly, Ala, Pro, Ser, Thr, Cys, Asn, Gln;

[0142] 4) Acidic: Asp, Glu;

[0143] 5) Basic: Lys, Arg, His; and

[0144] 6) Residues that affect the direction of the main chain: Pro, Gly.

[0145] 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.

[0146] The present disclosure encompasses pharmaceutically acceptable salts of polypeptides, including salts with a net positive charge, salts with a net negative charge, and salts with no net charge.

[0147] The term "pharmaceutically acceptable" can refer to a material (e.g., active ingredient or excipient) that is suitable for contact with the tissues and organs of an object without excessive stimulation, allergic response, immunogenicity, and toxicity, commensurate with a reasonable benefit / risk ratio, and effective for its intended use. The "pharmaceutically acceptable" excipient or carrier of a pharmaceutical composition is also compatible with the other ingredients of the composition. The term "pharmaceutically acceptable" can refer to a material, such as a carrier or diluent, that does not eliminate the biological activity or properties of the compound and is relatively nontoxic, i.e., the material can be applied to an individual without causing undesirable biological effects or interacting with any component of the composition comprising it in a harmful manner. A pharmaceutically acceptable excipient can refer to any pharmaceutically acceptable ingredient that is not therapeutically active and nontoxic to the object being administered, such as a disintegrant, binder, filler, solvent, buffer, tonicity agent, stabilizer, antioxidant, surfactant, carrier, diluent, excipient, preservative, or lubricant used in preparing a pharmaceutical product. Pharmaceutical compositions can facilitate administration of the compound to an organism and can be formulated in a conventional manner using one or more pharmaceutically acceptable inactive ingredients that facilitate processing of the active compound into a pharmaceutically usable formulation. Suitable formulations depend on the chosen route of administration, and a summary of pharmaceutical compositions can be found in, for example, 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 by reference. In some embodiments, the pharmaceutical composition can be formulated by dissolving the active substance (eg, an EPOR agonist or antagonist described herein) in an aqueous solution for injection into diseased tissues or diseased cells.In some embodiments, the pharmaceutical composition can be formulated by dissolving the active substance (eg, an EPOR agonist or antagonist described herein) in an aqueous solution for direct injection into diseased tissues or diseased cells.

[0148] The term "stringent hybridization conditions" may refer to hybridization in 5X SSC at a temperature of 42°C in 50% formamide, and filter washing in 0.2X SSC at a temperature of 60°C. (1X SSC is 0.15 M NaCl, 0.015 M sodium citrate.) Stringent hybridization conditions also encompass 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; hybridization with a denaturing agent, such as formamide, e.g., 50% (v / v) formamide with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinyl pyrrolidone / 50 mM sodium phosphate buffer, pH 6.5, and 750 mM sodium chloride, 75 mM sodium citrate at 42°C; or 50% 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 at 42°C, washes in 0.2X SSC (sodium chloride / sodium citrate) at 42°C and 50% formamide at 55°C, followed by a high stringency wash consisting of 0.1X SSC containing EDTA at 55°C.

[0149] The term "subject" can refer to an animal, 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), swine (e.g., pigs), equines (e.g., horses), canines (e.g., dogs), or felines (e.g., cats). Additional examples of mammals can include, but are not limited to, any member of the class mammals: humans, non-human primates, such as chimpanzees and other apes and monkey species; farm animals, such as cattle, horses, sheep, goats, pigs; domestic animals, such as rabbits, dogs, and cats; laboratory animals, including rodents, such as rats, mice, and guinea pigs, etc. In some cases, the mammal is a human. In some cases, the subject is an adult, child, or infant. In some cases, the subject can be an animal. In some cases, the animal can include humans and non-human animals. In one embodiment, the non-human animal can be a non-human mammal as described herein. In some cases, the subject is a companion animal. In some cases, the subject is a feline, canine, or rodent.

[0150] In the context of two polypeptides or polynucleotides, the terms "substantially homologous" or "substantially identical" can refer to two or more sequences or subsequences that, when compared and aligned for maximum correspondence, 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, as measured using a sequence comparison algorithm or by visual inspection. The terms "substantially homologous" or "substantially identical" can mean at least about 70% amino acid or nucleic acid residue identity. The terms "substantially homologous" or "substantially identical" can mean at least about 85% amino acid or nucleic acid residue sequence identity. Substantial homology or identity can exist over a sequence region that is at least about 20, 30, 40, 50, 100, 150 or 200 residues in length. The sequences may be substantially homologous or identical over the entire length of either or both compared biopolymers.

[0151] 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.

[0152] An example of a useful algorithm is PILEUP. PILEUP uses progressive alignments to generate multiple sequence alignments from a group of related sequences to display relationships and percentage sequence identities. It also draws a tree or dendrogram showing the clustering relationships used to generate the alignments. PILEUP uses a simplified form of the progressive alignment method of Feng and Doolittle, J. Mol. Evol., 35:351-360 (1987). The method used is similar to the method described by Higgins and Sharp, CABIOS, 5:151-153 (1989). This program can align up to about 300 sequences, with a maximum length of about 5,000 nucleotides or amino acids for each sequence. The multiple alignment process begins with aligning the two most similar sequences, generating a cluster of two aligned sequences. This cluster is then aligned with the next most relevant sequence or the cluster of aligned sequences. Two sequence clusters are aligned by simply expanding the alignment of two individual sequences. The final comparison is realized by a series of progressive comparisons. This program is run by specifying the amino acid or nucleotide coordinates of a specific sequence and its sequence comparison region and specifying program parameters. For example, the following parameters can be used to compare a reference sequence with other test sequences to determine the sequence identity percentage relationship: default gap weight (3.00), default gap length weight (0.10) and weighted end gap. Another algorithm that can be used to generate the multiple alignment of a sequence is Clustal W (see, for example, Thompson et al., Nucleic Acids Research, 22:4673-4680

[1994] ).

[0153] Another example of an algorithm suitable for determining sequence identity and sequence similarity percentages 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 satisfy some positive threshold score T when aligned with words of the same length in the database sequence. T is called the neighborhood word score threshold (Altschul 1990). These initial neighborhood word hits serve as seeds to initiate searches for longer HSPs containing them. This word hit is then extended in both directions along each sequence as far as possible to increase the cumulative alignment score. For nucleotide sequences, the cumulative score is calculated using the parameters M (reward score for a pair of matching residues, usually >0) and N (penalty score for non-matching residues, usually <0). For amino acid sequences, the cumulative score is calculated using a scoring matrix. Extension of the word hits in each direction is stopped when: the cumulative alignment score decreases by the amount X from its maximum achieved value; the cumulative score falls to zero or below due to the accumulation of one or more negative-scoring residue alignments; or 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 wordlength (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, e.g., a wordlength (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] ).

[0154] 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 smallest 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 similar to a reference sequence if the smallest sum probability in a comparison of the test polynucleotide to the reference polynucleotide is less than about 0.1, 0.01, or 0.001.

[0155] If one polypeptide differs from another polypeptide only in conservative amino acid substitutions, then the two polypeptides can be substantially homologous or identical. Two nucleic acid sequences can be substantially homologous or identical if two polynucleotides hybridize to each other under stringent conditions or under highly stringent conditions, as described herein.

[0156] 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 of, slow the progression of, 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 the 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 being sought by a researcher, veterinarian, medical doctor, or clinician.

[0157] The terms "treat," "treating," and "treatment" may include alleviating, improving, or eliminating a medical condition or one or more symptoms or complications associated with the condition, alleviating, improving, or eliminating one or more causes of the condition, preventing additional symptoms, inhibiting a disease or condition, such as preventing the development of the disease or condition, alleviating the disease or condition, causing regression of the disease or condition, alleviating a condition caused by the disease or condition, or prophylactically and / or therapeutically stopping the symptoms of the 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 alleviating or improving the severity of the disease, delaying the onset of the disease, inhibiting the progression of the disease, reducing the number of hospitalizations or hospital stays of a subject, improving the quality of life of a subject, reducing the number of symptoms associated with the disease, alleviating or improving the severity of symptoms associated with the disease, reducing the duration of symptoms associated with the disease, preventing the recurrence of symptoms associated with the disease, inhibiting the development or onset of disease symptoms, or inhibiting the progression of symptoms associated with the disease. In some embodiments, treating cancer may include reducing the size of a tumor or increasing the survival of a cancer patient. References to "treatment" of a medical condition may include prevention of the condition. The terms "prevent," "preventing," and "prevention" may include excluding, reducing the risk of development, and delaying the onset of a medical condition or one or more symptoms or complications associated with a condition.

[0158] Erythropoietin (EPO) analogs or engineered EPO

[0159] In some aspects, at least eight types of EPO analogs are provided herein, which can be generated or engineered. In some embodiments, an EPO analog can be referred to as an engineered EPO. An EPO analog or engineered EPO can bind to a heterologous EPOR and not to a homologous EPOR, and can be an agonist or antagonist of a heterologous EPOR. Other EPO analogs or engineered EPO can bind to a homologous EPOR and not to a heterologous EPOR, and can be an agonist or antagonist of a homologous EPOR. An EPO analog or engineered EPO can bind to a homologous EPOR and a heterologous EPOR, and can be an agonist of both, an antagonist of both, or an agonist of one and an antagonist of the other. The term EPO analog or engineered EPO can include the EPO shown in SEQ ID NO: 1.

[0160] Erythropoietin (EPO) is a pleiotropic cytokine glycoprotein that was originally identified as a regulator of red blood cell production in response to hypoxia. The mature human 165 amino acid long EPO protein sequence is represented by SEQ ID NO: 1

[0161] APPRLICDSRVLERYLLEAKEAENITTGCAEHCSLNENITVPDTKVNFYAWKRMEVGQQAVEVWQGLALLSEAVLRGQALLVNSSQPWEPLQLHVDKAVSGLRSLTTLLRALGAQKEAISPPDAASAAPLRTITADTFRKLFRVYSNFLR GKLKLYTGEACRTGDR (SEQ ID NO: 1). The amino acid sequence and nucleic acid sequence (including the signal peptide sequence) of human EPO are shown in Figure 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 can be determined by comparison with SEQ ID NO: 1.

[0162] 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 oligosaccharide chains and one O-linked oligosaccharide chain, which together account for approximately 40% of the total molecular weight of the glycoprotein. N-linked glycosylation occurs at asparagine residues (Asn) at positions 24, 38, and 83, while O-linked glycosylation occurs at the serine residue (Ser) at position 126.

[0163] Three of the helices (A, C, and D) participate in the two binding sites of the homologous EPOR. Helix B participates in the interaction with the heterologous EPOR. The interaction interface between EPO and the homologous EPOR has been marked in the crystal structure (Syed et al., Nature. 1998: 395 (6701): 511-6), which contains a high affinity site (site 1) and a low affinity site (site 2). Site 1 is characterized by a central hydrophobic binding pocket flanked by hydrophilic interactions at opposite ends, which contains 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 have been shown to reduce in vitro biological activity by less than a factor of 5, while mutations K45I, N147K, R150E, and G151A have been shown to reduce activity by less than a factor of 50. Mutations at position 1 significantly reduce affinity for the homologous EPOR. Position 2 comprises 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 have been shown to reduce in vitro biological activity to less than 1 / 5, while mutations R14Q, S100E, S100T, R103A, R103E, R103H, R103N, R103Q, S104I, and L108K have been shown to reduce activity to less than 1 / 50. EPO analogs or engineered EPOs with mutations at site 2 may retain high affinity binding to homologous EPOR but lose signaling activity. These EPO variants with mutations at sites 1 or 2 but not in helix B should be active with heterologous EPOR.

[0164] Helix B is not involved in binding to the homologous EPOR. Heterologous EPORs have EPOR chains and CD131 chains. CD131 can be a homodimer, resulting in a heterohexameric receptor and a higher-order dodecameric complex with the EPO receptor chain. Helix B of EPO may be crucial for EPOR / CD131 (heterologous EPOR) binding.

[0165] 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 Lys residues and is therefore unmodified. CEPO has been shown to be as active as EPO against heterologous EPORs but not homologous EPORs. Other modifications of the Lys residues in helices A, C, and D can be used to prepare EPO analogs or engineered EPO that interact with heterologous EPORs but not homologous EPORs. For example, using well-known PEGylation reagents, PEG can be attached to Lys residues in EPO to prepare chemically modified EPO analogs that have improved serum half-lives and preferentially activate heterologous EPORs over homologous EPORs. 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 of the Lys residues in helices A, C, and D. Similarly, other chemical modifications can be made to attach other moieties to Lys residues in EPO to produce other chemical derivatives that can bind to heterologous EPORs without binding to homologous EPORs. In some embodiments, one or more Lys residues on the EPO analogs or engineered EPO described herein can be carbamylated. In some embodiments, all Lys residues on the EPO analogs or engineered EPO described herein can be carbamylated. In some embodiments, no Lys residues on the EPO analogs or engineered EPO described herein can be carbamylated.

[0166] Peptide analogs of helix B also exhibit activity similar to CEPO. Activation of the heterologous EPOR leads to phosphorylation of the intracellular domain of CD131, but not EPOR itself. Activation of both homologous and heterologous EPORs leads to activation of JAK2 and STAT5. For example, the eleven-amino acid linear peptide QEQLERALNSS (SEQ ID NO: 2), which mimics the three-dimensional structure of the external aqueous face of the helix B peptide, is a peptide analog that activates the heterologous EPOR. Because the N-terminal residue is glutamine, this peptide can cyclize to form a cyclic peptide. Cyclic peptides also activate heterologous EPOR.

[0167] EPO has previously been expressed as a functional Fc fusion protein to enhance its half-life in vivo (Schriebl et al., Protein Expr Purif. 2006, 49(2):265-75; Shi et al., PLoS One, 2013 8(8):e72673). Other approaches include albumin fusion, PEGylation, or engineering more glycosylation sites, which 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 ways (e.g., PEGylation).

[0168] In some aspects, human EPO analogs that bind to heterologous EPOR (as antagonists) but do not bind to homologous EPOR can be generated or engineered. In some embodiments, these EPO analogs or engineered EPO 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 an important role in the interaction with heterologous EPOR and can be mutated / substituted. For example, alanine scanning and / or saturation mutagenesis can be used to mutagenesis the nucleic acid encoding helix B. Mutations that allow binding to heterologous EPOR and result in reduced activation of heterologous EPOR (but still bind to heterologous EPOR) in EPO can be combined with mutations that reduce the binding of EPO analogs to homologous EPOR as described above. The resulting EPO analogs or engineered EPO can antagonize heterologous EPOR, and their binding to homologous EPOR is reduced or may not be bound to it.

[0169] In some aspects, human EPO analogs as described herein or engineered EPO can include at least one amino acid replacement or mutation. In some embodiments, human EPO analogs as described herein or engineered EPO can include 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 replacements. For example, human EPO analogs as described herein or engineered EPO can include at least one amino acid replacement or mutation on 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 EPO described herein can comprise 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 comprising 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, can be substituted with any other amino acid 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 can be determined by alignment with SEQ ID NO: 1. For example, the human EPO analogs or engineered EPO described herein can comprise 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, or a combination thereof. In some embodiments, a human EPO analog or engineered EPO comprising at least one amino acid substitution or mutation described herein can be an agonist or antagonist of a heterologous EPOR.In some embodiments, a human EPO analog or engineered EPO comprising at least one amino acid substitution or mutation described herein can be an agonist or antagonist of a homologous EPOR.

[0170] In some embodiments, a human EPO analog or engineered EPO may comprise R103A. In some embodiments, a human EPO analog or engineered EPO may comprise K45D. In some embodiments, a human EPO analog or engineered EPO may comprise N147K. In some embodiments, a human EPO analog or engineered EPO may comprise R150E. In some embodiments, a human EPO analog or engineered EPO may comprise Q58A. In some embodiments, a human EPO analog or engineered EPO may comprise E62R. In some embodiments, a human EPO analog or engineered EPO may comprise E62A. In some embodiments, a human EPO analog or engineered EPO may comprise Q65A. In some embodiments, a human EPO analog or engineered EPO may comprise L69A. In some embodiments, a human EPO analog or engineered EPO may comprise E72R. In some embodiments, a human EPO analog or engineered EPO may comprise E72A. In some embodiments, a human EPO analog or engineered EPO may comprise R76E. In some embodiments, a human EPO analog or engineered EPO may comprise R76A. In some embodiments, a human EPO analog or engineered EPO may comprise L80A. In some embodiments, a human EPO analog or engineered EPO may comprise N83A. In some embodiments, a human EPO analog or engineered EPO may comprise S84A. In some embodiments, a human EPO analog or engineered EPO may comprise S85A. In some embodiments, a human EPO analog or engineered EPO may comprise K97A. In some embodiments, a human EPO analog or engineered EPO may comprise K116A. In some embodiments, a human EPO analog or engineered EPO may comprise K140A. In some embodiments, a human EPO analog or engineered EPO may comprise G151A. In some embodiments, a human EPO analog or engineered EPO may comprise K152A. In some embodiments, human EPO analogs or engineered EPO may comprise K154A. In some embodiments, human EPO analogs or engineered EPO may comprise K45D.

[0171] In some embodiments, the human EPO analog or engineered EPO may comprise N147K.

[0172] In some embodiments, the human EPO analog or engineered EPO may comprise R150E.

[0173] In some embodiments, human EPO analogs or engineered EPO may comprise K45D and R103A. In some embodiments, human EPO analogs or engineered EPO may comprise N147K and R103A. In some embodiments, human EPO analogs or engineered EPO may comprise R150E and R103A. In some embodiments, human EPO analogs or engineered EPO may comprise Q65A and E72R. In some embodiments, human EPO analogs or engineered EPO may comprise Q65A, E72R, and N83A. In some embodiments, human EPO analogs or engineered EPO may comprise K140A and K152A. In some embodiments, human EPO analogs or engineered EPO may comprise K140A, K152A, and K154A. In some embodiments, human EPO analogs or engineered EPO may comprise N24Q, N38Q, and N83Q. In some embodiments, human EPO analogs or engineered EPO may comprise E62A, Q65A, E72A, and R76A. In some embodiments, human EPO analogs or engineered EPO may comprise N24A, N38A, and N83A. In some embodiments, human EPO analogs or engineered EPO may comprise N24S, N38S, and N83S. In some embodiments, human EPO analogs or engineered EPO may comprise R103A and G151A. In some embodiments, human EPO analogs or engineered EPO may comprise K20A, K45A, and K52A. In some embodiments, human EPO analogs or engineered EPO may comprise K20A, K45A, K52A, K140A, K152A, and K154A. In some embodiments, human EPO analogs or engineered EPO may comprise K97A and K116A. In some embodiments, human EPO analogs or engineered EPO may comprise K20A, K45A, K52A, K97A, K116A, K140A, K152A, and K154A. In some embodiments, human EPO analogs or engineered EPO may comprise Q58A, Q65A, and E72R. In some embodiments, human EPO analogs or engineered EPO may comprise L80A, N83A, S84A, and S85A. In some embodiments, human EPO analogs or engineered EPO may comprise Q58A, Q65A, E72R, L80A, N83A, S84A, and S85A. In some embodiments, human EPO analogs or engineered EPO may comprise Q58A and L69A. In some embodiments, human EPO analogs or engineered EPO may comprise Q58A and L80A.In some embodiments, human EPO analogs or engineered EPO may comprise L69A and L80A. In some embodiments, human EPO analogs or engineered EPO may comprise Q58A, L69A and L80A.

[0174] In some embodiments, human EPO analogs or engineered EPO may comprise a fragment corresponding to SEQ ID Any of NOs: 1973-2019 has an amino acid sequence having 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 can comprise the amino acid sequence of any one of SEQ ID NOs: 1973-2019.

[0175] In some embodiments, human EPO analogs or engineered EPO may have a sequence comprising the sequence of SEQ ID any of NOs: 2020-2064 having a nucleotide sequence of an amino acid sequence of 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 can have the nucleotide sequence of any one of SEQ ID NOs: 2020-2064.

[0176] In some embodiments, an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein can be expressed at a concentration of less than about 600 nM, about 590 nM, about 580 nM, about 570 nM, about 560 nM, about 550 nM, about 540 nM, about 530 nM, about 520 nM, about 510 nM, about 500 nM, about 490 nM, about 480 nM, about 470 nM, about 460 nM, about 450 nM, about 440 nM, about 430 nM, about 420 nM, about 410 nM, about 400 nM, about 390 nM, about 380 nM, about 370 nM, about 360 nM, about 350 nM, about 340 nM, about 330 nM, about 320 nM , about 310nM, about 300nM, about 290nM, about 280nM, about 270nM, about 260nM, about 250nM, about 240nM, about 230nM, about 220nM, about 210nM, about 200nM, about 190nM, about 180nM, about 170nM, about 160nM, about 150nM, about 140nM M, about 130 nM, about 120 nM, about 110 nM, about 100 nM, about 90 nM, about 80 nM, about 70 nM, about 50 nM, about 50 nM, about 49 nM, about 48 nM, about 47 nM, about 46 nM, about 45 nM, about 44 nM, about 43 nM, about 42 nM, about 41 nM, about 40 nM, about 39 nM , about 38nM, about 37nM, about 36nM, about 35nM, about 34nM, about 33nM, about 32nM, about 31nM, about 30nM, about 29nM, about 28nM, about 27nM, about 26nM, about 25nM, about 24nM, about 23nM, about 22nM, about 21nM, about 20nM, about 19nM, about 18nM M, about 17 nM, about 16 nM, about 15 nM, about 14 nM, about 13 nM, about 12 nM, about 11 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 990 pM, about 980 pM, about 970 pM, about 960 pM, about 9 50pM, about 940pM, about 930pM, about 920pM, about 910pM, about 900pM, about 890pM, about 880pM, about 870pM, about 860pM, about 850pM, about 840pM, about 830pM, about 820pM, about 810pM, about 800pM, about 790pM, about 780pM, about 770 pM, about 760 pM, about 750 pM, about 740 pM, about 730 pM, about 720 pM, about 710 pM, about 700 pM, about 690 pM, about 680 pM, about 670 pM, about 660 pM, about 650 pM, about 640 pM, about 630 pM, about 620 pM, about 610 pM, about 600 pM,about 590 pM, about 580 pM, about 570 pM, about 560 pM, about 550 pM, about 540 pM, about 530 pM, about 520 pM, about 510 pM, about 500 pM, about 490 pM, about 480 pM, about 470 pM, about 460 pM, about 450 pM, about 440 pM, about 430 pM, about 420 pM, about 410 pM, about 400 pM, about 390 pM, about 380 pM, about 370 pM, about The invention also relates to a novel EPOR comprising a nucleic acid sequence of at least one EPOR1 protein and a nucleic acid sequence thereof. The invention also relates to a nucleic acid sequence of at least one EPOR1 protein and a nucleic acid sequence thereof. The invention also relates to a nucleic acid sequence of at least one EPOR1 protein and a nucleic acid sequence thereof. The invention also relates to a nucleic acid sequence of at least one EPOR1 protein and a nucleic acid sequence thereof.

[0177] In some embodiments, an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein can be expressed at a concentration of less than about 600 nM, about 590 nM, about 580 nM, about 570 nM, about 560 nM, about 550 nM, about 540 nM, about 530 nM, about 520 nM, about 510 nM, about 500 nM, about 490 nM, about 480 nM, about 470 nM, about 460 nM, about 450 nM, about 440 nM, about 430 nM, about 420 nM, about 410 nM, about 400 nM, about 390 nM, about 380 nM, about 370 nM, about 360 nM, about 350 nM, about 340 nM, about 330 nM, about 320 nM , about 310nM, about 300nM, about 290nM, about 280nM, about 270nM, about 260nM, about 250nM, about 240nM, about 230nM, about 220nM, about 210nM, about 200nM, about 190nM, about 180nM, about 170nM, about 160nM, about 150nM, about 140nM M, about 130 nM, about 120 nM, about 110 nM, about 100 nM, about 90 nM, about 80 nM, about 70 nM, about 50 nM, about 50 nM, about 49 nM, about 48 nM, about 47 nM, about 46 nM, about 45 nM, about 44 nM, about 43 nM, about 42 nM, about 41 nM, about 40 nM, about 39 nM , about 38nM, about 37nM, about 36nM, about 35nM, about 34nM, about 33nM, about 32nM, about 31nM, about 30nM, about 29nM, about 28nM, about 27nM, about 26nM, about 25nM, about 24nM, about 23nM, about 22nM, about 21nM, about 20nM, about 19nM, about 18nM M, about 17 nM, about 16 nM, about 15 nM, about 14 nM, about 13 nM, about 12 nM, about 11 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 990 pM, about 980 pM, about 970 pM, about 960 pM, about 9 50pM, about 940pM, about 930pM, about 920pM, about 910pM, about 900pM, about 890pM, about 880pM, about 870pM, about 860pM, about 850pM, about 840pM, about 830pM, about 820pM, about 810pM, about 800pM, about 790pM, about 780pM, about 770 pM, about 760 pM, about 750 pM, about 740 pM, about 730 pM, about 720 pM, about 710 pM, about 700 pM, about 690 pM, about 680 pM, about 670 pM, about 660 pM, about 650 pM, about 640 pM, about 630 pM, about 620 pM, about 610 pM, about 600 pM,about 590 pM, about 580 pM, about 570 pM, about 560 pM, about 550 pM, about 540 pM, about 530 pM, about 520 pM, about 510 pM, about 500 pM, about 490 pM, about 480 pM, about 470 pM, about 460 pM, about 450 pM, about 440 pM, about 430 pM, about 420 pM, about 410 pM, about 400 pM, about 390 pM, about 380 pM, about 370 pM, about The invention relates to a novel EPOR comprising a nucleic acid sequence of at least one EPOR sequence and a nucleic acid sequence thereof. The invention relates to a nucleic acid sequence of at least one EPOR sequence and a nucleic acid sequence thereof. The invention relates to a nucleic acid sequence of at least one EPOR sequence and a nucleic acid sequence thereof. The invention relates to a nucleic acid sequence of at least one EPOR sequence and a nucleic acid sequence thereof.

[0178] In some embodiments, the EPO analogs or engineered EPO described herein may have a lower binding affinity for a heterologous EPOR than a wild-type or native EPO protein. For example, an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein may have a lower binding affinity for a heterologous EPOR than a wild-type or native EPO protein that does not comprise one or more amino acid substitutions described herein. In some embodiments, the binding affinity of an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein can be 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 70%, at 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%.

[0179] In some embodiments, the EPO analogs or engineered EPO described herein may have a higher binding affinity for a heterologous EPOR than a wild-type or native EPO protein. For example, an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein may have a higher binding affinity for a heterologous EPOR than a wild-type or native EPO protein that does not comprise one or more amino acid substitutions described herein. In some embodiments, the binding affinity of an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein can be 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 70%, at 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%.

[0180] In some embodiments, the binding affinity level of the EPO analogs or engineered EPO described herein to the heterologous EPOR can be the same as that of the wild-type or native EPO protein. For example, the binding affinity of the heterologous EPOR of an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein can be the same or similar to that of the heterologous EPOR of a wild-type or native EPO protein that does not comprise one or more amino acid substitutions described herein. In some embodiments, the binding affinity level of the EPO analogs or engineered EPO described herein to the homologous EPOR can be the same as that of the wild-type or native EPO protein. For example, the binding affinity of the homologous EPOR of an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein can be the same or similar to that of the homologous EPOR of a wild-type or native EPO protein that does not comprise one or more amino acid substitutions described herein.

[0181] In some embodiments, the EPO analogs or engineered EPO described herein may have a lower binding affinity for the homologous EPOR than the wild-type or native EPO protein. For example, the binding affinity of an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein to the homologous EPOR may be lower than the binding affinity of a wild-type or native EPO protein that does not comprise one or more amino acid substitutions described herein to the homologous EPOR. In some embodiments, the binding affinity of an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein to a homologous EPOR can be 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 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%.

[0182] In some embodiments, the EPO analogs or engineered EPO described herein may have a higher binding affinity for the homologous EPOR than the wild-type or native EPO protein. For example, the binding affinity of an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein to the homologous EPOR may be higher than the binding affinity of a wild-type or native EPO protein that does not comprise one or more amino acid substitutions described herein to the homologous EPOR. In some embodiments, the binding affinity of an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein to a homologous EPOR can be 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 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%.

[0183] In some embodiments, the EPO analogs or engineered EPO described herein can bind to the homologous EPO receptor with a binding affinity higher than the binding affinity to the heterologous EPO receptor. In some embodiments, the EPO analogs or engineered EPO described herein can bind to the homologous EPO receptor with a binding affinity lower than the binding affinity to the heterologous EPO receptor. In some embodiments, the EPO analogs or engineered EPO described herein can bind to the heterologous EPO receptor with a binding affinity higher than the binding affinity to the homologous EPO receptor. In some embodiments, the EPO analogs or engineered EPO described herein can bind to the heterologous EPO receptor with a binding affinity lower than the binding affinity to the homologous EPO receptor.

[0184] In some embodiments, the EPO analogs or engineered EPO described herein can promote the activity of homologous EPOR or increase its activity level. In some embodiments, the EPO analogs or engineered EPO described herein can have no effect on the activity level of homologous EPOR. In some embodiments, the EPO analogs or engineered EPO described herein can inhibit the activity of homologous EPOR or reduce its activity level. In some embodiments, homologous EPOR activity can include but is not limited to phosphorylation of the intracellular domain of the homologous EPOR, Janus tyrosine kinase 2 (Jak2) or signal transducer and activator of transcription 5 (Stat5). In some embodiments, cognate EPOR activity can include, but is not limited to, Jak2, a Jak2 pathway, a Stat5 pathway, a mitogen-activated protein kinase (MAPK), a MAPK pathway, an extracellular signal-regulated kinase (ERK), an ERK pathway, phosphatidylinositol 3-kinase (PI3K), a PI3K pathway, v-Akt murine thymoma viral oncogene / protein kinase-B (Akt / PKB), an Akt / PKB pathway, mammalian target of rapamycin (mTOR), or activation of the mTOR pathway.

[0185] In some embodiments, the EPO analogs or engineered EPO described herein can promote the activity of a heterologous EPOR or increase its activity level. In some embodiments, the EPO analogs or engineered EPO described herein can have no effect on the activity level of a heterologous EPOR. In some embodiments, the EPO analogs or engineered EPO described herein can inhibit the activity of a heterologous EPOR or reduce its activity level. In some embodiments, heterologous EPOR activity can include, but is not limited to, phosphorylation of the intracellular domain of the heterologous EPOR, Janus tyrosine kinase 2 (Jak2), or signal transducer and activator of transcription 5 (Stat5). In some embodiments, heterologous EPOR activity can include, but is not limited to, Jak2, a Jak2 pathway, a Stat5 pathway, a mitogen-activated protein kinase (MAPK), a MAPK pathway, an extracellular signal-regulated kinase (ERK), an ERK pathway, phosphatidylinositol 3-kinase (PI3K), a PI3K pathway, v-Akt murine thymoma viral oncogene / protein kinase-B (Akt / PKB), an Akt / PKB pathway, mammalian target of rapamycin (mTOR), or activation of the mTOR pathway.

[0186] In some embodiments, the EPO analogs or engineered EPO described herein may not affect the activation or phosphorylation levels of Jak2, Stat5, mTOR, MAPK, ERK, PI3K, Akt / PKB of the intracellular domain of the homologous EPOR or heterologous EPOR compared to wild-type or native EPO protein. For example, when an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein is introduced into a cell or cell population, the activation levels of Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB or mTOR in the cell or cell population may be the same or similar to the levels in the cell or cell population into which the wild-type or native EPO protein not comprising one or more amino acid substitutions described herein is introduced. Any method known in the art can be used to measure the activation or phosphorylation of homologous EPOR, heterologous EPOR, Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB or mTOR. Examples of methods for measuring Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB, or mTOR activation levels 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 EPO analogs or engineered EPO described herein can increase or promote Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB or mTOR activation. For example, the level of Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB or mTOR activation in cells or cell populations introduced with an EPO analog or engineered EPO comprising one or more amino acid substitutions as described herein can be higher than the level in cells or cell populations introduced with wild-type or native EPO proteins that do not comprise one or more amino acid substitutions as described herein. In some embodiments, the level of Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB, or mTOR activation in cells or cell populations introduced with an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein can be 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 8%, 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 %, 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%.

[0188] In some embodiments, the EPO analogs or engineered EPO described herein can reduce or inhibit Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB or mTOR activation. For example, the level of Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB or mTOR activation in cells or cell populations introduced with an EPO analog or engineered EPO comprising one or more amino acid substitutions as described herein can be lower than that in cells or cell populations that do not comprise wild-type or native EPO proteins comprising one or more amino acid substitutions as described herein. In some embodiments, the level of Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB, or mTOR activation in a cell or cell population introduced with an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein can be 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 8%, 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 %, 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%.

[0189] In some embodiments, the EPO analogs or engineered EPO described herein can act as agonists of homologous EPOR and selectively bind to the homologous EPOR. In some embodiments, the EPO analogs or engineered EPO that are agonists of the homologous EPOR can have a higher binding affinity for the homologous EPOR than for the heterologous EPOR. For example, an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein can be an agonist of the homologous EPOR and have a higher binding affinity for the homologous EPOR than for the heterologous EPOR. In some embodiments, an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein can be an agonist of a homologous EPOR and have a homologous 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 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%.

[0190] In some embodiments, the EPO analogs or engineered EPO described herein can act as agonists of homologous EPOR and have binding specificity or selectivity for the homologous EPOR. In some embodiments, the EPO analogs or engineered EPO as agonists of homologous EPOR can have higher binding specificity or selectivity for the homologous EPOR than for the heterologous EPOR. For example, an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein can be an agonist of the homologous EPOR and have higher binding specificity or selectivity for the homologous EPOR than for the heterologous EPOR. In some embodiments, an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein can be an agonist of a homologous EPOR and have a homologous 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%, greater than a heterologous EPOR. , 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%.

[0191] In some embodiments, the EPO analogs or engineered EPO described herein can act as antagonists of homologous EPOR and selectively bind to the homologous EPOR. In some embodiments, the EPO analogs or engineered EPO that are antagonists of homologous EPOR can have a higher binding affinity for the homologous EPOR than for the heterologous EPOR. For example, an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein can be an antagonist of the homologous EPOR, and its binding affinity for the homologous EPOR is higher than its binding affinity for the heterologous EPOR. In some embodiments, an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein can be an antagonist of a homologous EPOR and have a homologous 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 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%.

[0192] In some embodiments, the EPO analogs or engineered EPO described herein can act as antagonists of homologous EPOR and have binding specificity or selectivity for the homologous EPOR. In some embodiments, the EPO analogs or engineered EPO as antagonists of homologous EPOR can have higher binding specificity or selectivity for the homologous EPOR than for the heterologous EPOR. For example, an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein can be an antagonist of the homologous EPOR and have higher binding specificity or selectivity for the homologous EPOR than for the heterologous EPOR. In some embodiments, an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein can be an antagonist of a homologous EPOR and has a homologous 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 , 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%.

[0193] In some embodiments, the EPO analogs or engineered EPO described herein can act as agonists of heterologous EPOR and selectively bind to the heterologous EPOR. In some embodiments, the EPO analogs or engineered EPO as agonists of heterologous EPOR can have a higher binding affinity for the heterologous EPOR than for the homologous EPOR. For example, an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein can be an agonist of the heterologous EPOR, and its heterologous EPOR binding affinity is higher than the homologous EPOR binding affinity. In some embodiments, an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein can be an agonist of a heterologous EPOR and has a heterologous 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 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%.

[0194] In some embodiments, the EPO analogs or engineered EPO described herein can act as agonists of heterologous EPOR and have binding specificity or selectivity for the heterologous EPOR. In some embodiments, the EPO analogs or engineered EPO as agonists of heterologous EPOR can have higher binding specificity or selectivity for the heterologous EPOR than for the homologous EPOR. For example, an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein can be an agonist of the heterologous EPOR and have higher binding specificity or selectivity for the heterologous EPOR than for the homologous EPOR. In some embodiments, an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein can be an agonist of a heterologous EPOR and has a heterologous 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 , 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%.

[0195] In some embodiments, the EPO analogs or engineered EPO described herein can act as antagonists to heterologous EPORs and selectively bind to heterologous EPORs. In some embodiments, the EPO analogs or engineered EPO that are antagonists to heterologous EPORs can have a higher binding affinity for the heterologous EPOR than for the homologous EPOR. For example, an EPO analog or engineered EPO that comprises one or more amino acid substitutions described herein can be an antagonist to heterologous EPOR, and its heterologous EPOR binding affinity is higher than its homologous EPOR binding affinity. In some embodiments, an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein can be an antagonist of a heterologous EPOR and has a heterologous 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 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%.

[0196] In some embodiments, the EPO analogs or engineered EPO described herein can act as antagonists of heterologous EPOR and have binding specificity or selectivity for the heterologous EPOR. In some embodiments, the EPO analogs or engineered EPO as antagonists of heterologous EPOR can have higher binding specificity or selectivity for the heterologous EPOR than for the homologous EPOR. For example, an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein can be an antagonist of heterologous EPOR and have higher binding specificity or selectivity for the heterologous EPOR than for the homologous EPOR. In some embodiments, an EPO analog or engineered EPO comprising one or more amino acid substitutions described herein can be an antagonist of a heterologous EPOR and has a heterologous 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 , 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%.

[0197] In some embodiments, the half-life of an EPO analog or engineered EPO described herein in human plasma can be from 1 hour to 5 days. In some embodiments, the half-life of an EPO analog or engineered EPO described herein can be from about 1 hour to about 120 hours. In some embodiments, the half-life of an EPO analog or engineered EPO described herein can be from about 1 hour to about 5 hours, from about 1 hour to about 10 hours, from about 1 hour to about 12 hours, from about 1 hour to about 24 hours, from about 1 hour to about 36 hours, from about 1 hour to about 48 hours, from about 1 hour to about 60 hours, from about 1 hour to about 72 hours, from about 1 hour to about 84 hours, from about 1 hour to about 96 hours, from about 1 hour to about 120 hours, from about 5 hours to about 10 hours, from about 5 hours to about 12 hours, from about 5 hours to about 24 hours, from about 5 hours to about 36 hours, from about 5 hours to about 5 hours. 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 From about 96 hours to about 96 hours, from about 36 hours to about 120 hours, from about 48 hours to about 60 hours, from about 48 hours to about 72 hours, from about 48 hours to about 84 hours, from about 48 hours to about 96 hours, from about 48 hours to about 120 hours, from about 60 hours to about 72 hours, from about 60 hours to about 84 hours, from about 60 hours to about 96 hours, from about 60 hours to about 120 hours, from about 72 hours to about 84 hours, from about 72 hours to about 96 hours, from about 72 hours to about 120 hours, from about 84 hours to about 96 hours, from about 84 hours to about 120 hours, or from about 96 hours to about 120 hours.In some embodiments, the half-life of the EPO analogs or engineered EPO described herein can be 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 half-life of the EPO analogs or engineered EPO described herein can be 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, about 96 hours, or about 120 hours. In some embodiments, the half-life of the EPO analogs or engineered EPO described herein can be at most 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.

[0198] The present disclosure also encompasses engineered EPORs comprising the extracellular domain (ECD) of EPOR. The ECD of EPOR comprises two domains, D1 and D2, and these two domains are required for EPO binding. In some embodiments, the Fc fusion protein of the ECD EPOR-Fc can bind to EPO. In some embodiments, the Fc fusion protein of the ECD EPOR-Fc can block EPOR activation. In some embodiments, the Fc fusion protein of the ECD EPOR-Fc can comprise a mutation. For example, the Fc fusion protein of the ECD EPOR-Fc can comprise a mutation at amino acid residue F93. In some embodiments, the Fc fusion protein of the ECD EPOR-Fc can comprise an F93A mutation. In some embodiments, the Fc fusion protein of the ECD EPOR-Fc comprising the F93A mutation may not bind to EPO. For example, a monomeric EPOR ECD comprising the F93A mutation or a dimeric EPOR-Fc comprising the F93A mutation may not bind to EPO.

[0199] The present disclosure also encompasses engineered heterologous EPORs comprising the extracellular domain (ECD) of CD131. The ECD of CD131 comprises four domains, D1, D2, D3, and D4. The D1 and D2 domains are responsible for dimerization on the distal side of the cell membrane. Without being bound by theory, the D3 and D4 domains may be regions that interact with EPOR to form heterologous EPORs. In some embodiments, a pestle-and-mortar technique may be used to generate heterologous Fc fusion proteins having EPOR ECD and CD131 ECD. Non-limiting examples of designs of heterologous Fc fusion proteins having EPOR ECD and CD131 ECD are shown in Table 3-3, and the sequences are shown in Figures 42A-42DIn some embodiments, EPO binding may require the D3 and D4 domains of CD131. For example, monomeric or dimeric EPOR with an F93A substitution may not bind to EPO, but a heterologous EPOR comprising a monomeric EPOR with an F93A mutation and a CD131 monomer may bind to EPO. It appears that the binding of EPO to heterologous EPOR is specific to the CD131 subunit. In some embodiments, heterodimeric EPOR (F93A) / CD131-Fc can be used to specifically block heterologous EPOR without blocking homologous EPOR.

[0200] Anti-EPOR, anti-CD131, and anti-EPO antibodies

[0201] 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.

[0202] In some embodiments, the antibody can be a monospecific antibody and binds to a single epitope. For example, a monospecific antibody can have multiple immunoglobulin variable domain sequences, each of which binds to the same epitope. In some embodiments, the antibody can be a bispecific antibody. A bispecific antibody can have specificity for no more than two antigens. The characteristics of a bispecific antibody can be that it has a first immunoglobulin variable domain sequence with binding specificity for a first epitope, and a second immunoglobulin variable domain sequence with binding specificity for a second epitope. In some embodiments, the first epitope and the second epitope can be on the same antigen, such as the same protein (or subunit of a multimeric protein). In some embodiments, the first epitope and the second epitope can overlap. In some embodiments, the first epitope and the second epitope do not overlap. In some embodiments, the first epitope and the second epitope can be on different antigens, such as different proteins (or different subunits of a multimeric protein). In some embodiments, a bispecific antibody can include a heavy chain variable domain sequence and a light chain variable domain sequence with binding specificity for the first epitope, and a heavy chain variable domain sequence and a light chain variable domain sequence with binding specificity for the 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 fragment thereof having binding specificity for a first epitope and a half antibody or fragment thereof having binding specificity for a second epitope. In some embodiments, a bispecific antibody may comprise a scFv or Fab or fragment thereof having binding specificity for a first epitope and a scFv or Fab or fragment thereof having binding specificity for a second epitope.

[0203] In some embodiments, the antibody can be a multispecific or multifunctional antibody. For example, a multispecific or multifunctional antibody can include multiple immunoglobulin variable domain sequences, wherein the first immunoglobulin variable domain sequence in the multiple sequences has binding specificity for the first epitope, and the second immunoglobulin variable domain sequence in the multiple sequences has binding specificity for the second epitope. In some embodiments, the first epitope and the second epitope are on the same antigen, such as the same protein (or subunit of a multimeric protein). In some embodiments, the first epitope and the second epitope may overlap. In some embodiments, the first epitope and the second epitope may not overlap. In some embodiments, the first epitope and the second epitope may be on different antigens, such as different proteins (or different subunits of a multimeric protein). In some embodiments, the multispecific antibody may include 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 include 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 can be separated, purified, recombinant, synthesized or any combination thereof. Bispecific or multispecific antibodies described herein can be prepared via any suitable method, and can be recombinant, synthesized or a combination thereof. In one aspect, a liquid composition or a lyophilized composition can be provided herein, comprising one or more bispecific or multispecific antibodies as described herein. In one embodiment, the composition can comprise a colony of bispecific or multispecific antibodies. In another embodiment, the composition can comprise a colony of two, three, four, five, six, seven, eight, nine, ten or more bispecific or multispecific antibodies as described above. Bispecific or multispecific antibodies as described herein can be used for in vitro assays, for example, identifying and / or purifying one or more tumor cells from mixed cultures (e.g., biological samples, such as biopsies or blood samples). Bispecific or multispecific antibodies as described herein can be used in in vivo animal models to test the therapeutic efficacy of bispecific or multispecific antibodies for tumors.

[0204] In some embodiments, antibodies can 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 can 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 can include a heavy chain and a light chain (referred to herein as a half antibody). In another example, an antibody can include two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequences, thereby forming two antigen-binding sites, such as Fab, Fab', F(ab')2, Fc, Fd, Fd', Fv, single-chain antibodies (e.g., scFv), single variable domain antibodies, diabodies (Dab) (divalent and bispecific), and chimeric (e.g., humanized) antibodies, which can be produced by modifying intact antibodies or antibodies synthesized de novo using recombinant DNA technology. These functional antibody fragments can retain the ability to selectively bind to their corresponding antigens. Antibodies and antibody fragments can be from any class of antibodies, including but not limited to IgG, IgA, IgM, IgD and IgE, and antibodies of any subclass (e.g., IgG1, IgG2, IgG3 and IgG4). Antibody preparations can be monoclonal or polyclonal. Antibodies can also be human antibodies, humanized antibodies, CDR-grafted antibodies or antibodies generated in vitro. Antibodies can have a heavy chain constant region selected from, for example, IgG1, IgG2, IgG3 or IgG4. Antibodies can also have a light chain selected from, for example, κ or λ. The term "immunoglobulin" (Ig) is used interchangeably with the term "antibody" in this article.

[0205] Non-limiting examples of antigen-binding fragments of antibodies may include: Fab fragments (monovalent fragments consisting of VL, VH, CL and CH1 domains); F(ab')2 fragments (bivalent fragments comprising two Fab fragments connected by a disulfide bond in the hinge region); Fd fragments, which consist of VH and CH1 domains; Fv fragments, which consist of the VL and VH domains of a single arm of an antibody; diabody (dAb) fragments, which consist of a VH domain; Camelid or camelized variable domains; single-chain Fv (scFv) (see, e.g., 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 can be obtained using conventional techniques known to those skilled in the art, and the fragments can be screened for utility in the same manner as intact antibodies. For example, single-chain antibodies (scFV) can be engineered (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 generate multivalent antibodies that are specific for different epitopes of the same target protein. In some embodiments, antibodies can include complete molecules and functional fragments thereof. The constant region of an antibody can be changed or mutated to modify one or more properties of the antibody (e.g., to increase or decrease one or more of the following: Fc receptor binding, antibody glycosylation, the number of cysteine ​​residues, effector cell function, or complement function). Methods for changing the constant region of an antibody are known in the art. In some embodiments, antibodies with altered function (e.g., altered affinity for effector ligands such as FcRs on cells 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 388,151A1, U.S. Pat. No. 5,624,821, and U.S. Pat. No. 5,648,260, the entire contents of which are incorporated by reference).

[0206] In some embodiments, the antibodies, antigen-binding fragments thereof, or functional fragments thereof described herein may include non-antibody scaffolds. Non-limiting examples of non-antibody scaffolds include affibodies, affilins, anticalins, atrimers, avimers, bicyclic peptides, Cys-knots, DARPins, FN3 scaffolds (e.g., adnectins, centyrins, pronectins, Tn3), fynomers, Kunitz domains, or OBodies.

[0207] In some embodiments, the antibodies described herein, their antigen-binding fragments, or their functional fragments can be derivatized or connected to another functional molecule (e.g., another peptide or protein). As used herein, a "derivatized" antibody is a modified antibody. Derivatization methods can include, but are not limited to, adding fluorescent moieties, radionucleotides, toxins, enzymes, or affinity ligands (such as biotin). For example, antibodies can be functionally connected to one or more other molecular entities, such as another antibody (e.g., bispecific antibody or double antibody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and / or a protein or peptide that can mediate the association of an antibody or antibody portion with another molecule (such as a streptavidin core region or a polyhistidine tag), for example, by chemical coupling, gene fusion, non-covalent association, or other methods. One type of derivatized antibody can be produced by cross-linking two or more antibodies (of the same type or different types, for example, to produce a bispecific antibody). Suitable cross-linkers may include heterobifunctional cross-linkers having two different reactive groups separated by an appropriate spacer (e.g., m-maleimidobenzoyl-N-hydroxysuccinimide ester) or homobifunctional cross-linkers (e.g., disuccinimidyl suberate). Such linkers are available from Pierce Chemical Company, Rockford, IL, USA.

[0208] In some embodiments, the antibody may also be a single domain antibody. A single domain antibody may include an antibody whose complementary determining region is part of a single domain polypeptide. Non-limiting examples may include heavy chain antibodies, antibodies that are naturally free of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies, and single domain scaffolds other than those derived from antibodies. A single domain antibody may be any single domain antibody in the prior art, or any future single domain antibody. Single domain antibodies may be derived from any species, including but not limited to mice, humans, camels, llamas, fish, sharks, goats, rabbits, and cattle. In some embodiments, a single domain antibody may be a naturally occurring single domain antibody, referred to as a heavy chain antibody that is free of light chains. Such single domain antibodies are disclosed, for example, in WO 94 / 04678. In some embodiments, the variable domains derived from heavy chain antibodies that are naturally free of light chains are referred to herein as VHH or nanobodies. Such VHH molecules may be derived from antibodies produced in camelids (e.g., camels, llamas, dromedaries, alpacas, and guanacos). Species other than camelids can also produce heavy chain antibodies naturally devoid of light chains.

[0209] The VH and VL regions can be subdivided into regions of high variability, termed "complementarity determining regions" (CDRs), interspersed with more conserved regions, termed "framework regions" (FRs or FWs). The extent of the framework regions and CDRs has been precisely defined by many 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., Antibody Engineering Lab Manual (Ed.: Duebel, S. and Kontermann, R., Springer-Verlag, Heidelberg). In some embodiments, CDR can include the amino acid sequence that gives antigen specificity and binding affinity in the antibody variable region. In some embodiments, the antibody can 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, any one of many known schemes can be used to determine the boundaries of the amino acid sequence of a given CDR, including Kabat et al., (1991), "Sequences of Proteins of Immunological Interest," 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD ("Kabat" numbering scheme); Al-Lazikani et al., (1997) JMB 273, those described in 927-948 ("Chothia" numbering scheme).

[0210] The antibodies described herein can be produced recombinantly, for example, using phage display or by using combinatorial approaches. Phage display and combinatorial methods for generating antibodies are known in the art (e.g., Ladner et al., U.S. Patent No. 5,223,409; Kang et al., International Publication No. WO 92 / 18619; Dower et al., International Publication No. WO 91 / 17271; Winter et al., International Publication No. WO 92 / 20791; Markland et al., International Publication No. WO 92 / 15679; Breitling et al., International Publication No. WO 93 / 01288; McCafferty et al., International Publication No. WO 92 / 01047; Garrard et al., International Publication No. WO 92 / 09690; Ladner et al., International Publication No. WO 90 / 02809; 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 entire contents of which are incorporated herein by reference).

[0211] In some embodiments, the antibodies described herein can 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 antibody can be a rodent antibody (mouse or rat antibody). Methods for producing rodent antibodies are known in the art.

[0212] In some embodiments, the antibodies described herein, their antigen-binding fragments or their functional fragments can be humanized antibodies or humanized antigen-binding fragments. As used herein, "humanized" antibodies refer to non-human (e.g., mouse) antibody forms that are specific chimeric immunoglobulins, immunoglobulin chains or fragments thereof containing minimal sequences derived from non-human immunoglobulins. In some embodiments, humanized antibodies can be human immunoglobulins (recipient antibodies) in which the residues of the complementary determining regions (CDRs) of the recipient are replaced by residues of CDRs of non-human species (donor antibodies) (such as mice, rats or rabbits) with the desired specificity, affinity and biological activity. In some embodiments, humanized antibodies can have at least one or two, but usually all three recipient CDRs (heavy and / or light immunoglobulin chains) replaced by donor CDRs. In some embodiments, antibodies can be replaced by at least a portion of non-human CDRs, or only some CDRs can be replaced by non-human CDRs. In some embodiments, the minimum number of CDRs required for antigen binding can be replaced. In some embodiments, the Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. In addition, humanized antibodies can be included in the residue that is not found in the CDR or framework sequence of receptor antibodies or imports, but are included to further improve or optimize antibody performance.In general, humanized antibodies can include at least one and generally two variable domains, wherein all or substantially all CDR districts correspond to those of non-human immunoglobulins, and all or substantially all FR districts are those of human immunoglobulin consensus sequences.Humanized antibodies can also best include at least a portion of immunoglobulin constant region or domain (Fc), generally at least a portion of human immunoglobulin.Antibody can have the Fc district modified as described in, for example, WO 99 / 58572.Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five or six) changed relative to the original antibody, also referred to as one or more CDRs "derived from" from the one or more CDRs of the original antibody.Humanized antibodies can, for example, be produced by modeling antibody variable domains and using genetic engineering techniques (such as CDR transplants or CDR replacements) to produce antibodies, wherein one, two or all CDRs of immunoglobulin chains can be substituted.Various techniques for producing humanized antibodies are described in, for example, 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 For example, human monoclonal antibodies can be generated using transgenic mice carrying human immunoglobulin genes rather than mouse systems. Splenocytes of these transgenic mice immunized with an antigen of interest can be used to generate hybridomas that secrete human mAbs with specific affinity for epitopes 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, immunocompetent transgenic mice can be used. In some embodiments, the immunocompetent transgenic mice can contain human antibody heavy chains, human antibody light chains, or a combination thereof. In some embodiments, the immunocompetent transgenic mice can contain human antibody heavy chains, human antibody lambda light chains, human antibody kappa light chains, or a combination thereof. In some embodiments, one or more specific amino acids can be substituted, deleted, or added to the humanized antibody.Criteria for selecting amino acids from donors 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 hereby incorporated by reference. Other techniques for humanizing antibodies are described in Padlan et al., EP 519596A1, published on December 23, 1992.

[0213] In some embodiments, the antibodies described herein, their antigen-binding fragments, or their functional fragments may comprise a CDR-grafted scaffold domain. In some embodiments, the scaffold domain may be based on a fibronectin domain, such as a fibronectin type III domain. In some embodiments, the overall folding of the fibronectin type III (Fn3) domain may be closely related to the overall folding of the minimum functional antibody fragment (the variable domain of the antibody heavy chain). The Fn3 end has three loops; the positions of the BC, DE, and FG loops correspond roughly to the positions of the CDR1, 2, and 3 of the antibody VH domain. In some embodiments, Fn3 may not have a disulfide bond; and therefore, unlike antibodies and their fragments, Fn3 may be stable under reducing conditions (see, for example, WO 98 / 56915; WO 01 / 64942; WO 00 / 34784). The Fn3 domain may be modified (e.g., using the CDRs or hypervariable loops described herein) or mutated, for example, to select a domain that binds to an antigen / marker / cell as described herein. In some embodiments, the scaffold domain (e.g., folded domain) can be based on an antibody, such as a "minibody" scaffold produced by deleting three β chains from the heavy chain variable domain of 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, a minibody 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, which is a six-stranded β-folded sandwich of 74 residues 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 mutated, for example, to select a domain that binds to a marker / antigen / cell as described herein. Another exemplary scaffold domain is a beta sandwich structure derived from the extracellular domain of CTLA-4 (see, e.g., WO 00 / 60070).Other exemplary scaffold domains may 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; trefoil structures, zinc finger domains, DNA binding proteins, particularly monomeric DNA binding proteins, RNA binding proteins, enzymes, such as proteases (particularly inactivated proteases), RNA enzymes, molecular chaperones, such as thioredoxin and heat shock proteins; and intracellular signaling domains (such as SH2 and SH3 domains). See, for example, US20040009530 and US 7,501,121, which are incorporated herein by reference. In some embodiments, scaffold domains may be evaluated and selected by, for example, 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, salinity, organic solvents, and oxidant concentrations. In some embodiments, the scaffold domain can be a small, stable protein domain, such as a protein of fewer than 100, 70, 50, 40, or 30 amino acids. The domain can contain one or more disulfide bonds, or can chelate a metal, such as zinc.

[0214] In some embodiments, antibodies as described herein, their antigen-binding fragments or their functional fragments may be included in a variable region or portion thereof, such as a CDR, generated in a non-human organism (e.g., a rat or mouse). In some embodiments, antibodies as described herein, their antigen-binding fragments or their functional fragments may be chimeric antibodies, CDR-grafted antibodies or humanized antibodies. In some embodiments, antibodies as described herein, their antigen-binding fragments or their functional fragments may be generated in a non-human organism and modified. For example, antibodies, their antigen-binding fragments or their functional fragments generated in a non-human organism (e.g., a rat or mouse) may be modified in a variable framework or constant region to reduce antigenicity and / or immunogenicity in the human body. In some embodiments, chimeric antibodies can be produced by recombinant DNA techniques known in the art (see Robinson et al., International Patent Publication PCT / US86 / 02269; Akira et al., European Patent Publication 184,187; Taniguchi, M., European Patent Publication 171,496; Morrison et al., European Patent Publication 173,494; Neuberger et al., International Application WO 86 / 01533; Cabilly et al., U.S. Patent No. 4,816,567; Cabilly et al., European Patent Publication 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).

[0215] In some embodiments, the antibodies described herein, their antigen-binding fragments or their functional fragments can selectively bind to the epitope on the antigen of the target protein or the antigen of the target protein. In some embodiments, the target can include erythropoietin (EPO) protein, the EPO receptor subunit of homologous EPOR or heterologous EPOR, the CD131 subunit of heterologous EPOR or a combination thereof. In some embodiments, the target can include heterologous EPOR. For example, the antibodies described herein, their antigen-binding fragments or their functional fragments can selectively bind to heterologous EPOR comprising EPO receptor subunits and CD131 subunits. In this embodiment, the antibodies described herein, their antigen-binding fragments or their functional fragments can be combined with the EPO receptor subunits and CD131 subunits of heterologous EPOR. In some embodiments, the antibodies described herein, their antigen-binding fragments or their functional fragments can be non-naturally present. In some embodiments, the antibodies described herein, their antigen-binding fragments or their functional fragments can be separated and / or purified. In some embodiments, the antibodies described herein, their antigen-binding fragments or their functional fragments can be used for in vitro assays (e.g., binding assays, functional assays, etc.).

[0216] In some embodiments, the antibodies described herein or their functional fragments can bind to the target and can act as antagonists. In one example, an anti-EPO antibody can bind to an EPO protein and can prevent the formation of a complex between the EPO protein and the homologous EPOR. In another example, an anti-EPO antibody can bind to an EPO protein and can prevent the formation of a complex between the EPO protein and the heterologous EPOR. In another example, an anti-EPOR antibody can bind to an EPO receptor subunit and can prevent the formation of a complex of the homologous EPOR, the formation of a complex of the heterologous EPOR, the formation of a complex between the EPO protein and the homologous EPOR, or the formation of a complex between the EPO protein and the heterologous EPOR. In another example, an anti-CDC131 antibody can bind to the CDC131 subunit of the heterologous EPOR and can prevent the formation of a complex of the heterologous EPOR or the formation of a complex between the EPO protein and the heterologous EPOR. In some embodiments, preventing the formation of a complex of the homologous EPOR or the formation of a complex between the EPO protein and the homologous EPOR can prevent the activation or function of the homologous EPOR. In some embodiments, preventing the formation of a complex of a heterologous EPOR or a complex between an EPO protein and a heterologous EPOR can prevent the activation or function of the heterologous EPOR. In some embodiments, an anti-EPO antibody can bind to an EPO protein and inhibit or reduce the activity level of a homologous EPOR or a heterologous EPOR without affecting the binding of the EPO protein to the homologous EPOR or the heterologous EPOR. In some embodiments, an anti-EPOR antibody can bind to the EPO receptor subunit of a homologous EPOR or a heterologous EPOR and inhibit or reduce the activity level of a homologous EPOR or a heterologous EPOR without affecting the formation of a complex of the homologous EPOR or the heterologous EPOR or the formation of a complex between an EPO protein and a homologous EPOR or an EPO protein and a heterologous EPOR. In some embodiments, an anti-CD131 antibody can bind to the CD131 subunit of a heterologous EPOR and inhibit or reduce the activity level of a heterologous EPOR without affecting the formation of a complex of the heterologous EPOR or the binding of the EPO protein to the heterologous EPOR.

[0217] In some embodiments, the antibodies described herein or their functional fragments can bind to a target and can act as an agonist. In one example, an anti-EPOR antibody can bind to the EPO receptor subunit of a homologous EPOR in a manner that simulates the binding of EPO to a homologous EPOR. In another example, an anti-EPOR antibody can bind to the EPO receptor subunit of a heterologous EPOR in a manner that simulates the binding of EPO to a heterologous EPOR. In another example, an anti-CD131 antibody can bind to the CD131 subunit of a heterologous EPOR in a manner that simulates the binding of EPO to a heterologous EPOR. In some embodiments, simulating the binding of EPO to a homologous EPOR can result in activation of the homologous EPOR. In some embodiments, simulating the binding of EPO to a heterologous EPOR can result in activation of the heterologous EPOR. In some embodiments, anti-EPO antibodies can promote or increase the activity of a homologous EPOR or a heterologous EPOR without affecting the binding affinity of the EPO protein to the homologous EPOR or the heterologous EPOR. In some embodiments, anti-EPOR antibodies can promote or increase the activity of homologous EPOR or heterologous EPOR without affecting the binding affinity of the EPO protein to the homologous EPOR or heterologous EPOR or the binding affinity of the homologous EPOR (e.g., between the two EPO receptor subunits of the homologous EPOR) or the heterologous EPOR (e.g., between the EPO receptor subunit of the heterologous EPOR and the CD131 subunit). In some embodiments, anti-CD131 antibodies can promote or increase the activity of heterologous EPOR without affecting the binding affinity of the EPO protein to the heterologous EPOR or the binding affinity of the heterologous EPOR (e.g., between the EPO receptor subunit of the heterologous EPOR and the CD131 subunit).

[0218] In some embodiments, homologous EPOR activity or heterologous EPOR activity may include, but is not limited to, phosphorylation of the intracellular domain of the homologous EPOR, the intracellular domain of the heterologous EPOR, Janus tyrosine kinase 2 (Jak2), or signal transducer and activator of transcription 5 (Stat5). In some embodiments, homologous EPOR activity or heterologous 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 homologous EPOR, heterologous EPOR, Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB, or mTOR. In some embodiments, the antibodies or functional fragments thereof described herein can inhibit 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 antibodies or functional fragments thereof described herein can promote the activation or phosphorylation of homologous EPOR, heterologous EPOR, Jak2, Stat5 or mTOR. In some embodiments, the antibodies or functional fragments thereof described herein can promote 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 antibodies or functional fragments thereof described herein may not affect the activation or phosphorylation of homologous EPOR, heterologous EPOR, Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB or mTOR. In some embodiments, the antibodies or functional fragments 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 homologous EPOR, heterologous 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 Jak2, Stat5, MAPK, ERK, PI3K, Akt / PKB, or mTOR activation levels include, but are not limited to, Western blot, flow cytometry assay, cell proliferation assay, apoptosis assay, or enzyme-linked immunosorbent assay (ELISA).

[0219] In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof described herein can bind to a target and can act as an agonist of a heterologous EPOR. In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof described herein can be agonists of a heterologous EPOR and can selectively bind to a heterologous EPOR. In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof can be agonists of a heterologous EPOR, and their binding affinity for a heterologous EPOR can be higher than their binding affinity for a homologous EPOR. In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof described herein can be agonists of heterologous EPOR, and their heterologous EPOR binding affinity can be 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 , 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%.

[0220] In some embodiments, the anti-EPO antibody, anti-EPOR antibody, or functional fragment thereof can be an agonist of a heterologous EPOR and can have binding specificity or selectivity for the heterologous EPOR. In some embodiments, the anti-EPO antibody, anti-EPOR antibody, or functional fragment thereof can be an agonist of a heterologous EPOR, and its specificity or selectivity for the heterologous EPOR can be higher than its specificity or selectivity for the homologous EPOR. For example, the anti-EPO antibody, anti-EPOR antibody, or functional fragment thereof can be an agonist of a heterologous EPOR, and its binding specificity or selectivity for the heterologous EPOR can be higher than its binding specificity or selectivity for the homologous EPOR. In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof described herein can be agonists of heterologous EPOR, and their heterologous EPOR binding specificity or selectivity 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 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%.

[0221] In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof described herein can bind to a target and can act as an antagonist of a heterologous EPOR. In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof described herein can be antagonists of a heterologous EPOR and can selectively bind to a heterologous EPOR. In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof can be antagonists of a heterologous EPOR, and their binding affinity for a heterologous EPOR can be higher than their binding affinity for a homologous EPOR. In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof described herein can be antagonists of heterologous EPOR, and their heterologous EPOR binding affinity can be 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 , 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%.

[0222] In some embodiments, the anti-EPO antibody, anti-EPOR antibody, or functional fragment thereof can be an antagonist of a heterologous EPOR and can have binding specificity or selectivity for the heterologous EPOR. In some embodiments, the anti-EPO antibody, anti-EPOR antibody, or functional fragment thereof can be an antagonist of a heterologous EPOR, and its specificity or selectivity for the heterologous EPOR can be higher than its specificity or selectivity for the homologous EPOR. For example, the anti-EPO antibody, anti-EPOR antibody, or functional fragment thereof can be an antagonist of a heterologous EPOR, and its binding specificity or selectivity for the heterologous EPOR can be higher than its binding specificity or selectivity for the homologous EPOR. In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof described herein can be antagonists of heterologous EPOR, and their heterologous EPOR binding specificity or selectivity 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 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%.

[0223] In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof described herein can bind to a target and can act as an agonist of a homologous EPOR. In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof described herein can be agonists of a homologous EPOR and can selectively bind to a homologous EPOR. In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof can be agonists of a homologous EPOR, and their binding affinity for the homologous EPOR can be higher than their binding affinity for a heterologous EPOR. In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof described herein can be agonists of homologous EPOR, and their homologous EPOR binding affinity can be 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 , 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%.

[0224] In some embodiments, the anti-EPO antibody, anti-EPOR antibody, or functional fragment thereof may be an agonist of a homologous EPOR and may have binding specificity or selectivity for the homologous EPOR. In some embodiments, the anti-EPO antibody, anti-EPOR antibody, or functional fragment thereof may be an agonist of a homologous EPOR, and its specificity or selectivity for the homologous EPOR may be higher than its specificity or selectivity for the heterologous EPOR. For example, the anti-EPO antibody, anti-EPOR antibody, or functional fragment thereof may be an agonist of a homologous EPOR, and its binding specificity or selectivity for the homologous EPOR may be higher than its binding specificity or selectivity for the heterologous EPOR. In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof described herein can be agonists of homologous EPOR and have a homologous 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 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%.

[0225] In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof described herein can bind to a target and can act as an antagonist of a homologous EPOR. In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof described herein can be antagonists of a homologous EPOR and can selectively bind to a homologous EPOR. In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof can be antagonists of a homologous EPOR, and their binding affinity for the homologous EPOR can be higher than their binding affinity for the heterologous EPOR. In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof described herein can be antagonists of homologous EPOR, and their homologous EPOR binding affinity can be 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 , 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%.

[0226] In some embodiments, the anti-EPO antibody, anti-EPOR antibody, or functional fragment thereof may be an antagonist of a homologous EPOR and may have binding specificity or selectivity for the homologous EPOR. In some embodiments, the anti-EPO antibody, anti-EPOR antibody, or functional fragment thereof may be an antagonist of a homologous EPOR, and its specificity or selectivity for the homologous EPOR may be higher than its specificity or selectivity for the heterologous EPOR. For example, the anti-EPO antibody, anti-EPOR antibody, or functional fragment thereof may be an antagonist of the homologous EPOR, and its binding specificity or selectivity for the homologous EPOR may be higher than its binding specificity or selectivity for the heterologous EPOR. In some embodiments, the anti-EPO antibodies, anti-EPOR antibodies, or functional fragments thereof described herein can be antagonists of homologous EPOR, and their homologous EPOR binding specificity or selectivity 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 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%.

[0227] In some aspects, the antibodies described herein are specific for EPO, heterologous EPOR or homologous EPOR, and include all forms described above. Antibodies can be engineered for use in specific organisms. The organism can be a human, a canine, or commercially valuable livestock, such as a pig, horse, dog, cat, chicken, or other bird. The engineering of such antibodies can include, for example, CDR splicing, humanization, humaneering, chimerization, or isolation of human (or other organism) antibodies using any library technology or monoclonal technology known in the art.

[0228] Some examples of antibodies with alternative scaffolds may include, but are not limited to, nanobodies, affibodies, minibodies, evibodies, and domain antibodies. Some examples of alternative scaffolds that can be used to generate antibodies may 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 domains; C-type lectin-like domains; lipocalin family proteins; ankyrin repeat proteins; the Z domain of protein A; γ-crystallin; Tendamistat; neocarzinostatin; CBM4-2; T cell receptor; Im9; designer AR proteins; designer T PR 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 β-lactamase; GFP; thioredoxin; staphylococcal 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 562 ; Transferrin; 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.

[0229] Bispecific or bifunctional antibodies can comprise two different heavy chain / light chain pairs and two different binding sites. Bispecific antibodies can be produced by a variety of methods, including but not limited to hybridoma fusion or Fab' fragment ligation. See, for example, Songsivilai & Lachmann Clin. Exp. Immunol 79:315-321 (1990), Kostelny et al., J. Immunol. 148:1547-1553 (1992), which are incorporated by reference in their entirety for all purposes.

[0230] Bispecific antibody molecules can be classified into five different structural groups: (i) bispecific immunoglobulin G (BsIgG); (ii) IgG with additional antigen-binding moieties attached; (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, knob-in-hole common LC, knob-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 ertuinomab (Neovii Biotech, Fresenius Biotech), which targets CD3 and HER2. In some embodiments, BsIgG comprises a heavy chain engineered for heterodimerization. For example, a "knob-in-hole" strategy, a SEED platform, a common heavy chain (e.g., in a κλ-body) and a heterodimer Fc region can be used to engineer the heavy chain to achieve heterodimerization. See Spiess et al., Mol. Immunol. 67 (2015): 95-106. Strategies that have been used to avoid heavy chain pairing of homodimers in BsIgG include knob-in-hole, duobody, azymetric, charge pair, HA-TF, SEEDbody, and differential protein A affinity. See ibid. BsIgG can be produced by expressing component antibodies separately in different host cells and then being purified / assembled into BsIgG. BsIgG can also be produced by expressing component antibodies in a single host cell. BsIgG can be purified using affinity chromatography, for example, using protein A and sequential pH elution. The IgG of additional antigen-binding portion thereof is another form of bispecific antibody molecule. For example, monospecific IgG can be engineered to have bispecificity by attaching additional antigen-binding unit to monospecific IgG (for example, at the N-terminus or C-terminus of heavy chain or light chain). Exemplary additional antigen-binding unit includes single domain antibodies (for example, variable heavy chain or variable light chain), engineered protein scaffolds and paired antibody variable domains (for example, single chain variable fragment or variable fragment). Referring to the same.Examples of additional 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 IgG-scFv is MM-141 (Merrimack Pharmaceuticals), which binds to IGF-1R and HER3. Examples of DVD-Ig include ABT-981 (AbbVie), which binds to IL-1α and IL-1β; and ABT-122 (AbbVie), which binds to TNF and IL-17A.

[0231] Bispecific antibody fragment (BsAb) is for lacking some or all of the bispecific antibody molecular forms in antibody constant domain.For example, some BsAb lack Fc district.In some embodiments, bispecific antibody fragment comprises heavy chain and the light chain district connected by peptide linker, thus allows the effective expression of BsAb in single host cell.Exemplary bispecific antibody fragment includes but is not limited to nanobody, nanobody-HAS, BiTE, diabody, DART, TandAb, sc diabody, sc diabody-CH3, diabody-CH3, three bodies (triple body), miniantibody (miniantibody), miniantibody, TriBi miniantibody, scFv-CH3 KIH, Fab-scFv, scFv-CH-CL-scFv, F (ab ') 2, F (ab ') 2-scFv2, scFv-KIH, Fab-scFv-Fc, tetravalent HCAb, sc diabody-Fc, diabody-Fc, series connection scFv-Fc and inner antibody. For example, the BiTE format includes a series of scFvs, wherein the component scFv binds to the surface antigens on CD3 and cancer cells on T cells. Bispecific fusion proteins include antibody fragments that are connected to other proteins, for example, to increase additional specificity and / or functionality. An example of a bispecific fusion protein is immTAC, which includes an anti-CD3 scFv connected to an affinity-matured T cell receptor that recognizes an HLA-presenting peptide. In some embodiments, docking-and-lock (DNL) methods can be used to generate bispecific antibody molecules with higher valence. In addition, fusion with albumin-binding protein or human serum albumin can extend the serum half-life of antibody fragments. In some embodiments, chemical binding (for example, chemical conjugation of antibodies and / or antibody fragments) can be used to produce BsAb molecules. Exemplary bispecific antibody conjugates include CovX-body formats, in which low molecular weight drugs are conjugated to a single reactive lysine in each Fab arm or antibody or its fragment in a site-specific manner. In some embodiments, conjugation improves the serum half-life of low molecular weight drugs. An exemplary CovX-body is CVX-241 (NCT01004822), which comprises an antibody conjugated to two short peptides that inhibit VEGF or Ang2. In some cases, the bispecific antibody may further comprise a linker. In some cases, 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 a knob and hole. In some cases, 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 may 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 connecting peptide can be (GGGGS)3 or (GGGGS)4. The linkers described herein can be used in multispecific antibodies. In this embodiment, the multispecific antibody can have more than one linker. In this embodiment, the linkers can be the same. Alternatively, the linkers can be different.

[0232] Antibody molecules can be produced by (e.g., at least one or more components) recombinant expression in a host system. Exemplary host systems include eukaryotic cells (e.g., mammalian cells, e.g., Chinese hamster ovary celI, CHO cells, or insect cells, e.g., SF9 or S2 cells) and prokaryotic cells (e.g., Escherichia coli). Bispecific antibody molecules can be produced by expressing components separately in different host cells and then purifying / assembling. Alternatively, antibody molecules can be produced by expressing components in a single host cell. Purification of bispecific antibody molecules can be carried out by various methods, such as affinity chromatography, for example, using protein A and sequential pH elution. In other embodiments, affinity tags can be used for purification, such as histidine-containing tags, myc tags or streptavidin tags.

[0233] In one aspect, the antibody can be part of a conjugate molecule comprising all or part of the antibody and a prodrug. The term "prodrug" refers to a precursor or derivative form of a pharmaceutically active substance. Compared to the parent drug, the prodrug may be less cytotoxic to the cell and can be enzymatically activated or converted into 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, β-lactam-containing prodrugs, optionally substituted phenoxyacetamide-containing prodrugs, and optionally substituted phenylacetamide-containing prodrugs, 5-fluorocytosine and other 5-fluorouridine prodrugs that can be converted into more active non-cytotoxic drugs. Examples of cytotoxic drugs that can be derivatized into prodrug forms include, but are not limited to, those cytotoxic agents described above. See, for example, U.S. Patent No. 6,702,705.

[0234] In some aspects, anti-EPOR, anti-CD131 or anti-EPO antibodies can include an antigen binding domain or antigen binding fragment. In some embodiments, the antigen binding domain or antigen binding fragment can include 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) can 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 can 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.

[0235] In some embodiments, the light chain variable region (VL) can 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 can comprise any one of the VL sequences listed in Table 5. In some embodiments, the VL can 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 can comprise any one of the VL sequences listed in Table 7. In some embodiments, the VL can 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 can comprise any one of the VL sequences listed in Table 9.

[0236] In some embodiments, VH may comprise a VH complementarity determining region 1 (VH-CDR1), a VH-CDR2, or a VH-CDR3. In some embodiments, VH may comprise a VH complementarity determining region 1 (VH-CDR1), a VH-CDR2, and a VH-CDR3.

[0237] In some embodiments, the VH-CDRl can 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-CDRl sequences listed in Table 14. 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 SEQ ID NOs: 2068-2255. In some embodiments, the VH-CDR1 may comprise the sequence of any one of SEQ ID NOs: 2068-2255. In some embodiments, an anti-EPOR antibody that binds to an EPO receptor subunit can comprise a VH-CDR1 sequence that is 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% identical to any one of the VH-CDR1 sequences listed in Table 14.

[0238] In some embodiments, the VH-CDRl can 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-CDRl sequences listed in Table 15. 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 SEQ ID NOs: 2820-2948. In some embodiments, the VH-CDR1 may comprise the 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 that is 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% identical to any one of the VH-CDR1 sequences listed in Table 15.

[0239] In some embodiments, the VH-CDRl can 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-CDRl sequences listed in Table 16. 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 SEQ ID NOs: 3336-3471. In some embodiments, the VH-CDR1 may comprise the sequence of any one of SEQ ID NOs: 3336-3471. In some embodiments, an anti-EPOR antibody that binds to the EPO receptor subunit and the CD131 subunit of a heterologous EPOR can comprise a VH-CDR1 sequence that is 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% identical to any one of the VH-CDR1 sequences listed in Table 16.

[0240] In some embodiments, the VH-CDR2 can 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, 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 SEQ ID NOs: 2256-2443. In some embodiments, the VH-CDR2 may comprise the 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 that is 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% identical to any one of the VH-CDR2 sequences listed in Table 14.

[0241] In some embodiments, the VH-CDR2 can 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, 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 SEQ ID NOs: 2949-3077. In some embodiments, the VH-CDR2 may comprise the 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 that is 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% identical to any one of the VH-CDR2 sequences listed in Table 15.

[0242] In some embodiments, the VH-CDR2 can 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, 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 SEQ ID NOs: 3472-3607. In some embodiments, the VH-CDR2 may comprise the sequence of any one of SEQ ID NOs: 3472-3607. In some embodiments, an anti-EPOR antibody that binds to the EPO receptor subunit and the CD131 subunit of a heterologous EPOR can comprise a VH-CDR2 sequence that is 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% identical to any one of the VH-CDR2 sequences listed in Table 16.

[0243] In some embodiments, the VH-CDR3 can 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 the 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 that is 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% identical to any one of the VH-CDR3 sequences listed in Table 4.

[0244] In some embodiments, the VH-CDR3 can 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 the 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 heterologous 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.

[0245] In some embodiments, the VH-CDR3 can 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 the sequence of any one of SEQ ID NOs: 1331-1466. In some embodiments, an anti-EPOR antibody that binds to the EPO receptor subunit and the CD131 subunit of a heterologous EPOR can comprise a VH-CDR3 sequence that is 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% identical to any one of the VH-CDR3 sequences listed in Table 8.

[0246] In some embodiments, the VL may comprise a VL complementarity determining region 1 (VL-CDR1), a VL-CDR2, or a VL-CDR3. In some embodiments, the VL may comprise a VL complementarity determining region 1 (VL-CDR1), a VL-CDR2, and a VL-CDR3.

[0247] In some embodiments, the VL-CDR1 can 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, the VL-CDR1 can 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, the VL-CDR1 can comprise the sequence of any one of SEQ ID NOs: 2444-2631. In some embodiments, an anti-EPOR antibody that binds to an EPO receptor subunit can comprise a VL-CDR1 sequence that is 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% identical to any one of the VL-CDR1 sequences listed in Table 14.

[0248] In some embodiments, the VL-CDR1 can 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, the VL-CDR1 can 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, the VL-CDR1 can comprise the 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 that is 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% identical to any one of the VL-CDR1 sequences listed in Table 15.

[0249] In some embodiments, the VL-CDRl can 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-CDRl sequences listed in Table 16. In some embodiments, the VL-CDR1 can 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, the VL-CDR1 can comprise the 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 heterologous EPOR can comprise a VL-CDR1 sequence that is 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% identical to any one of the VL-CDR1 sequences listed in Table 16.

[0250] In some embodiments, the VL-CDR2 can 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 can 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 can comprise the 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 that is 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% identical to any one of the VL-CDR2 sequences listed in Table 14.

[0251] In some embodiments, the VL-CDR2 can 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 the 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 that is 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% identical to any one of the VL-CDR2 sequences listed in Table 15.

[0252] In some embodiments, the VL-CDR2 can 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 the 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 heterologous EPOR can comprise a VL-CDR2 sequence that is 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% identical to any one of the VL-CDR2 sequences listed in Table 16.

[0253] In some embodiments, the VL-CDR3 can 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 the 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 that is 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% identical to any one of the VL-CDR3 sequences listed in Table 4.

[0254] In some embodiments, the VL-CDR3 can 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 the sequence of any one of SEQ ID NOs: 944-1072. In some embodiments, an anti-CD131 antibody that binds to the CD131 subunit of a heterologous 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.

[0255] In some embodiments, the VL-CDR3 can 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 the 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 heterologous EPOR can comprise a VL-CDR3 sequence that is 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% identical to any one of the VL-CDR3 sequences listed in Table 8.

[0256] In some embodiments, an anti-EPOR antibody that binds to an EPO receptor subunit of a heterologous EPOR may comprise: a VH-CDR1 comprising a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 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; a VH-CDR2 comprising 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; NO:2256-2443 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; and a VH-CDR3 comprising a VH-CDR3 identical to SEQ ID NO:2256-2443; Any of NOs: 63-250 has 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.In some embodiments, an anti-EPOR antibody that binds to an EPO receptor subunit of a heterologous EPOR may comprise: a 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%, 99.1%, 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; a VL-CDR2 comprising 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; NO:2632-2819 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; and a VL-CDR3 comprising a VL-CDR3 identical to SEQ ID NO:2632-2819; A sequence of NOs:251-438 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.

[0257] In some embodiments, the anti-CD131 antibody may comprise: a VH-CDR1 comprising a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 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; a VH-CDR2 comprising 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; NO:2949-3077 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; and a VH-CDR3 comprising a VH-CDR3 identical to SEQ ID NO:2949-3077; 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 among one of NOs:815-943.In some embodiments, the anti-CD131 antibody may comprise: a 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%, 99.1%, 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; a VL-CDR2 comprising 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; NO:3207-3335 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; and a VL-CDR3 comprising a VL-CDR3 identical to SEQ ID NO:3207-3335; A sequence among NOs:944-1072 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.

[0258] In some embodiments, an anti-EPOR antibody that binds to an EPO receptor subunit and a CD131 subunit of a heterologous EPOR may comprise: a VH-CDR1 comprising a sequence having at least 100%, 99.9%, 99.8%, 99.7%, 99.6%, 99.5%, 99.4%, 99.3%, 99.2%, 99.1%, 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; a VH-CDR2 comprising 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; NOs:3472-3607 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; and a VH-CDR3 comprising a VH-CDR3 identical to SEQ ID NOs:3472-3607; Any of NOs: 1331-1466 has 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.In some embodiments, an anti-EPOR antibody that binds to an EPO receptor subunit and a CD131 subunit may comprise: a 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%, 99.1%, 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; a VL-CDR2 comprising 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; NOs:3744-3879 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; and a VL-CDR3 comprising a VL-CDR3 identical to SEQ ID NOs:3744-3879; 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 among NOs:1467-1602.

[0259] In some embodiments, an anti-EPOR antibody that binds to an EPO receptor subunit of a heterologous EPOR can 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 an EPO receptor subunit of a heterologous EPOR can 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 an EPO receptor subunit of a heterologous EPOR may comprise: a VH comprising the amino acid sequence of any one of SEQ ID NOs: 439-626; and a VL comprising the amino acid sequence of any one of SEQ ID NOs: 627-814.

[0260] In some embodiments, the 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, the 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.

[0261] In some embodiments, an anti-EPOR antibody that binds to the EPO receptor subunit and the CD131 subunit of a heterologous EPOR can 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 the EPO receptor subunit and the CD131 subunit of a heterologous EPOR can 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 an EPO receptor subunit and a CD131 subunit of a heterologous EPOR EPO receptor subunit may comprise: a VH comprising the amino acid sequence of any one of SEQ ID NOs: 1603-1738; and a VL comprising the amino acid sequence of any one of SEQ ID NOs: 1739-1874.

[0262] In some embodiments, anti-EPOR antibodies, anti-CD131 antibodies or anti-EPO antibodies may comprise a VH sequence and a kappa chain variable region (VK) sequence. In some embodiments, anti-EPOR antibodies, anti-CD131 antibodies or anti-EPO antibodies may comprise a VH sequence and a lambda chain variable region. In some embodiments, the 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, the anti-EPOR antibody, anti-CD131 antibody, or anti-EPO antibody may comprise the VH sequence of any one of SEQ ID NOs: 1739-1955. For example, an anti-EPOR antibody, an anti-CD131 antibody, or an anti-EPO antibody can 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 of the VH sequences of any one of SEQ ID NOs: 1739-1955. In some embodiments, the 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 the VK sequence of any one of SEQ ID NOs: 1956-1972.For example, an anti-EPOR antibody, an anti-CD131 antibody, or an anti-EPO antibody can comprise a VK sequence that 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%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, or at least 20% sequence identity to any VK sequence of any one of SEQ ID NOs: 1956-1972.

[0263] In some aspects, anti-EPOR antibodies, anti-CD131 antibodies or anti-EPO antibodies can be combined with heterologous EPOR or homologous EPOR or EPO (respectively) with an affinity of about 1 pM to about 100 nM, about 2.0 nM to about 5.1 nM, about 45 nM to about 300 nM or about 2.0 nM to about 300 nM. In some embodiments, anti-EPOR antibodies, anti-CD131 antibodies or anti-EPO antibodies can be combined 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, any method known in the art can be used to measure binding affinity. 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 can be used to measure binding affinity. In some embodiments, binding affinity can be screened using a suitable bioassay.

[0264] In some embodiments, the binding affinity of the anti-EPO antibodies, anti-EPOR antibodies, and anti-CD131 antibodies described herein can be less than about 600 nM, about 590 nM, about 580 nM, about 570 nM, about 560 nM, about 550 nM, about 540 nM, about 530 nM, about 520 nM, about 510 nM, about 500 nM, about 490 nM, about 480 nM, about 470 nM, about 460 nM, about 450 nM, about 440 nM, about 430 nM, about 420 nM, about 410 nM, about 400 nM, about 390 nM, about 380 nM, about 370 nM, about 360 nM, about 350 nM, about 340 nM, about 330 nM, about 320 nM. , about 310nM, about 300nM, about 290nM, about 280nM, about 270nM, about 260nM, about 250nM, about 240nM, about 230nM, about 220nM, about 210nM, about 200nM, about 190nM, about 180nM, about 170nM, about 160nM, about 150nM, about 140nM M, about 130 nM, about 120 nM, about 110 nM, about 100 nM, about 90 nM, about 80 nM, about 70 nM, about 50 nM, about 50 nM, about 49 nM, about 48 nM, about 47 nM, about 46 nM, about 45 nM, about 44 nM, about 43 nM, about 42 nM, about 41 nM, about 40 nM, about 39 nM , about 38nM, about 37nM, about 36nM, about 35nM, about 34nM, about 33nM, about 32nM, about 31nM, about 30nM, about 29nM, about 28nM, about 27nM, about 26nM, about 25nM, about 24nM, about 23nM, about 22nM, about 21nM, about 20nM, about 19nM, about 18nM M, about 17 nM, about 16 nM, about 15 nM, about 14 nM, about 13 nM, about 12 nM, about 11 nM, about 10 nM, about 9 nM, about 8 nM, about 7 nM, about 6 nM, about 5 nM, about 4 nM, about 3 nM, about 2 nM, about 1 nM, about 990 pM, about 980 pM, about 970 pM, about 960 pM, about 9 50pM, about 940pM, about 930pM, about 920pM, about 910pM, about 900pM, about 890pM, about 880pM, about 870pM, about 860pM, about 850pM, about 840pM, about 830pM, about 820pM, about 810pM, about 800pM, about 790pM, about 780pM, about 770 pM, about 760 pM, about 750 pM, about 740 pM, about 730 pM, about 720 pM, about 710 pM, about 700 pM, about 690 pM, about 680 pM, about 670 pM, about 660 pM, about 650 pM, about 640 pM, about 630 pM, about 620 pM, about 610 pM, about 600 pM,about 590 pM, about 580 pM, about 570 pM, about 560 pM, about 550 pM, about 540 pM, about 530 pM, about 520 pM, about 510 pM, about 500 pM, about 490 pM, about 480 pM, about 470 pM, about 460 pM, about 450 pM, about 440 pM, about 430 pM, about 420 pM, about 410 pM, about 400 pM, about 390 pM, about 380 pM, about 370 pM, about 360 pM, about 350 pM, about 340 pM, about 330 pM, about 320 pM, about 310 pM, about 300 pM, about 290 pM, about 280 pM, about 270 pM, about 260 pM, about 250 pM, about 240 pM, about 230 pM, about 220 pM, about 210 pM, about 200 pM, about 190 pM, about 180 pM, about 170 pM, about 160 pM, or any integer therebetween. In some embodiments, the binding affinity of the anti-EPO antibodies, anti-EPOR antibodies, and anti-CD131 antibodies described herein can be less than 150 pM, about 140 pM, about 130 pM, about 120 pM, about 110 pM, about 100 pM, about 95 pM, about 90 pM, about 85 pM, about 80 pM, about 75 pM, about 70 pM, about 65 pM, about 60 pM, about 55 pM, about 50 pM, about 45 pM, about 40 pM, about 35 pM, about 30 pM, about 25 pM, about 20 pM, about 15 pM, about 1 0 pM, about 9 pM, about 8 pM, about 7 pM, about 6 pM, about 5 pM, about 4 pM, about 3 pM, about 2 pM, about 1 pM, about 0.9 pM, about 0.8 pM, about 0.7 pM, about 0.6 pM, about 0.5 pM, about 0.4 pM, about 0.3 pM, about 0.2 pM, about 0.1 pM, about 0.09 pM, about 0.08, about 0.07 pM, about 0.06 pM, about 0.05 pM, about 0.04 pM, about 0.03 pM, about 0.02 pM, about 0.01 pM, or any integer therebetween.

[0265] In some cases, the anti-EPO antibodies, anti-EPOR antibodies, or anti-CD131 antibodies described herein can have an antagonistic effect. In some embodiments, the anti-EPO antibodies described herein can bind to EPO and inhibit or block the EPO / EPOR interaction. For example, the anti-EPO antibodies can bind to EPO and inhibit EPO from binding to homologous EPOR or heterologous 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 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 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%.

[0266] In some embodiments, the anti-EPOR antibodies described herein can bind to EPOR subunits of a homologous EPOR or a heterologous EPOR and inhibit or block homologous EPOR complex formation, heterologous EPOR complex formation, EPO / homologous EPOR interaction, or EPO / heterologous EPOR interaction. For example, an anti-EPOR antibody can bind to an EPOR subunit and inhibit the formation of a homologous EPOR or a heterologous EPOR. For example, an anti-EPOR antibody can bind to an EPOR subunit of a homologous EPOR or a heterologous EPOR and inhibit the binding of a homologous EPOR or a heterologous EPOR 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 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 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 %, 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%.

[0267] In some embodiments, the anti-CD131 antibodies described herein can bind to CD131 and inhibit or block heterologous EPOR complex formation or EPO / heterologous EPOR interaction. For example, anti-CD131 antibodies can bind to CD131 and inhibit the formation of heterologous EPOR. For example, anti-CD131 antibodies can bind to the CD131 subunit of heterologous EPOR and inhibit heterologous 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 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 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%.

[0268] In some cases, the anti-EPO antibodies, anti-EPOR antibodies, or anti-CD131 antibodies described herein can have an agonistic effect. In some embodiments, the anti-EPO antibodies described herein can bind to EPO and enhance or promote the EPO / EPOR interaction. In some embodiments, in the case of anti-EPO antibodies, the EPO / EPOR interaction is enhanced 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 110%, at least about 111%, at least about 112%, at least about 113%, at least about 1 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%.

[0269] In some embodiments, the anti-EPOR antibodies described herein can bind to the EPOR subunits of a homologous EPOR or a heterologous EPOR and enhance or promote homologous EPOR complex formation, heterologous EPOR complex formation, EPO / homologous EPOR interaction, or EPO / heterologous EPOR interaction. In some embodiments, with the anti-EPOR antibodies, homologous EPOR complex formation, heterologous EPOR complex formation, EPO / homologous EPOR interaction, or EPO / heterologous EPOR interaction is enhanced 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 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 At least about 71%, at least about 72%, 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%.

[0270] In some embodiments, the anti-CD131 antibodies described herein can bind to CD131 and enhance or promote heterologous EPOR complex formation or EPO / heterologous EPOR interaction. In some embodiments, with the anti-CD131 antibodies, heterologous EPOR complex formation or EPO / heterologous EPOR interaction is enhanced 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 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%.

[0271] In some embodiments, affinity maturation can be used in the case of the antibodies disclosed herein to obtain anti-EPOR antibodies, anti-CD131 or anti-EPO antibodies with the desired affinity. When anti-EPOR antibodies, anti-CD131 or anti-EPO antibodies are obtained from animals (e.g., transgenic animals carrying human antibody libraries), the antibodies produced in the transgenic animals can undergo affinity maturation. Alternatively, chain shuffling methods and / or mutations of nucleic acids encoding VH and VL can be used to affinity mature antibodies from transgenic animals or from other technologies (such as display technologies), followed by screening and / or selection of antibodies with greater affinity.

[0272] The most widely used method for minimizing the immunogenicity of non-human antibodies while retaining specificity and affinity can include transplanting the CDRs of non-human antibodies onto a human framework selected due to structural homology with the non-human framework (Jones et al., 1986, Nature 321:522-5; U.S. Patent No. 5,225,539; hereby incorporated by reference in its entirety). Incorporating some non-human residues at key positions of the framework can improve the affinity of the CDR-grafted antibody (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 hereby incorporated by reference in their entirety). Exemplary methods for antibody humanization by CDR transplantation are disclosed in, for example, U.S. Patent No. 6,180,370, which is hereby incorporated by reference in its entirety.

[0273] Improvements to traditional CDR transplantation methods can use various hybrid selection methods, wherein in the selection for antigen binding in a continuous number of rounds, a part of non-human antibodies is combined with a library of complementary human antibody sequences, and most non-human sequences are gradually replaced by human sequences in the process. For example, in chain shuffling technology (Marks et al., 1992, Biotechnology 10: 779-83, which is hereby incorporated by reference in its entirety for all purposes), the affinity based on non-human chains will be sufficient to constrain the selection of human partners to the same epitope on the antigen, and a chain of non-human antibodies can be combined with the natural human antibody library of another chain. Selected human partners can then be used to guide the selection of the remaining non-human chains.

[0274] Other methods may include chain replacement technology, in which the non-human CDR3 is retained and only the remainder of the V region (including the framework and CDR1 and 2) is replaced individually in sequential steps (see, 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 hereby incorporated by reference in their entirety for all purposes).

[0275] These techniques can be used to prepare antibodies suitable for use in non-human subjects by engineering CDRs into the framework regions of the subject species using methods analogous to CDR grafting methods used to prepare antibodies for use in humans.

[0276] The present disclosure encompasses pharmaceutically acceptable salts of anti-EPOR antibodies, anti-CD131 antibodies, or anti-EPO antibodies, including salts with a net positive charge, salts with a net negative charge, and salts with no net charge, and includes, but is not limited to, salts of anti-EPOR antibodies, anti-CD131 antibodies, or anti-EPO antibodies (including fragments thereof) as compounds, in pharmaceutical compositions, in their therapeutic and diagnostic uses, and in their manufacture.

[0277] In some embodiments, the half-life of anti-EPO antibodies, anti-EPOR antibodies, and anti-CD131 antibodies described herein in human plasma can be 1 minute to 1 hour. In some embodiments, the half-life of anti-EPO antibodies, anti-EPOR antibodies, and anti-CD131 antibodies described herein can be 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 half-life of anti-EPO antibodies, anti-EPOR antibodies, and anti-CD131 antibodies described herein can be 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 half-life of anti-EPO antibodies, anti-EPOR antibodies, and anti-CD131 antibodies described herein in human plasma can be 1 hour to 5 days. In some embodiments, the half-life of anti-EPO antibodies, anti-EPOR antibodies, and anti-CD131 antibodies described herein can be about 1 hour to about 120 hours.In some embodiments, the half-life of the anti-EPO antibodies, anti-EPOR antibodies, and anti-CD131 antibodies described herein can be 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 ... 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 In some embodiments, the half-life of the anti-EPO antibodies, anti-EPOR antibodies, and anti-CD131 antibodies described herein can be 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, 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 half-life of the anti-EPO antibodies, anti-EPOR antibodies, and anti-CD131 antibodies described herein can be 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 half-life of anti-EPO antibodies, anti-EPOR antibodies, and anti-CD131 antibodies described herein can be 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 half-life of anti-EPO antibod...

Claims

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

2. The composition of claim 1, wherein 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 λ chain variable regions.

3. The composition of claim 1 or 2, wherein the prevention of the formation of the EPO protein-heterologous EPO receptor complex, the formation of the heterologous EPO receptor between the EPO receptor subunit and the CD131 subunit, or the activation of the heterologous EPO receptor inhibits immune tolerance, wherein the heterologous EPO receptor comprises the EPO receptor subunit and the CD131 subunit.

4. The composition of claim 3, wherein the prevention of the formation of the EPO protein-heterologous EPO receptor complex, the formation of the heterologous EPO receptor between the EPO receptor subunit and the CD131 subunit, or the activation of the heterologous EPO receptor promotes the differentiation of multiple naive T cells into multiple effector T cells, wherein the heterologous EPO receptor comprises the EPO receptor subunit and the CD131 subunit.

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. The composition of claim 3, wherein the prevention of the formation of the EPO protein-heterologous EPO receptor complex, the formation of the heterologous EPO receptor between the EPO receptor subunit and the CD131 subunit, or the activation of the heterologous EPO receptor inhibits the differentiation of multiple naive T cells into multiple regulatory T cells, wherein the heterologous EPO receptor comprises the EPO receptor subunit and the CD131 subunit.

7. A composition as described in 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. A composition as described in claim 1 or 2, wherein the prevention of the formation of the EPO protein-heterologous EPO receptor complex, the formation of the heterologous EPO receptor between the EPO receptor subunit and the CD131 subunit, or the activation of the heterologous EPO receptor increases multiple progenitor cell depletion of T cells, wherein the heterologous EPO receptor comprises the EPO receptor subunit and the CD131 subunit.

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

10. The composition of claim 1 or 2, wherein the prevention of the formation of the EPO protein-heterologous EPO receptor complex, the formation of the heterologous EPO receptor between the EPO receptor subunit and the CD131 subunit, or the activation of the heterologous EPO receptor stimulates an immune response in cancer, wherein the heterologous EPO receptor comprises the EPO receptor subunit and the CD131 subunit.

11. A composition as described in claim 1 or 2, wherein the prevention of the formation of the EPO protein-heterologous EPO receptor complex, the formation of the heterologous EPO receptor between the EPO receptor subunit and the CD131 subunit, or the activation of the heterologous EPO receptor sensitizes cancer cells to immune checkpoint inhibitors, wherein the heterologous EPO receptor comprises the EPO receptor subunit and the CD131 subunit.

12. The composition of claim 11, wherein the immune checkpoint inhibitor comprises a cytotoxic T lymphocyte-associated protein 4 (CTLA-4) inhibitor, a programmed death 1 (PD-1) inhibitor, or a programmed 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. The composition of claim 1 or 2, wherein the prevention of the formation of the EPO protein-heterologous EPO receptor complex, the formation of the heterologous EPO receptor between the EPO receptor subunit and the CD131 subunit, or the activation of the heterologous EPO receptor reduces tumor growth, wherein the heterologous EPO receptor comprises the EPO receptor subunit and the CD131 subunit.

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

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

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

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

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

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

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

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

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

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

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

32. The composition of any one of claims 2-20, 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 2-20, 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 2-20, 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 the antibody or the functional fragment thereof of the composition as described in any one of claims 1-34.

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

37. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a composition as described in any one of claims 1-35.

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

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

40. The method of claim 38, wherein said suppressing immune tolerance comprises increasing the subject's immune response to a viral or bacterial infection.

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

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

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

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

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

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

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

48. A method for treating cancer, wherein the method comprises administering a composition or a derivative thereof to a subject having cancer or at risk of having cancer, wherein the composition or the derivative thereof inhibits the activity of the subject's allogeneic erythropoietin (EPO) receptor.

49. The method of claim 48, wherein the heterologous EPO receptor is expressed on myeloid cells.

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

51. The composition of claim 50, wherein 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, or VH and kappa chain variable regions (VK), or VH and λ chain variable regions.

52. The composition of claim 50 or claim 51, wherein the heterologous EPO receptor is expressed on myeloid cells.

53. A composition as described in any one of claims 50-52, wherein the promotion of the formation of the EPO protein-heterologous EPO receptor complex, the formation of the heterologous EPO receptor between the EPO receptor subunit and the CD131 subunit, or the activation of the heterologous EPO receptor between the EPO receptor subunit and the CD131 subunit induces antigen-specific immune tolerance.

54. A composition as described in claim 52, wherein the promotion of the formation of the EPO protein-heterologous EPO receptor complex, the formation of the heterologous EPO receptor between the EPO receptor subunit and the CD131 subunit, or the activation of the heterologous EPO receptor between the EPO receptor subunit and the CD131 subunit inhibits the differentiation of multiple naive T cells into multiple effector T cells.

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. A composition as described in claim 52, wherein the promotion of the formation of the EPO protein-heterologous EPO receptor complex, the formation of the heterologous EPO receptor between the EPO receptor subunit and the CD131 subunit, or the activation of the heterologous EPO receptor between the EPO receptor subunit and the CD131 subunit promotes the differentiation of multiple naive T cells into multiple regulatory T cells.

57. A composition as described in 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. A composition as described in any one of claims 50-57, wherein the antibody or the functional fragment thereof does not affect the activity of the homologous EPO receptor.

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

60. A composition as described in any one of claims 50-59, wherein when administered to a subject with an autoimmune disease or a subject with a transplanted organ, the promotion of the formation of the EPO protein-heterologous EPO receptor complex, the formation of the heterologous EPO receptor between the EPO receptor subunit and the CD131 subunit, or the activation of the heterologous EPO receptor between the EPO receptor subunit and the CD131 subunit reduces the immune response.

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

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

63. A composition as described in any one of claims 50-59, wherein when administered to a subject suffering from systemic chronic inflammation, the promotion of the formation of the EPO protein-heterologous EPO receptor complex, the formation of the heterologous EPO receptor between the EPO receptor subunit and the CD131 subunit, or the activation of the heterologous EPO receptor between the EPO receptor subunit and the CD131 subunit reduces systemic chronic inflammation.

64. A composition as described in any one of claims 50-63, wherein the antibody or the functional fragment thereof is IgG, IgM, IgE, IgA, IgD, a derivative thereof, or a combination thereof.

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

66. The composition of any one of claims 50-65, wherein the antigen binding domain comprises Fab, Fab', (Fab')2, variable fragment (Fv), single-chain variable fragment (scFv), scFv-Fc, Fab-Fc, VHH, non-antibody scaffold, or a combination thereof.

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

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

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

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

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

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

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

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

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

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

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

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. 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. A composition as described in any 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. The composition of claim 82, wherein the tumor-associated antigen is selected from 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 the antibody or the functional fragment thereof of the composition as described in any one of claims 50-85.

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

88. A method of treating a disease or condition in a subject in need thereof, the method comprising administering to the subject a composition as described in any one of claims 50-86.

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

90. The method of claim 88, wherein the subject has received or is about to receive an organ transplant or a foreign therapeutic protein.

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

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

93. 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 homologous EPO receptor comprising at least two EPO receptor subunits or a heterologous EPO receptor comprising an EPO receptor subunit and a CD131 subunit.

95. The composition of claim 91, wherein the EPO receptor is a heterologous 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. A composition as described in any one of claims 91-96, 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. A composition as described in claim 98, wherein the complex is capable of preventing EPO receptor activity.

100. A composition as described in claim 91, wherein the composition or a derivative thereof comprises an engineered erythropoietin (EPO) protein, wherein the engineered EPO protein inhibits the activity of a heterologous erythropoietin (EPO) receptor in myeloid cells.

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

102. The composition of claim 100 or 101, wherein the engineered EPO protein comprises 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.

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

104. A composition as described in claim 101 or 102, wherein the composition or a derivative thereof promotes an immune response.

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

108. A composition as described in 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. A composition as described in claim 101 or 102, wherein the composition or its derivative increases a plurality of progenitor-depleted T cells.

110. The composition of claim 109, wherein the plurality of progenitor-depleted 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 immune checkpoint inhibitors.

113. The composition of claim 112, wherein the immune checkpoint inhibitor comprises a cytotoxic T lymphocyte-associated protein 4 (CTLA-4) inhibitor, a programmed death 1 (PD-1) inhibitor, or a programmed 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. 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 102-117, 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. A composition as described in any of claims 102-130, wherein the position is determined by comparison 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. A composition as described in any of claims 101-133, wherein the engineered EPO protein has a lower binding affinity for a heterologous EPO receptor than a corresponding wild-type EPO protein without the at least one amino acid substitution.

135. A composition as described in any of claims 101-134, wherein the heterologous EPO receptor activity comprises phosphorylation of the intracellular domain of the heterologous 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. A composition as described in any one of claims 101-135, wherein the heterologous 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. A composition as described in any of claims 101-136, wherein the engineered EPO protein has a higher binding affinity for the homologous EPO receptor than the corresponding wild-type EPO protein without the at least one amino acid substitution.

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

139. A composition as described in any of claims 101-136, wherein the engineered EPO protein binds to the homologous EPO receptor with a binding affinity that is lower than the binding affinity to the heterologous EPO receptor.

140. A composition as described in any of claims 101-136, wherein the engineered EPO protein does not affect and does not inhibit the activity of the homologous EPO receptor.

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

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

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 the engineered EPO protein of the composition of any one of claims 101-143.

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

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

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

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

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

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

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

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

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

154. The composition of claim 153, wherein the engineered EPO protein comprises at least one amino acid modification and / or at least one amino acid substitution, wherein the at least one amino acid modification and / or at least one amino acid substitution comprises: 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.

155. The composition of claim 153 or 154, wherein the promoting of the heterologous EPO receptor activity reduces an immune response when administered to a subject suffering from an autoimmune disease or a subject having a transplanted organ.

156. The composition of claim 155, wherein the transplant 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 when administered to a subject suffering from systemic chronic inflammation, the promoting activity of the heterologous EPO receptor reduces systemic chronic inflammation.

159. A composition as described in any one of claims 153-158, wherein the promoting the heterologous EPO receptor activity induces antigen-specific immune tolerance.

160. A composition as described in any one of claims 153-158, wherein the promoting the heterologous EPO receptor activity inhibits the differentiation of multiple naive T cells into multiple 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. A composition as described in any one of claims 153-158, wherein the promotion of the heterologous EPO receptor activity promotes the differentiation of multiple naive T cells into multiple regulatory T cells.

163. A composition as described in claim 162, 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).

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

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

166. A composition as described in any one of claims 153-166, wherein the amino acid residue positions are determined by comparison with SEQ ID NO:

1.

167. The composition of any one of claims 153-166, 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. A composition as described in any of claims 153-169, wherein the engineered EPO protein has a higher binding affinity for the heterologous EPO receptor than a corresponding wild-type EPO protein without the at least one amino acid substitution.

171. A composition as described in any of claims 153-170, wherein the heterologous EPO receptor activity includes phosphorylation of the intracellular domain of the heterologous 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. A composition as described in any one of claims 153-171, wherein the heterologous 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. A composition as described in any of claims 153-172, wherein the engineered EPO protein has a lower binding affinity for a homologous EPO receptor comprising at least two EPO receptor subunits than a corresponding wild-type EPO protein without the at least one amino acid substitution.

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

175. A composition as described in any of claims 153-174, wherein the engineered EPO protein does not affect and does not inhibit the activity of the homologous EPO receptor.

176. A composition as described in any of claims 153-175, wherein the homologous EPO receptor activity includes phosphorylation of the intracellular domain of the homologous 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 homologous 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. The composition of any one of claims 153-177, wherein the half-life of the engineered EPO is at least 5 hours.

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

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

181. The composition of claim 180, wherein the immune cell comprises a macrophage, a dendritic cell, a T cell, a natural killer cell, or a B cell.

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

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

184. A composition comprising a nucleic acid sequence encoding the EPO protein of the composition described in any one of claims 153-183.

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

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

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

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

189. A composition comprising an engineered erythropoietin (EPO) protein, wherein the engineered EPO protein promotes homologous erythropoietin (EPO) receptor activity and reduces the effect on heterologous EPO receptor activity, wherein the homologous EPO receptor comprises at least two EPO receptor subunits, and the heterologous EPO receptor comprises an EPO receptor subunit and a CD131 subunit.

190. The composition of claim 189, wherein the engineered EPO protein comprises 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.

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

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

193. The composition of claim 190, wherein the engineered EPO protein comprises at least one amino acid substitution comprising 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. A composition as described in any of claims 189-192, wherein the position is determined by comparison with SEQ ID NO:

1.

197. A composition as described in any one of claims 189-193, wherein the engineered EPO further comprises an amino acid modification comprising carbamylation or pegylation.

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

199. A composition as described in any of claims 189-198, wherein the engineered EPO protein has a higher binding affinity for the homologous EPO receptor than a corresponding wild-type EPO protein without the at least one amino acid substitution.

200. A composition as described in any of claims 189-199, wherein the homologous EPO receptor activity includes phosphorylation of the intracellular domain of the homologous 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. A composition as described in any of claims 189-200, wherein the homologous 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. A composition as described in any of claims 189-201, wherein the engineered EPO protein has the same level of binding affinity for the heterologous EPO receptor as compared to a corresponding wild-type EPO protein without the at least one amino acid substitution.

203. A composition as described in any of claims 189-202, wherein the heterologous EPOR activity comprises phosphorylation of the intracellular domain of the homologous 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. A composition as described in any one of claims 189-203, wherein the heterologous 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. A composition as described in any one of claims 202-204, 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 multiple naive T cells into multiple 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 multiple naive T cells into multiple regulatory T cells.

209. A composition as described in claim 208, 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).

210. A composition as described in any one of claims 202-204, wherein the engineered EPO protein does not affect the immune response.

211. A composition as described in any of claims 189-210, wherein the half-life of the engineered EPO is at least 5 hours.

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

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

214. A composition comprising a nucleic acid sequence encoding the EPO protein of the composition described in any one of claims 189-213.

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

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

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

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

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

220. 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. 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 condition, the composition comprising a compound, a pharmaceutically acceptable salt, solvate or stereoisomer thereof, wherein the compound inhibits erythropoietin (EPO) receptor activity in myeloid cells of the subject.

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

226. A composition as described in claim 224 or 225, wherein the heterologous EPO receptor comprises an EPO subunit and a CD131 subunit.

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

228. A composition as described in any one of claims 224 to 227, wherein the compound is an inhibitor of promotion of hypoxia inducible factor (HIF), IL-1α, IL-1β, TNF-α, IL-6, estrogen receptor, phospholipase C-γ1, or 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 composition of any one of claims 224 to 230, wherein the compound is CAY10585 (LW6), nigromycin, chrysin, dimethyl bisphenol A, echinomycin, 2-methoxyestradiol (2ME2), SYP-5, PX-478 2HCl, KC7F2, GN44028, velutin, FM19G11, PT2399, PT2385, benzyltrimethicone, HIF-2a-IN-1, HIF-2a-IN-2, HIF-2a-IN-3, HIF-2a-IN-4, TC-S 700, IDF-11774, paeoniflorin, emetine hydrochloride, glucosamine, PX12, vitexine, BAY 87-2243, rificiguat (YC-1), vorinostat, tansiramycin, silybin, diallyl trisulfide (DATS), gephyranthes diene (GEX1A), celastrol, phenylethyl isothiocyanate (PEITC), gliotoxin, 1-isothiocyanato-4R-methylsulfinylbutane, acriflavine, emodin, cardiac glycosides, 3,3'-diindolylmethane (DIM), pyruvic acid-B (PAB), methyl psoralen, andrographolide, isoliquiritigenin, wondonin, thymoquinone, or curcumin.

232. The composition of any one of claims 224 to 231, wherein the compound is CAY10585 (LW6), nigromycin, chrysin, dimethyl bisphenol A, echinomycin, 2-methoxyestradiol (2ME2), SYP-5, PX-478 2HCl, KC7F2, GN44028, veglutidin, FM19G11, PT2399, PT2385, bezutifan, HIF-2a-IN-1, HIF-2a-IN-2, HIF-2a-IN-3, HIF-2a-IN-4, TC-S 700, IDF-11774, paeoniflorin, emetine hydrochloride, glucosamine, PX12, vitexine, BAY 87-2243, riviciguat (YC-1), vorinostat or tanspiramycin.

233. The composition of any one of claims 224 to 232, wherein the compound is nigromycin, echinomycin, PT2399, beztifan, vorinostat, or tanspiramycin.

234. The composition of any one of claims 224 to 231, wherein the compound is silybin, diallyl trisulfide (DATS), gessolide (GEX1A), celastrol, phenylethyl isothiocyanate (PEITC), gliotoxin, 1-isothiocyanato-4R-methylsulfinylbutane, acriflavine, emodin, cardiac glycosides, 3,3'-diindolylmethane (DIM), pyrimethicone-B (PAB), methyl psoralen, andrographolide, isoliquiritigenin, wondonin, thymoquinone, or curcumin.

235. A composition for administration to a subject having cancer or a chronic infectious condition, the composition comprising a compound, a pharmaceutically acceptable salt, solvate or stereoisomer thereof, wherein the compound inhibits erythropoietin (EPO) receptor activity, thereby reversing the subject's resistance to immune checkpoint blockade.

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

237. A composition as described in claim 235 or 236, wherein the heterologous EPO receptor comprises an EPO subunit and a CD131 subunit.

238. The composition of any one of claims 235 to 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 inhibitor of CTLA-4, PD-1 or PD-L1 is nivolumab, pembrolizumab, cemiplizumab, atezolizumab, avelumab, durvalumab, ipilimumab, lirilumab and BMS-986016.

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

241. A composition as described in any one of claims 235 to 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 to 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 to 242, wherein the compound is an inhibitor of hypoxia inducible factor (HIF).

244. The composition of any one of claims 235 to 243, wherein the compound is CAY10585 (LW6), nigromycin, chrysin, dimethyl bisphenol A, echinomycin, 2-methoxyestradiol (2ME2), SYP-5, PX-478 2HCl, KC7F2, GN44028, velutin, FM19G11, PT2399, PT2385, bezutifan, HIF-2a-IN-1, HIF-2a-IN-2, HIF-2a-IN-3, HIF-2a-IN-4, TC-S 700, IDF-11774, paeoniflorin, emetine hydrochloride, glucosamine, PX12, vitexine, BAY 87-2243, rificiguat (YC-1), vorinostat, tansiramycin, silybin, diallyl trisulfide (DATS), gephyranthes diene (GEX1A), celastrol, phenylethyl isothiocyanate (PEITC), gliotoxin, 1-isothiocyanato-4R-methylsulfinylbutane, acriflavine, emodin, cardiac glycosides, 3,3'-diindolylmethane (DIM), pyruvic acid-B (PAB), methyl psoralen, andrographolide, isoliquiritigenin, wondonin, thymoquinone, or curcumin.

245. The composition of any one of claims 235 to 244, wherein the compound is CAY10585 (LW6), nigromycin, chrysin, dimethyl bisphenol A, echinomycin, 2-methoxyestradiol (2ME2), SYP-5, PX-478 2HCl, KC7F2, GN44028, veglutidin, FM19G11, PT2399, PT2385, bezutifan, HIF-2a-IN-1, HIF-2a-IN-2, HIF-2a-IN-3, HIF-2a-IN-4, TC-S 700, IDF-11774, paeoniflorin, emetine hydrochloride, glucosamine, PX12, vitexine, BAY 87-2243, riviciguat (YC-1), vorinostat or tanspiramycin.

246. The composition of any one of claims 235 to 245, wherein the compound is nigromycin, echinomycin, PT2399, beztifan, vorinostat, or tanspiramycin.

247. The composition of any one of claims 235 to 244, wherein the compound is silybin, diallyl trisulfide (DATS), gessolide (GEX1A), celastrol, phenylethyl isothiocyanate (PEITC), gliotoxin, 1-isothiocyanato-4R-methylsulfinylbutane, acriflavine, emodin, cardiac glycosides, 3,3'-diindolylmethane (DIM), pyrimethicone-B (PAB), methyl psoralen, andrographolide, isoliquiritigenin, wondonin, thymoquinone, or curcumin.

248. A composition for administration to a subject, the composition comprising a compound, a pharmaceutically acceptable salt, solvate or stereoisomer thereof, wherein the compound promotes the activity of a heterologous erythropoietin (EPO) receptor, wherein the heterologous EPO receptor comprises an EpoR subunit and a CD131 subunit, such that the subject's immune tolerance to the antigen is increased; and wherein the compound has no substantial effect on the activity of a homologous EPO receptor, wherein the homologous EPO receptor comprises at least two EPO receptor subunits.

249. The composition of claim 248, wherein the heterologous EPO receptor is on a macrophage, a monocyte, a dendritic cell, a basophil, a neutrophil, or an 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 factor, 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 rosastat, valdostat, enadostat, dedostat, molistat, dimethyloxalylglycine, dapnostat, prolyl hydroxylase inhibitor 1, TM6089, TRC160334, PHD-1-IN-1, MK-8617, JNJ-42041935, TP0463518, IOX (JICL38), IOX4, IOX3 (FG-2216), notoginseng, 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 rosastat, valdostat, enadostat, dedostat, molistat, dimethyloxalylglycine, daptostat, prolyl hydroxylase inhibitor 1, TM6089, TRC160334, PHD-1-IN-1, MK-8617, JNJ-42041935, TP0463518, IOX (JICL38), IOX4, IOX3 (FG-2216), notoginseng, HIF-PHD-IN-1, AKB-6899, VH298, M1001, ML228, or dimethyloxalylglycine (DMOG).

255. The composition of any one of claims 248 to 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 an autoantigen.

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 for an immunosuppressive state.

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

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

263. A composition for administration to a subject having cancer, the composition comprising an RNA interference (RNAi) molecule, wherein the RNAi binds to an RNA molecule selected from 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; wherein after the RNAi is administered to the subject, the subject's tumor mass is reduced.

264. The composition of claim 263, wherein the tumor mass is reduced to less than 0.5 cm 3 .

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

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

267. A composition for administration to a subject suffering from cancer, the composition comprising an RNA interference (RNAi) molecule, wherein the RNAi binds to an RNA molecule selected from 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; wherein after the RNAi is administered to the subject, the subject's immune response is enhanced by inducing more effector T cells (T cells). eff ) cells to increase.

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

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

270. The composition of any one of claims 263 to 268, wherein the RNAi reduces the EPO level of the 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 two-fold.

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

274. The composition of claim 273, wherein the nanoparticles are lipid nanoparticles.

275. The composition of any one of claims 263 to 274, wherein the RNAi molecule is a siRNA molecule, a miRNA molecule, an antisense RNA molecule, or a 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 a double-stranded or single-stranded siRNA molecule.

280. The composition of claim 279, wherein the single-stranded siRNA molecule comprises a residue 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, SEQ ID NO:37, SEQ ID NO:38 : The nucleic acid sequence of at least one of 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 has at least 80%, 85%, 90% or 95% identity.

281. The composition of claim 280, wherein the single-stranded siRNA molecule comprises a residue 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, SEQ ID NO:37, SEQ ID NO:38 : The nucleic acid sequence of at least one of 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 has 100% identity.

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

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