Synthetic antibody agonists of erythropoietin receptor

Abstract

An object of the present invention is erythropoietin receptor (EPOR) binding sites as well as EPOR agonists and antagonists for the diagnosis and treatment of diseases, in particular kidney disease and anemia or genetic syndromes caused by cancer treatment. More specifically, the present invention relates to synthetic EPOR antibody constructs, in particular diabodies.

Description

[0001] The present invention relates to erythropoietin receptor (EPOR) binding sites, as well as EPOR agonists and antagonists, for use in the diagnosis and treatment of anemia or genetic syndromes caused by diseases, particularly kidney diseases and cancer. More specifically, the present invention relates to synthetic EPOR antibody constructs, particularly bispecific antibodies. These EPOR agonists provide a new treatment option for patients suffering from chronic kidney disease, treatment-related anemia, and hereditary EPO deficiency. Background Art

[0002] Human erythropoietin (EPO) is a growth factor that promotes erythropoiesis. It binds to the human EPO receptor (hEPOR) on erythroid progenitor cells and activates the intracellular signaling cascades involved in erythropoiesis. Naturally, EPO is produced by peritubular cells of the kidney in response to hypoxia. Patients with chronic kidney disease (CKD) typically have reduced EPO production, leading to anemia (a deficiency of red blood cells). Therefore, patients need to receive frequent injections of recombinant EPO or other erythropoiesis-stimulating agents (ESAs) to maintain normal erythropoiesis. The most common ESAs are epoetin alfa and darbepoetin alfa, which are administered to patients at dosing intervals of 2 to 3 times per week and 1 to 2 weeks once, respectively.

[0003] Erythropoiesis begins with the lineage commitment of hematopoietic stem and progenitor cells (HSPCs) to the erythroid lineage. When HSPCs differentiate into erythroid progenitor cells, they begin to express EPOR and respond to circulating EPO, which promotes further proliferation and ultimately maturation into differentiated red blood cells. The binding of hEPO to hEPOR occurs through high-affinity and low-affinity binding sites (sites 1 and 2, respectively), inducing a conformational change in the receptor, which in turn activates downstream signaling cascades. It is thought that unactivated hEPOR is expressed on the cell surface as a preformed dimer in the absence of ligand. hEPO binding occurs through asymmetric association, first interacting with the high-affinity hEPO site 1 and then the low-affinity site 2, which stimulates the repositioning of hEPOR monomers within the dimeric complex, subsequently activating the JAK2 receptor-associated kinase.

[0004] In addition to CKD, anemia is also a common complication, affecting approximately 40% of cancer patients and approximately 90% of chemotherapy patients. Although recombinant hEPO is an effective treatment for cancer-related anemia, its adverse effects on disease recurrence and patient survival have raised concerns and hindered its use in many cancer patients. The harmful effects of hEPO on cancer patients are at least partially mediated through non-erythropoietic signaling pathways, such as the Ephrin B4 receptor (EPHB4) and the common β subunit of the IL-3R (CD131). hEPO can also promote cancer growth by increasing oxygen delivery to hypoxic regions within tumors. The pleiotropic effects of EPO not only highlight the need and opportunity to develop therapies that specifically stimulate erythropoiesis of EPOR, but also the need and opportunity to selectively inhibit the EPO signaling mechanisms that promote cancer.

[0005] There is still a need to identify EPOR agonists and antagonists that can address the above challenges to aid in the diagnosis and treatment of diseases, particularly cancer anemia and other disease conditions. Summary of the Invention

[0006] The present invention provides novel epitopes on hEPOR. The epitopes provide selective activation of the erythropoietic-specific effects of hEPOR without adverse effects, such as but not limited to activation of other cell surface receptors that may have carcinogenic effects, such as EPHB4 and CD131.

[0007] The present invention also provides antibody and polypeptide sequences that can selectively bind to human EPOR with equally high affinity and bind to murine EPOR with very low affinity (e.g., see Figure 1 A, Figure 2 and Figure 9 ). The antibody and polypeptide sequences can act as hEPO agonists and antagonists.

[0008] One embodiment of the present invention is the use of hEPOR agonists as diagnostic and therapeutic agents for the treatment of diseases, including CKD, cancer-related anemia or treatment-related anemia, and hereditary anemia.

[0009] One aspect of the present invention relates to highly selective hEPOR agonists, and in a preferred embodiment, includes synthetic binding molecules, such as antibodies, diabodies, higher-order molecules (e.g., tetravalents). In a more preferred embodiment of the present invention, a synthetic diabody that acts as an hEPOR agonist is provided.

[0010] One aspect of the present invention relates to highly selective hEPOR antagonists, and in a preferred embodiment, includes synthetic binding molecules, such as antibodies. In a more preferred embodiment of the present invention, a synthetic antibody that acts as an hEPOR antagonist is provided.

[0011] One aspect of the present invention relates to the hEPOR activation region as described herein. According to one embodiment and with reference to NCBI Reference Sequence: NP_000112.1, as Figure 9 shown in E (SEQ ID NO:29), the human hEPOR activation region according to the present invention includes a first region that includes residues 82, 84 to 88, and 90 (numbering reference SEQ ID NO:29). According to another embodiment, the human hEPOR activation region according to the present invention includes a second region that includes residues 119, 121, 123, 125, 128, 129, 131, 134, and 136 of hEPOR. According to one embodiment, the human hEPOR activation region according to the present invention includes residues 82, 84 to 88, 90, 119, 121, 123, 125, 128, 129, 131, 134, and 136 of hEPOR.

[0012] Another aspect of the present invention relates to an antibody that binds to the hEPOR activation region as described herein. According to one embodiment, the preferred antibody according to the present invention binds to residues 82, 84 to 88, and 90 of hEPOR. According to one embodiment, the preferred antibody according to the present invention binds to residues 119, 121, 123, 125, 128, 129, 131, 134, and 136 of human EPOR. According to one embodiment, the more preferred antibody according to the present invention binds to residues 82, 84 to 88, 90, 119, 121, 123, 125, 128, 129, 131, 134, and 136 of human EPOR. According to another embodiment, the functional epitope of the antibody according to the present invention includes residues 82, 84 to 88, 90, 119, 121, 123, 125, 128, 129, 131, 134, and 136 of human EPOR.

[0013] According to one embodiment, the antibody of the present invention contacts the EPO activation site of EPOR.

[0014] According to one embodiment, the hEPOR activation region of the present invention comprises the amino acid sequence of hEPOR, which sequence comprises QEDEPWL (SEQ ID NO:1). According to one embodiment, the hEPOR activation region of the present invention comprises the amino acid sequence of hEPOR, which sequence comprises PERTSGPHV (SEQ ID NO:2). According to a preferred embodiment, the hEPOR activation region of the present invention comprises a first amino acid sequence of hEPOR and a second amino acid sequence of hEPOR, the first amino acid sequence comprising QEDEPWL (SEQ ID NO:1) and the second amino acid sequence comprising PERTSGPHV (SEQ ID NO:2).

[0015] According to another embodiment, the antibody further comprises a CDR-L1 having a contiguous amino acid sequence X1X2X3X4X5, wherein: X1 is S, D or T; X2 is V or an aliphatic amino acid; X3 and X4 are D, E, G, H, K, N, Q, R or S; and X5 is A or an aliphatic amino acid.

[0016] According to another embodiment, the antibody further comprises a CDR-L2 having a contiguous amino acid sequence X1X2X3X4X5X6X7, wherein: X1 is S or T; X2 is A, D or an aliphatic amino acid; X3 and X4 are D, E, G, H, K, N, Q, R or S; X5 is L, D or an aliphatic amino acid; X6 is Y or a polar amino acid; and X7 is S, D or T.

[0017] According to another embodiment, the antibody further comprises a CDR-L3 having a contiguous amino acid sequence X1X2X3X4X5X6, wherein: X1 is S, F, T, A, I or P; X2 is S, P, T, C, A, F, Y, G, R or D; X3 is Y, R, P, S, D, R, H, F, N, I, G, P, E, Q or T; X4 is S, F, A, G, V, Y, P, T or N; X5 is L, P or an aliphatic amino acid; and X6 is I, F or a hydrophobic amino acid.

[0018] According to another embodiment, the antibody further comprises a CDR-L3 having a contiguous amino acid sequence X1X2X3X4X5, wherein: X1 is A, D, I, S, T, G, V or P; X2 is Y, N, L, D, H, F, S or V; X3 is W, S, G, R, L, P, K or E; X4 is L, P or an aliphatic amino acid; and X5 is I, F or a hydrophobic amino acid.

[0019] According to another embodiment, the antibody further comprises a CDR-H1 having a contiguous amino acid sequence X1X2X3X4X5X6, wherein: X1 is L or an aliphatic amino acid; X2 is Y, S, N, G, D, H, R, F, T, Q, K, P, E or I; X3 is S, A, F, Y, R, N, G, T or H; X4 is Y, F, H, S or N; X5 is Y, A, F, V, L, G, P, T or an aliphatic or aromatic amino acid; and X6 is I, M or a hydrophobic amino acid.

[0020] According to another embodiment, the antibody further comprises a CDR-H1 having a contiguous amino acid sequence X1X2X3X4X5X6, wherein: X1 is L, F or an aliphatic amino acid; X2 is Y, S, N, G, D, H, R, F, T, D, P or I; X3 is S, A, F, Y, R, N, G, T, D or H; X4 is Y, S or F; X5 is Y, A, F, V, L, G, P, T, S or an aliphatic or aromatic amino acid; and X6 is I, M or a hydrophobic amino acid.

[0021] According to another embodiment, the antibody further comprises a CDR-H2 having a contiguous amino acid sequence X1X2X3X4X5X6X7X8X9X10, wherein: X1 is S, Y or T; X2 is I or an aliphatic amino acid; X3 is S, Y, A or a polar amino acid; X4 is P or an aliphatic amino acid; X5 is Y, H, F or a polar amino acid; X6 is Y, S, H or a polar amino acid; X7 is S, T, D or G; X8 is Y, F or a polar amino acid; X9 is T, D or an S amino acid; and X10 is Y, S or a polar amino acid.

[0022] According to another embodiment, the antibody further comprises a CDR-H3 having a contiguous amino acid sequence X1X2X3X4X5X6, wherein: X1 is H, R or N; X2 is G, A, V or S; X3 is Y, F or H; X4 is G, S, I, V, A, T or an aliphatic amino acid; X5 is A, G or an aliphatic amino acid; and X6 is M, L or a hydrophobic amino acid.

[0023] According to another embodiment, the antibody further comprises a CDR-H3 having a contiguous amino acid sequence X1X2X3X4X5X6, wherein: X1 is H, N or T; X2 is G, A or S; X3 is Y, F or H; X4 is G, S, A, T or an aliphatic amino acid; X5 is A or an aliphatic amino acid; and X6 is L or a hydrophobic amino acid.

[0024] According to another embodiment, the antibody further comprises a CDR-L1 having a contiguous amino acid sequence X1X2X3X4X5, wherein: X1 is S or D; X2 is V; X3 is S or D; X4 is S; and X5 is A.

[0025] According to another embodiment, the antibody further comprises a CDR-L2 having a contiguous amino acid sequence X1X2X3X4X5X6X7, wherein: X1 is S; X2 is A or D; X3 and X4 are D or S; X5 is L or D; X6 is Y; and X7 is S or D.

[0026] According to another embodiment, the antibody further comprises a CDR-L3 having a contiguous amino acid sequence X1X2X3X4X5X6, wherein: X1 is S; X2 is S; X3 is D, H, N, E, Y or Q; X4 is S or F; X5 is L; and X6 is I or F.

[0027] According to another embodiment, the antibody further comprises a CDR-H1 having a contiguous amino acid sequence X1X2X3X4X5X6, wherein: X1 is L; X2 is R, D, T, Q, K, S, Y, E or H; X3 is S; X4 is Y; X5 is Y; and X6 is M.

[0028] According to another embodiment, the antibody further comprises a CDR-H2 having a contiguous amino acid sequence X1X2X3X4X5X6X7X8X9X10, wherein: X1 is S; X2 is I; X3 is S or A; X4 is P; X5 is Y or H; X6 is Y or H; X7 is S, D or G; X8 is Y; X9 is T, D or S amino acid; and X10 is Y.

[0029] According to another embodiment, the antibody further comprises a CDR-H3 having a contiguous amino acid sequence X1X2X3X4X5X6, wherein: X1 is H; X2 is G; X3 is Y; X4 is G or S; X5 is A; and X6 is L or M.

[0030] According to one embodiment, there is provided an antibody comprising: (a) a CDR-L1 as described herein; (b) a CDR-L2 as described herein; and (c) a CDR-L3 as described herein. According to one embodiment, the antibody of the present invention comprises: (a) a CDR-H1 as described herein; (b) a CDR-H2 as described herein; and (c) a CDR-H3 as described herein.

[0031] According to one embodiment, one aspect of the present invention is a synthetic diabody, the synthetic diabody comprising: (a) a CDR-L1 as described herein; (b) a CDR-L2 as described herein; and (c) a CDR-L3 as described herein. According to yet another embodiment of the present invention, there is provided a synthetic diabody, the synthetic diabody comprising: (a) a CDR-H1 as described herein; (b) a CDR-H2 as described herein; and (c) a CDR-H3 as described herein.

[0032] According to yet another embodiment of the present invention, there is provided a synthetic bispecific antibody, which comprises: (a) CDR-L1 as described herein; (b) CDR-L2 as described herein; (c) CDR-L3 as described herein; (d) CDR-H1 as described herein; (e) CDR-H2 as described herein; and (e) CDR-H3 as described herein.

[0033] According to yet another embodiment of the present invention, there is provided a higher-order molecule, such as a tetravalent body, which molecule comprises: (a) CDR-L1 as described herein; (b) CDR-L2 as described herein; (c) CDR-L3 as described herein; (d) CDR-H1 as described herein; (e) CDR-H2 as described herein; and (e) CDR-H3 as described herein.

[0034] According to yet another embodiment of the present invention, there is provided a bispecific antibody of the present invention having a linker that promotes the formation of bispecific antibodies, which linker is preferably an 18A (±2A) amino acid linker, more preferably a linker having any amino acid combination of 5 to 15 amino acids, still more preferably having the sequences GGGGG (SEQ ID NO:12), IKGGGGGEV (SEQ ID NO:13), LKVLSRGVV (SEQ ID NO:14), ISARAGSLV (SEQ ID NO:15), SKSRAGGEV (SEQ ID NO:16), LKGGRGGKV (SEQID NO:17), NKGGGGAKV (SEQ ID NO:18), IKGSSRDDI (SEQ ID NO:19), MKHAGRGGV (SEQ IDNO:20), SKGGGGGEV (SEQ ID NO:21), MKHAGRGGV (SEQ ID NO:22), LECSDCSGI (SEQ ID NO:23), and more preferably a linker having the sequence GGGGG (SEQ ID NO:12).

[0035] According to one embodiment, an antibody of the present invention comprises CDR-L1, which CDR-L1 comprises the contiguous amino acid sequence SVSSA (SEQ ID NO:3).

[0036] According to one embodiment, an antibody of the present invention comprises CDR-L2, which CDR-L2 comprises the contiguous amino acid sequence SASSLYS (SEQ ID NO:4).

[0037] According to one embodiment, the antibody of the present invention comprises CDR-L3, and the CDR-L3 comprises the continuous amino acid sequence SSYSLI (SEQ ID NO:5). According to one embodiment, the antibody of the present invention comprises CDR-L3, and the CDR-L3 comprises the continuous amino acid sequence AYWPI (SEQ ID NO:6). According to one embodiment, the antibody of the present invention comprises CDR-L3, and the CDR-L3 comprises the continuous amino acid sequence SSYSLF (SEQ ID NO:24). According to one embodiment, the antibody of the present invention comprises CDR-L3, and the CDR-L3 comprises the continuous amino acid sequence SSDSLF (SEQ ID NO:25). According to one embodiment, the antibody of the present invention comprises CDR-L3, and the CDR-L3 comprises the continuous amino acid sequence SSNFLI (SEQ ID NO:30) or the continuous amino acid sequence SSQFLI (SEQ ID NO:36).

[0038] According to one embodiment, the antibody of the present invention comprises CDR-H1, and the CDR-H1 comprises the continuous amino acid sequence LYSYYI (SEQ ID NO:7). According to one embodiment, the antibody of the present invention comprises CDR-H1, and the CDR-H1 comprises the continuous amino acid sequence LSSYYI (SEQ ID NO:8) or the continuous amino acid sequence LESYYM (SEQ ID NO:34) or the continuous amino acid sequence LRSYYM (SEQ ID NO:38) or the continuous amino acid sequence LESYYI (SEQ ID NO:37) or the continuous amino acid sequence LDSYYI (SEQ ID NO:35).

[0039] According to one embodiment, the antibody of the present invention comprises CDR-H2, and the CDR-H2 comprises the continuous amino acid sequence SISPYYSYTY (SEQ ID NO:9). According to one embodiment, the antibody of the present invention comprises CDR-H2, and the CDR-H2 comprises the continuous amino acid sequence SISPHYGYTY (SEQ ID NO:26) or the continuous amino acid sequence SIAPYHGYTY (SEQ ID NO:31).

[0040] According to one embodiment, the antibody of the present invention comprises a CDR-H3, which CDR-H3 comprises the contiguous amino acid sequence HGYGAM (SEQ ID NO:10). According to one embodiment, the antibody of the present invention comprises a CDR-H3, which CDR-H3 comprises the contiguous amino acid sequence HSYAAL (SEQ ID NO:11). According to one embodiment, the antibody of the present invention comprises a CDR-H3, which CDR-H3 comprises the contiguous amino acid sequence HGFGAM (SEQ ID NO:27). According to one embodiment, the antibody of the present invention comprises a CDR-H3, which CDR-H3 comprises the contiguous amino acid sequence HGYSAM (SEQ ID NO:28) or the contiguous amino acid sequence HGYGAL (SEQ ID NO:32).

[0041] According to yet another embodiment of the present invention, the antibody further comprises Figure 6 any one of A to Figure 6 E, or Figure 13 or Figure 16 the CDR-L1, CDR-L2 and CDR-L3 of the antibody of

[0042] According to yet another embodiment, the antibody or antigen-binding portion comprises CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3, wherein CDR-L1 comprises SEQ ID NO:3, CDR-L2 comprises SEQ ID NO:4, CDR-L3 comprises SEQ ID NO:30, CDR-H1 comprises SEQ ID NO:38, CDR-H2 comprises SEQ ID NO:31, and CDR-H3 comprises SEQ ID NO:32. Preferably, the antibody is Ab 19429.

[0043] According to yet another embodiment, the antibody or antigen-binding portion comprises CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2 and CDR-H3, wherein CDR-L1 comprises SEQ ID NO:3, CDR-L2 comprises SEQ ID NO:4, CDR-L3 comprises SEQ ID NO:25, CDR-H1 comprises SEQ ID NO:38, CDR-H2 comprises SEQ ID NO:26, and CDR-H3 comprises SEQ ID NO:28. Preferably, the antibody is Ab 19113.

[0044] According to yet another embodiment, the antibody or antigen-binding portion comprises CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3, wherein CDR-L1 comprises SEQ ID NO:3, CDR-L2 comprises SEQ ID NO:4, CDR-L3 comprises SEQ ID NO:30, CDR-H1 comprises SEQ ID NO:35, CDR-H2 comprises SEQ ID NO:31, and CDR-H3 comprises SEQ ID NO:32. Preferably, the antibody is Ab19429 H1D2.

[0045] According to yet another embodiment, the antibody or antigen-binding portion comprises CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3, wherein CDR-L1 comprises SEQ ID NO:3, CDR-L2 comprises SEQ ID NO:4, CDR-L3 comprises SEQ ID NO:36, CDR-H1 comprises SEQ ID NO:38, CDR-H2 comprises SEQ ID NO:31, and CDR-H3 comprises SEQ ID NO:32. Preferably, the antibody is Ab19429 L3Q3.

[0046] According to yet another embodiment, the antibody or antigen-binding portion comprises CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3, wherein CDR-L1 comprises SEQ ID NO:3, CDR-L2 comprises SEQ ID NO:4, CDR-L3 comprises SEQ ID NO:5, CDR-H1 comprises SEQ ID NO:7, CDR-H2 comprises SEQ ID NO:9, and CDR-H3 comprises SEQ ID NO:10. Preferably, the antibody is Ab 4636.

[0047] According to yet another embodiment, the antibody or antigen-binding portion comprises CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3, wherein CDR-L1 comprises SEQ ID NO:3, CDR-L2 comprises SEQ ID NO:4, CDR-L3 comprises SEQ ID NO:6, CDR-H1 comprises SEQ ID NO:8, CDR-H2 comprises SEQ ID NO:9, and CDR-H3 comprises SEQ ID NO:11. Preferably, the antibody is Ab 4635.

[0048] According to yet another embodiment, the antibody or antigen-binding portion includes CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3, where CDR-L1 includes SEQ ID NO:3, CDR-L2 includes SEQ ID NO:4, CDR-L3 includes SEQ ID NO:5, CDR-H1 includes SEQ ID NO:38, CDR-H2 includes SEQ ID NO:9, and CDR-H3 includes SEQ ID NO:10. Preferably, the antibody is Ab 14949.

[0049] According to yet another embodiment, the antibody or antigen-binding portion includes CDR-L1, CDR-L2, CDR-L3, CDR-H1, CDR-H2, and CDR-H3, where CDR-L1 includes SEQ ID NO:3, CDR-L2 includes SEQ ID NO:4, CDR-L3 includes SEQ ID NO:25, CDR-H1 includes SEQ ID NO:37, CDR-H2 includes SEQ ID NO:26, and CDR-H3 includes SEQ ID NO:28. Preferably, the antibody is Ab 19113H1E2.

[0050] According to yet another embodiment, CDR-L1 is located approximately at residues 28 to 38, CDR-L2 is located approximately at residues 56 to 65; and CDR-L3 is located approximately at residues 107 to 116. According to another embodiment, CDR-H1 is located approximately at residues 30 to 39, CDR-H2 is located approximately at residues 55 to 66; and CDR-H3 is located approximately at residues 107 to 116.

[0051] Another embodiment of the invention relates to an anti-hEPOR antibody that selectively binds to human EPOR and simian EPOR, but not to EPOR from other non-human mammalian species. Such antibodies with high species-specific binding affinity are particularly useful for preclinical studies as well as diagnostic and therapeutic applications.

[0052] Also contemplated are variants of the synthetic antibody that have a higher binding affinity for hEPOR and simian EPOR than for EPOR from other non-human species.

[0053] Various forms of antibodies, diabodies, other binding molecules, and their variants are contemplated herein. For example, the antibody mutant can be a full-length antibody (e.g., having a human immunoglobulin constant region) or an antibody fragment (e.g., Fab or F(ab')2) or a diabody. In addition, the compounds of the invention can be labeled with a detectable label, immobilized on a solid phase, and / or conjugated with a heterologous compound (e.g., a cytotoxic agent).

[0054] The diagnostic and therapeutic uses of the compounds of the present invention are contemplated. In one diagnostic application, the present invention provides a method for determining the presence of a target protein, the method comprising exposing a sample suspected of containing the protein to a compound of the present invention and determining the binding of the compound to the sample. For this use, the present invention may include a kit comprising a compound of the present invention and instructions for using it to detect the protein.

[0055] The present invention also provides a composition comprising a compound of the present invention and a pharmaceutically acceptable carrier or diluent. Such a composition for therapeutic use is sterile and may be lyophilized. The use of the compounds of the present invention in the preparation of a medicament for treating the indications described herein is also contemplated.

[0056] The present invention also provides a method for treating a mammal, the method comprising administering to the mammal an effective amount of an embodiment of the present invention. The mammal to be treated in this method may be a non-human mammal, such as a primate or a rodent (e.g., a mouse or a rat or a rabbit) suitable for collecting preclinical data. The non-human mammal may be healthy (e.g., in a toxicology study) or may be suffering from a disease that requires treatment with the target composition or compound. In one embodiment, the mammal has a disease or is at risk of developing a disease. In a specific embodiment, the disease is kidney disease, cancer-related anemia or treatment-related anemia, and hereditary anemia syndromes. The amount of the composition or compound administered will be a therapeutically effective amount for treating the disease. In a dose escalation study, multiple doses of the compound or composition may be administered to the mammal. In another embodiment, a therapeutically effective amount of the composition or compound is administered to a human patient to treat the patient's disease. In a preferred embodiment, the antibody of the present invention for treating kidney disease, cancer-related anemia or treatment-related anemia, and hereditary anemia is a bispecific antibody. Thus, the compounds of the present invention can be used to manufacture a medicament for treating kidney disease, cancer-related anemia or treatment-related anemia, and hereditary anemia. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The foregoing and other objects, features and advantages of the present invention will become apparent from the following description in conjunction with the accompanying drawings. The patent or patent application file contains at least one color drawing. After request and payment of the necessary fees, the Patent Office may provide a copy of this patent or patent application publication with color drawings.

[0058] Figure 1 A to Figure 1C shows an embodiment of the present invention. A: Natural binding molecule constructs derived from the bispecific antibody phage library described herein, having binding activity against human EPOR and mouse EPOR, and showing activity in the TF-1 proliferation assay. Subsequently, these natural binding molecule constructs were cloned into the VL-VH bispecific antibody Fc form, expressed and purified in mammalian cells. All bispecific antibody-Fc proteins were used for ELISA with immobilized human (black bars) or mouse (white bars) EPOR. In addition, the ability of the bispecific antibody-Fc proteins to induce the proliferation of the human erythroid TF-1 cell line was determined and normalized to EPO (silver bars). B: Schematic diagrams of the bispecific antibody forms used for each construct, showing the variable heavy and light chains, the linker, and the constant heavy chain. C: Natural human EPOR-binding molecule constructs obtained by selection using library F described previously were expressed and purified as IgG, similar to the prior art (References 1, 2). The ability of these IgG (solid lines) to induce the proliferation of TF-1 cells was tested at a series of concentrations and compared to EPO (dashed line).

[0059] Figure 2 A and Figure 2 B shows the binding of the natural bispecific antibody-Fc protein to TF-1 cells using flow cytometry. As a negative control, goat anti-human Alexa488-conjugated secondary antibody alone was used.

[0060] Figure 3 A to Figure 3 E shows the characteristics of a preferred embodiment of the present invention. A: The CDR amino acid sequences of two preferred embodiments 4636 and 4635 according to the IMGT nomenclature. B: Titration ELISA of the binding of the two preferred embodiments to immobilized human EPOR. The EC50 values calculated from the two curves for 4636 and 4635 were 1.5 and 0.56, respectively. C: ELISA was also performed to detect the specificity of 4636 and 4635 for two receptors (EPHB4 and CD131), which have previously been shown to bind to EPO. D: Epitope binding was performed using competitive biolayer interferometry ("BLI"). First, human EPOR was immobilized and then used to capture the indicated saturated (200 nM) D-Fc in solution phase. After washing away the unbound D-Fc, a second competitive D-Fc was added, and the ability of this second competitive D-Fc to bind to EPOR was measured and normalized. E: Competitive phage ELISA was performed by measuring the binding of the indicated phage-D-Fc to immobilized EPOR at the indicated EPO concentrations.

[0061] Figure 4 A and Figure 4Panel B shows the functional activities of the preferred embodiments of the present invention. A: Embodiments (4636 and 4635) of the present invention stimulate the proliferation of the human erythroid UT-7 / EPO cell line (Reference 3). B: Embodiments (4636 and 4635) of the present invention induce phosphorylation of JAK2, STAT3, STAT5, ERK, and AKT, which are downstream effectors of EPOR, in a dose-dependent manner (the dosage of D-Fc is 1 nM, 10 nM, and 100 nM, and the dosage of EPO is 1 IU / mL, 10 IU / mL, and 100 lU / mL).

[0062] Figure 5 A through Figure 5 Panel D shows the 4636 and 4635 heavy and light chain randomization schemes for the affinity mature phage library.

[0063] Figure 6 A through Figure 6 Panel D shows the amino acid CDR sequences of the 4636 ( Figure 6 A and Figure 6 B), 4635 ( Figure 6 C and Figure 6 D) heavy and light chain affinity mature clones and their phage ELISA binding data.

[0064] Figure 7 A through Figure 7 Panel D shows the preferred embodiments of the 4636 affinity mature screening. A: Comparison of the binding ability of each embodiment with the 4636 parental D-Fc protein. B: EC50 values calculated from the curves in Figure 7 A. C: Stimulation of UT7 / EPO cell proliferation by the preferred embodiments compared to the parental 4636. D: EC50 values calculated from the curves in Figure 7 C.

[0065] Figure 8 A through Figure 8Panel D shows the structural features of the preferred embodiment 14949 in Fab form in complex with EPOR. A: Structure (1EER) of an EPO molecule binding to two asymmetric EPORs via site 1 and site 2 to trigger activation. B: The 14949-Fab / EPOR ternary structure was reoriented using Molecule Operating Environment (MOE) software to accommodate the preferred 18 Å diabody linker connecting the VH and VL domains in the most energetically favorable state. The CH1 domain is not shown. The EPO was superimposed to show how site 2 is blocked, but site 1 is not. C: The 14949-Fab / EPOR ternary structure was compared to a ternary structure (1EBA) formed by EPO and a previously identified peptide antagonist of EPOR that induces receptor dimerization but does not trigger activation (reference 4). D: Structure of EPOR in complex with a previously discovered diabody is shown and compared to the EPO-EPOR structure (reference 5).

[0066] Figure 9 A through Figure 9 Panel E shows the paratope of 14949 and the corresponding hEPOR epitope. A: Ribbon diagram of the interaction interface between hEPOR (dark gray) and the heavy (light gray) and light (gray) chains of 14949, with binding residues labeled. B: Expanded view of the paratope shows the interacting residues (dark gray) in the heavy (left panel) and light (right panel) chains. C: Sequences of all CDR regions of 14949 (underlined) and the interacting residues shown in B (gray shading). All amino acid positions are numbered according to IMGT nomenclature. D: Expanded view of the hEPOR epitope shows the interacting residues (gray). E: Sequence alignment comparison of hEPOR with M. fascicularis and M. musculus. Residues at sites 1 and 2 that bind EPO are indicated in bold (*), shaded (*), or both. 14949 binding residues identical to hEPOR are shown in light gray shading, and non-conserved residues are shown in dark gray shading.

[0067] Figure 10 A through Figure 10E compared the binding of molecule 14949 according to the present invention with EPO, prior art ABT-007, prior art bispecific antibodies 305, 310 and 330, and with scFv-Fc-10, -29 and -15. A: In this structure, the hEPOR residues (light gray) interacting with hEPO were compared with the residues of the 14949 epitope (medium gray). Both high affinity (left panel) and low affinity (right panel) are shown. The common residues at both sites are shown in dark gray. B: The prior art ABT-007 epitope was compared with the 14949 epitope, where the specific binding residues are shown in light gray and medium gray, respectively. Dark gray indicates the hEPOR residues that interact with both ABT-007 and 14949 Fab. C: Comparison of the 14949 epitope (medium gray) with the epitopes of three previously identified bispecific antibodies 305 (4Y5V), 310 (4Y5X) and 330 (4Y5Y) (light gray). The residues that interact with 14949 as well as 305, 310 and 330 are shown in dark gray. D: The 14949 epitope was also compared with the epitopes of three prior art scFv-Fc agonist molecules based on mutagenesis screening. Residues that, when mutated to alanine, result in a >50% reduction in scFv-Fc binding to hEPOR also constitute the 14949 epitope and are shown in dark gray. Residues that only affect scFv-Fc binding (>50%) but do not belong to the 14949 epitope are shown in light gray, while the 14949 Fab specific amino acids are shown in medium gray. E: A diagram summarizing all the structural comparisons in A to D.

[0068] Figure 11 A to Figure 11 C shows different forms of a preferred 14949 embodiment and their functional characteristics. A: Schematic diagrams of different forms, including the bispecific antibody-Fc form with VL and VH linked to the CH2 domain and CH3 domain. Also shown are the inter-bispecific antibody and intra-bispecific antibody forms, where the latter has a (GGGGS)3 covalent linker. B: Titration ELISA experiments showing similar binding for different forms. C: Proliferation of UT7 / EPO cells was measured at different doses of protein to determine whether the form affects agonist activity.

[0069] Figure 12 A to Figure 12 D shows a randomization scheme using 14949 as a template. A: CDR-L scheme for sub-library AP229. B: CDR-H scheme for sub-library AP229. C: CDR-L scheme for sub-library AP230. D: CDR-H scheme for sub-library AP230.

[0070] Figure 13Lists the sequences of affinity - matured clones that bind to hEPOR in phage ELISA, whose binding is blocked by the saturated 14949D - Fc protein. The randomized positions in the affinity - matured libraries AP229 and AP230 are shown in gray. The production yields are listed.

[0071] Figure 14 A to Figure 14 C show the characterization of the affinity - matured clones, which have higher yields than the parental 14949 (except for 19432 which has a cryptic N - glycosylation site). A: A UT7 / EPO cell proliferation assay was performed to detect the activity of these clones. EPO and 15033D - Fc were used as positive and negative controls respectively. B: A non - specific ELISA was performed using a panel of representative antigens to detect non - specific binding of high - value clones. C: Affinity measurements were performed using BLI to compare the dissociation constants (KD) of the parental 14949 with 19429 and 19113.

[0072] Figure 15 Shows the results of the UT7 / EPO cell proliferation assay, which measures and compares the agonistic activities of the affinity - matured 19113 and 19429 VL - VH constructs in the bis - antibody - Fc and IgG forms. The activity of IgG1 - form ABT - 007 was also tested in this assay. hEPO and 15033D - Fc were used as positive and negative controls respectively.

[0073] Figure 16 A and Figure 16 B, Figure 16 B' show the analysis of variants at positions that were not previously randomized in the AP229 and AP230 sub - libraries. A: A table showing the mutations tested. B, B': A set of UT7 / EPO cell proliferation assays showing the activities of the variants shown in A. In each experiment, EPO and 15033D - Fc were used as positive and negative controls respectively.

[0074] Figure 17 A and Figure 17 B show the fractionation and analysis of various species based on molecular weight after protein A purification of the 19429 - H1D2 / L3Q3 supernatant. A: A size - exclusion chromatogram showing the peaks eluted from the column. There are three main peaks, labeled 1, 2, and 3. B: Then, the materials in peaks 1, 2, and 3 were used for a UT7 / EPO cell proliferation assay to measure the agonistic activity of each peak. EPO and 15033D - Fc were used as controls.

[0075] Figure 18 A and Figure 18Panel B shows the fractionation and analysis of various substances based on molecular weight after purification of the 19113-H1E2 supernatant by Protein A. A: The size exclusion chromatogram shows the peaks eluted from the column. Trastuzumab was used as a control. 19113-H1E2 had three main peaks, labeled 1, 2, and 3, respectively. B: Then, the materials in peaks 1, 2, and 3 were used for the UT7 / EPO cell proliferation assay to measure the agonist activity of each peak, with EPO and 15033D-Fc used as controls. Detailed Description

[0076] In the present disclosure, a number of terms and abbreviations are used. Definitions of such terms and abbreviations are provided below.

[0077] As used herein, those skilled in the relevant art will generally understand that the term "erythropoietin" or its abbreviation "EPO" refers to the erythropoietin protein, or when used to refer to a nucleic acid, refers to the nucleic acid encoding EPO. As used herein, those skilled in the relevant art will generally understand that the term "erythropoietin receptor" or its abbreviation "EPOR" refers to the EPO receptor, or when used to refer to a nucleic acid, refers to the nucleic acid encoding EPOR.

[0078] As used herein, those skilled in the relevant art will generally understand that the term "comprising" generally means the presence of the recited features, integers, steps, or components in a claim, but does not exclude the presence or addition of one or more other features, integers, steps, components, or combinations thereof.

[0079] As used herein, those skilled in the relevant art will generally understand that the term "treatment" generally refers to a method of obtaining a beneficial or desired result. Beneficial or desired results can include, but are not limited to, preventing or precluding, alleviating or ameliorating one or more symptoms or conditions, reducing the severity of a disease, stabilizing (i.e., not worsening) a disease state, preventing the spread of a disease, delaying or slowing the progression of a disease, improving or alleviating a disease state, and remission (whether partial or complete), whether detectable or not. "Treatment" can also refer to an extension of survival as compared to the expected survival of a subject not receiving treatment.

[0080] As used herein, those skilled in the relevant art will generally understand that the term "therapeutically effective amount" refers to an amount sufficient to effect treatment when administered to a subject in need of treatment. In embodiments of the present invention, a therapeutically effective amount can include, but is not limited to, an amount that eliminates or reduces the disease impact (e.g., tumor burden) in a subject.

[0081] As used herein, those of ordinary skill in the relevant art will generally understand that the term "amino acid sequence" refers to the amino acid sequence of a naturally occurring or non-naturally occurring protein molecule. The terms "amino acid sequence" and similar terms, such as "polypeptide" or "protein", do not mean to limit the amino acid sequence to the complete, native amino acid sequence associated with the protein molecule. An amino acid sequence can be said to have an amino (N) terminus and a carboxyl (C) terminus. Individual amino acids in a peptide or polypeptide can be referred to as "residues", and these residues are numbered sequentially in ascending order from the N-terminus towards the C-terminus. Amino acids that are typically located near the N-terminus are generally referred to as N-terminal amino acids, while amino acids that are typically located near the C-terminus are referred to as C-terminal amino acids. Those of ordinary skill in the relevant art should understand that referring to an amino acid residue as an N-terminal or C-terminal amino acid residue may vary depending on the protein.

[0082] As used herein, those of ordinary skill in the relevant art will generally understand that the term "aliphatic amino acid" can include one of the following amino acids: alanine, glycine, isoleucine, leucine, proline, valine, or methionine; the term "polar amino acid" can include one of the following amino acids: serine, threonine, cysteine, asparagine, glutamine, or tyrosine; and the term "hydrophobic amino acid" can include one of the following amino acids: glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan.

[0083] As used herein, those of ordinary skill in the relevant art will generally understand that the terms "coding nucleic acid molecule", "coding DNA sequence", "coding RNA sequence", "coding mRNA sequence", "oligonucleotide having a nucleotide sequence encoding a gene", "polynucleotide having a nucleotide sequence encoding a gene", "coding DNA", "coding RNA", and similar terms generally refer to the order or sequence of nucleotides along a single-stranded or double-stranded nucleic acid including the coding region of a gene, or in other words, to the nucleic acid sequence encoding a gene product. The order of these nucleotides determines the order of amino acids along the polypeptide chain. The coding region can exist in the form of cDNA, genomic DNA, or RNA. The oligonucleotide or polynucleotide can be single-stranded (e.g., the sense strand) or double-stranded (e.g., the antisense strand and the sense strand). If desired, appropriate control elements (e.g., enhancer / promoter, splice junction, polyadenylation signal, etc.) can be placed in proximity to the gene coding region to enable proper initiation of transcription and / or proper processing of the primary RNA transcript. Additionally, the coding region used in an expression vector can contain endogenous enhancer / promoter, splice junction, intervening sequence, polyadenylation signal, etc., or a combination of endogenous and exogenous control elements.

[0084] Those skilled in the art will understand that nucleic acid molecules are said to have a "5'-end" and a "3'-end" because mononucleotides are joined by phosphodiester bonds to form oligonucleotides or polynucleotides such that the 5'-phosphate of one mononucleotide pentose ring is attached to the 3'-oxygen of its adjacent one in one direction. Thus, for the ends of an oligonucleotide or polynucleotide, if its 5'-phosphate is not joined to the 3'-oxygen of the previous mononucleotide pentose ring, it is called the "5'-end", and if its 3'-oxygen is not joined to the 5'-phosphate of the next mononucleotide pentose ring, it is called the "3'-end". As used herein, even a nucleic acid sequence located within a larger oligonucleotide or polynucleotide can be said to have a 5'-end and a 3'-end. In a linear or circular nucleic acid molecule, discrete elements are said to be located "upstream" or "5'", "downstream" or "3'". Since DNA molecules are usually in a double helix structure, DNA molecules are considered to have a "sense" strand and an "antisense" strand. The sense strand and the antisense strand are said to be reverse complementary because the 3'-end of the sense strand can complementarily bind to the 5'-end of the antisense strand, and the 5'-end of the antisense strand can complementarily bind to the 3'-end of the sense strand. The "sense" strand of a DNA molecule is usually copied into messenger RNA (mRNA) during transcription. Thus, the mRNA produced during transcription has the same sequence as the sense strand through the transcription of the antisense strand, and thus the final protein may be based on the sense version of the DNA molecule. The term "antisense strand" is used to refer to the nucleic acid strand complementary to the "sense" strand. Sometimes the designation (-) (i.e., "negative") is used to refer to the antisense strand, and sometimes the designation (+) (i.e., "positive") is used to refer to the sense strand.

[0085] As used herein, the term "homology" refers to the degree of complementarity. There may be partial homology or complete homology (i.e., identity). When applied to polypeptides, the term "substantially homologous" as used herein means that two peptide sequences, when optimally aligned (e.g., aligned by the programs GAP or BESTFIT using default gap weights), have at least 80% sequence identity, preferably at least 90% sequence identity, more preferably at least 95% sequence identity or more (e.g., 99% sequence identity). Amino acid sequences may differ by conservative amino acid substitutions. Those skilled in the art will understand that the term "conservative amino acid substitution" refers to the general interchangeability of residues having chemically similar side chains. For example, the group of amino acids having aliphatic side chains can include glycine, alanine, valine, leucine, and isoleucine; the group of amino acids having aliphatic hydroxyl side chains can include serine and threonine; the group of amino acids having amide-containing side chains can include asparagine and glutamine; the group of amino acids having aromatic side chains can include phenylalanine, tyrosine, and tryptophan; the group of amino acids having basic side chains can include lysine, arginine, and histidine; the group of amino acids having sulfur-containing side chains can include cysteine and methionine.

[0086] As used herein, the term "fragment" when referring to a single-chain amino acid sequence means a polypeptide that may have deleted the amino (N)-terminal portion and / or the carboxyl (C)-terminal portion compared to the native protein, but the remaining amino acid sequence of the fragment is the same as the amino acid sequence of the native protein. Those skilled in the art will understand that the term "fragment" may also refer to a part of a multi-chain protein molecule (such as an antibody fragment).

[0087] When used with respect to an entity, the terms "naturally occurring" or "native" as used herein mean that the entity can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including a virus) that can be isolated from a natural source and that has not been modified is naturally occurring. Those skilled in the art will understand that the term "synthetic" refers to a compound that is not naturally occurring.

[0088] As used herein, the term "target" refers to a structure to be identified, detected, characterized, or amplified, such as a nucleic acid or protein molecule. Thus, a "target" needs to be found among other structures.

[0089] When the term "isolated" is used with respect to a nucleic acid or a peptide, such as "isolated oligonucleotide", "isolated polynucleotide", or "isolated polypeptide", it means a nucleic acid or amino acid sequence that has been identified and separated from at least one contaminant that is normally associated with it in its natural source. The isolated compound exists in a form or environment different from the form or environment in which it is found in nature. In contrast, a non-isolated compound (such as a nucleic acid or amino acid sequence) exists in the state in which it exists in nature. For example, a given DNA sequence (such as a gene) exists on a host cell chromosome adjacent to neighboring genes; an RNA sequence (e.g., a particular mRNA sequence encoding a specific protein) is mixed in a cell with many other mRNAs encoding multiple proteins.

[0090] As used herein, the term "portion" when referring to a nucleotide sequence or an amino acid sequence means a fragment of that sequence.

[0091] As used herein, the term "purified" or "purification" means the removal of contaminants from a sample. For example, EPOR agonists are purified by removing contaminating non-immunoglobulins; they are also purified by removing immunoglobulins that do not bind to EPOR. The removal of non-immunoglobulins and / or the removal of immunoglobulins that do not bind to EPOR results in an increase in the percentage of EPOR agonists in the sample.

[0092] The terms "recombinant protein" or "recombinant polypeptide" as used herein refer to a protein molecule expressed from a recombinant DNA molecule.

[0093] A variety of techniques known in the art are used to detect antibody binding relevant to the present invention. These techniques include, but are not limited to, RIA (radioimmunoassay), ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoradiometric assay, gel diffusion precipitation reaction, immunodiffusion assay, SEC (size exclusion chromatography), BLI (biolayer interferometry), in situ immunoassay (e.g., using colloidal gold, enzyme, or radioisotope labeling), Western blot, precipitation reaction, agglutination assay (e.g., gel agglutination assay, hemagglutination assay, etc.), complement fixation assay, immunofluorescence assay, protein A assay, and immunoelectrophoresis assay, etc.

[0094] As used herein, the term "Western blot" refers to the analysis of proteins (or polypeptides) immobilized on a support such as nitrocellulose or a membrane. The proteins are placed on an acrylamide gel to separate the proteins, and then the proteins are transferred from the gel to a solid support, such as nitrocellulose or nylon membrane. Then the immobilized proteins are exposed to an antibody reactive with the target antigen. The binding of the antibody can be detected by various methods, including using radiolabeled antibodies, enzyme-linked antibodies, etc.

[0095] The term "epitope" as used herein refers to the part of an antigen that contacts an antibody (i.e., the antigenic determinant). When a protein or protein fragment is used to immunize a host animal, many regions of the protein may induce the production of antibodies that specifically bind to a particular region or three-dimensional structure on the protein; these regions or structures are called epitopes. Epitopes may compete with the intact antigen (i.e., the "immunogen" used to initiate the immune response) for binding to the antibody. Those skilled in the art should understand that a "paratope", also known as an antigen-binding site, is the part of an antibody that recognizes and binds to an antigenic epitope. Each paratope consists of six complementarity-determining regions - three from each of the light and heavy chains. The ends of each arm of the Y-shaped antibody have the same paratope.

[0096] The term "conformational epitope" refers to an epitope in which discontinuous amino acids are clustered together in a three-dimensional conformation. In a conformational epitope, the interaction points occur between amino acid residues on proteins that are separated from each other. In one embodiment, the epitope is the epitope described in the examples of this specification.

[0097] The term "sample" as used herein has the broadest meaning. Samples suspected of containing nucleic acid or amino acid sequences may include cells, chromosomes isolated from cells (e.g., spreads of metaphase chromosomes), genomic DNA (in solution or bound to a solid support), RNA (in solution or bound to a solid support), cDNA (in solution or bound to a solid support), etc. Samples suspected of containing proteins may include cells, a part of a tissue, an extract containing one or more proteins, etc.

[0098] As used herein, the term "response" refers to the generation of a detectable signal (e.g., accumulation of a reporter molecule, increase in ion concentration, accumulation of a detectable chemical product (e.g., an antibody)) when used for a determination or other outcome.

[0099] As used herein, the terms "agonist" and "agonistic" refer to or describe a molecule that, when bound to a receptor, is capable of directly or indirectly initiating, activating, stimulating, or inducing one or more aspects of a response (e.g., a physiological response) or other biological activity. In a preferred embodiment, the compounds of the invention (e.g., bispecific antibodies) can include agonists of EPO, as they mimic one or more properties of EPO. As used herein, the terms "antagonist" and "antagonistic" refer to or describe a molecule that, when bound to a receptor, is capable of directly or indirectly counteracting or blocking one or more aspects of a physiological response or other biological activity. An antagonist is the opposite of an agonist. In another preferred embodiment of the invention, the compounds of the invention (e.g., full antibodies) can include EPO antagonists, as they inhibit the action of EPO.

[0100] As used herein, the term "antibody" or "Ab" is used in the broadest sense and specifically encompasses a single anti - EPOR monoclonal antibody (including agonists, antagonists, and neutralizing or blocking antibodies) and anti - EPOR antibody compositions having multi - epitope specificity. The "antibodies" used herein include intact immunoglobulin or antibody molecules, polyclonal antibodies, multispecific antibodies (i.e., bispecific antibodies formed from at least two intact antibodies), immunoglobulin or antibody fragments (e.g., Fab, F(ab')2 or Fv), and synthetic bispecific antibodies, provided that they exhibit any of the desired agonistic or antagonistic properties described herein. A variety of procedures known in the art can be used to generate such antibodies against a particular antigen or against its derivatives, fragments, analogs, homologs, or orthologs.

[0101] Antibodies are generally proteins or polypeptides that exhibit binding specificity for a particular antigen. Natural antibodies are generally heterotetrameric glycoproteins composed of two identical light chains (L) and two identical heavy chains (H). Typically, each light chain is linked to a heavy chain by a covalent disulfide bond, and the number of disulfide bonds between the heavy chains varies among different immunoglobulin isotypes. Each heavy and light chain also has evenly spaced intrachain disulfide bonds. One end of each heavy chain has a variable domain (VH), followed by a number of constant domains. One end of each light chain has a variable domain (VL) and the other end has a constant domain; the constant domain of the light chain aligns with the first constant domain of the heavy chain, and the variable domain of the light chain aligns with the variable domain of the heavy chain. Specific amino acid residues are thought to form the interface between the variable domains of the light and heavy chains. Based on the amino acid sequence of the constant domain, the antibody light chains of any vertebrate species can be grouped into one of two distinct types, called kappa and lambda. Immunoglobulins can be classified into different classes based on the amino acid sequence of the heavy chain constant domain. There are five main classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), such as IgG-1, IgG-2, IgG-3, and IgG-4; IgA-1 and IgA-2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu.

[0102] As used herein, "antibody fragment" includes a portion of a full antibody, generally the antigen-binding or variable region of a full antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, diabodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments.

[0103] The term "diabody" refers to a small antibody fragment having two antigen-binding sites, wherein each antigen-binding site comprises a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH and VL). In a diabody, each VL domain is linked to one VH domain in a single-chain Fv (scFv) fragment by a short linker. By using a short linker (e.g., six to fifteen amino acids or 18 Å in a preferred embodiment of the invention) to pair the two domains on the same chain, the domains are forced to pair with the complementary domain on the other chain and generate two antigen-binding sites. Diabodies are described more fully, for example, in EP 404,097, WO 93 / 11161, and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0104] As used herein, the term "linker" refers to a linking sequence that promotes bispecific antibody formation, preferably an 18A (±2A) amino acid linker, more preferably a linker having any amino acid combination of 5 to 15 amino acids, more preferably having the sequences GGGGG, IKGGGGGEV, LKVLSRGVV, ISARAGSLV, SKSRAGGEV, LKGGRGGKV, NKGGGGAKV, IKGSSRDDI, MKHAGRGGV, SKGGGGGGEV, MKHAGRGGV, LECSDCSGI, and even more preferably having the sequence GGGGG.

[0105] As used herein, the term "variable domain" describes certain portions of an antibody that are different in sequence between antibodies and are used for the binding and specificity of each particular antibody to its particular antigen. However, this variability is not usually evenly distributed throughout the variable domain of the antibody. It is typically concentrated in three segments within the variable domains of the light and heavy chains, called complementarity-determining regions ("CDRs"), "hypervariable regions", or "hypervariable domains". The more conserved portions of the variable domain are called framework regions ("FRs"). The variable domains of the native heavy and light chains each comprise four FR regions, mostly adopting a β-sheet structure, connected by three CDRs that form loops that, in some cases, form part of the β-sheet structure. The CDRs in each chain are held tightly together by the FR regions and, together with the CDRs in the other chain, form the antigen-binding site of the antibody. The constant domains do not directly participate in the binding of the antibody to the antigen but exhibit various effector functions, such as the antibody participating in antibody-dependent cell cytotoxicity.

[0106] As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies comprising the population are identical except for possible minor natural variations. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, compared to conventional (polyclonal) antibody preparations, which typically comprise different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen.

[0107] The modifier "monoclonal" indicates that the property of the antibody is obtained from a substantially homogeneous population of antibodies and should not be construed as requiring that the antibody be produced by any particular method.

[0108] Those skilled in the art will understand that modifications to the antibodies of the present invention are contemplated herein. The antibodies of the present invention can be modified by conjugation, tagging, or labeling by methods known in the art, and the antibodies of the present invention can be conjugated, tagged, or modified with any known diagnostic or therapeutic agent, including but not limited to cytotoxic agents (such as immunotoxin conjugates), prodrugs, drugs (such as pharmaceutically active substances), or other effector molecules effective in the treatment of diseases, as well as known reporter molecules. Such modified antibodies, also referred to as their immunochemical derivatives, include but are not limited to: (a) labeled (e.g., radiolabeled, enzyme-labeled, fluorescent dye, or chemiluminescent compound) monoclonal antibodies of the present invention, preferably humanized mAbs, for diagnosing or detecting tumors and tumor spread (e.g., metastasis) using known imaging techniques; and (b) immunotoxin conjugates of the mAbs of the present invention, preferably humanized mAbs, wherein the mAbs of the present invention are conjugated with known cytotoxic, radioactive, radiolabeled, prodrug, or drug moieties (e.g., radioimmunotherapy). Those skilled in the art will understand that the terms "cytotoxic agent," "cytotoxin," or "cytotoxicity" as used herein generally refer to substances that inhibit or prevent cell function and / or cause cell destruction, including but not limited to radioisotopes, chemotherapeutic agents, and toxins, such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant, or animal origin, including fragments and / or variants thereof. Those skilled in the art will also understand that the term "prodrug" as used in this application generally refers to a precursor or derivative form of a pharmaceutically active substance that is less cytotoxic to target cells than the pharmaceutically active substance and is capable of being activated or converted into a more pharmaceutically active substance.

[0109] Those skilled in the art will appreciate that the antibodies of the present invention can also be prepared by recombinant DNA methods, such as the method described in U.S. Pat. No. 4,816,567, which is hereby incorporated by reference. The DNA encoding the monoclonal antibodies of the present invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to the genes encoding the murine heavy and light antibody chains). Once isolated, the DNA can be placed into an expression vector and then transfected into a host cell, such as simian COS cells, Chinese hamster ovary (CHO) cells or myeloma cells that do not produce immunoglobulins, to synthesize the monoclonal antibodies in the recombinant host cells. The DNA can also be modified, for example, by replacing the homologous murine sequences with the coding sequences of the human heavy and light chain constant domains (U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)) or by covalently linking all or part of the immunoglobulin coding sequences to the coding sequences of non-immunoglobulin polypeptides. Such non-immunoglobulin polypeptides can replace the constant domains of the antibodies of the present invention, or can replace the variable domains of one of the antigen-binding sites of the antibodies of the present invention to produce chimeric bivalent antibodies.

[0110] Those skilled in the art will understand that "phage display" refers to a technique for generating and selecting new proteins that bind to ligands (such as antigens). Using phage display technology, large libraries of protein variants can be generated and sequences that bind to a target antigen with high affinity can be rapidly screened out. Methods for generating peptide libraries and screening these libraries have been disclosed in many patents (such as U.S. Pat. No. 5,723,286, U.S. Pat. No. 5,432,018, U.S. Pat. No. 5,580,717, U.S. Pat. No. 5,427,908, and U.S. Pat. No. 5,498,530). It should also be understood that antibody phage display libraries can also be created. (Smith et al., Science (1985), 228:1315; Skerra and Pluckthun, Science (1988), 240:1038). Libraries of antibodies or antigen-binding polypeptides have been prepared in a variety of ways, including by inserting random DNA sequences or by altering individual genes by cloning related gene families. Methods for using phage display to display antibodies or antigen-binding fragments have been described in U.S. Pat. Nos. 5,750,373, 5,733,743, 5,837,242, 5,969,108, 6,172,197, 5,580,717, and 5,658,727. Antibody phage display libraries are then screened to express antibodies or antigen-binding proteins with desired properties. Phage display technology has several advantages over traditional hybridoma and recombinant methods for preparing antibodies with desired properties. This technology can develop large antibody libraries with diverse sequences in a shorter time without the use of animals. The preparation of hybridomas or humanized antibodies can easily take several months. In addition, since immunization is not required, phage antibody libraries can be generated against toxic or poorly antigenic antigens (Hogenboom, Immunotechniques (1988), 4:1 - 20). Commercially available, for example, developed by Cambridge Antibody Technology and Morphosys (Vaughan et al. (1996) Nature Biotech 14:309; Knappik et al. (1999) J. Mol. Biol. 296:57).

[0111] Generating diverse libraries of antibodies or antigen-binding proteins is important for isolating high-affinity antibodies. Libraries with limited CDR diversification have been generated using a variety of methods. See, e.g., Tomlinson, Nature Biotech. (2000), 18:989-994. The CDR3 regions have received attention in part because they are often found to be involved in antigen binding. The CDR3 regions on the heavy chain vary widely in size, sequence, and structural conformation. Others have also generated diversity by randomizing the CDR regions of the variable heavy and light chains using all 20 amino acids at each position. Using all 20 amino acids is thought to result in a large number of variant antibody sequences, thus increasing the chance of identifying novel antibodies (Barbas, PNAS 91:3809 (1994); Yelton, D.E., J. Immunology, 155:1994 (1995); Jackson, J.R., J. Immunology, 154:3310 (1995) and Hawkins, R.E., J. Mol. Biology, 226:889 (1992)).

[0112] Once a binding molecule with the desired binding and functional activity has been identified, a preferred embodiment of the invention involves generating binding mutants, preferably by introducing one or more amino acid alterations (e.g., substitutions) in one or more hypervariable or CDR regions of the binding molecule. One method is to use phage display for affinity maturation (Hawkins et al. J. Mol. Biol. 254:889-896 (1992) and Lowman et al. Biochemistry 30(45):10832-10837 (1991)). In this technique, many hypervariable sites are mutated to generate possible amino acid substitutions at each site. The resulting antibody mutants are displayed and screened for their biological activity (e.g., binding affinity). Those skilled in the art will understand that this process is commonly referred to as affinity maturation.

[0113] Those skilled in the art should understand that the compositions of the present invention (including but not limited to synthetic antibodies) can be formulated into pharmaceutical compositions for administration in a conventional manner using standard pharmaceutical formulation chemical components and methods, all of which are readily available to those skilled in the art. Those skilled in the art should also understand that such pharmaceutical compositions can include one or more excipients, carriers, stabilizers, or other pharmaceutically acceptable inactive compounds, such as but not limited to wetting agents or emulsifiers, pH buffering substances, etc. Pharmaceutically acceptable salts can also be included therein. For a detailed discussion of pharmaceutically acceptable excipients, carriers, and auxiliary substances, see Remington's Pharmaceutical Sciences (Mack Pub. Co. N.J. 1991), which is incorporated herein by reference. Such pharmaceutical compositions can be made into injectable or oral formulations. The antibodies of the present invention can be administered by injection, including but not limited to intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, transdermal injection, or nasal injection. The therapeutically effective dose may vary depending on body weight, and the administration time and duration will be determined by a specific clinical study protocol.

[0114] The following description and the embodiments described therein are provided by way of illustration of one or more examples of specific embodiments of the principles and aspects of the present invention. These examples are provided to explain these principles and the present invention, and not to limit these principles and the present invention. Throughout the specification and the drawings, the same components are labeled with the same reference numerals.

[0115] The present invention relates to diagnosing and treating critical diseases or cancer-related anemia by selective activation or signal transduction of EPOR, without having an adverse effect on the recurrence of diseases (such as cancer, kidney disease) and patient survival. More preferably, the embodiments of the present invention are directed to specific synthetic binding molecules, including antibodies, diabodies, etc., as treatments for diseases (including cancer-related anemia). More preferably, the present invention relates to a method of activating the EPOR activation site by binding to an agonist of the present invention. The present invention can be applied alone or in combination with other therapeutic reagents to a variety of disease states, including but not limited to cancer-related anemia and kidney disease.

[0116] The two antigen-binding sites of the synthetic agonist (such as an antibody or diabody) dimerize EPOR, which is crucial for its phosphorylation and activation of the downstream signaling pathways leading to erythropoiesis. The EPOR agonist exhibits excellent erythropoiesis-promoting function in vitro and has a persistent activity in vivo. These EPOR agonists may provide a new treatment option for patients with chronic kidney disease or treatment-related anemia.

[0117] Examples

[0118] Generation, Preparation, and Characterization of EPOR-Binding Molecules

[0119] To select EPOR agonists, a diabody library was constructed by modifying the phagemid vector encoding the human framework scFv6. In a preferred embodiment, the C-terminus of the light chain variable (VL) domain can be linked to the N-terminus of the heavy chain variable (VH) domain via a linker (preferably a Gly5 linker (e.g., GGGGG)), resulting in a diabody linked to the M13 gene-3 minor coat protein via a modified IgG hinge sequence. In a preferred embodiment, four of the six CDRs (three heavy chain CDRs and CDR-L3) were diversified as previously described for library F1, although the length diversity of CDR-H3 was reduced. The library contained 4.2×10 9 unique clones and was used for selection as described previously.

[0120] Using the diabody-phage library described herein, binding selection was performed on the recombinant extracellular domain ("ECD") of human EPOR ("hEPOR-ECD"). Phage antibodies that specifically bound the antigen (e.g., ECD) by ELISA but not the control protein were sequenced by PCR amplification, and unique sequence clones were identified. Sequencing of individual binding clones identified 14 unique clones that bound to human EPOR and murine EPOR in vitro as purified diabody-Fc (fragment crystallizable region) proteins (see Figure 1 A and Figure 1 B). Attachment of the Fc region was also thought to extend the half-life of the diabody in serum. Flow cytometry analysis using human erythroleukemia TF-1 cells demonstrated that these 14 D-Fcs also bound to cell surface-expressed EPOR ( Figure 2 A and Figure 2 B). However, among the 14 D-Fcs, only 7 (D-Fc-1 to D-Fc-7) stimulated the proliferation of TF-1 cells, as expected for EPOR activation (see Figure 1 A). Interestingly, all D-Fcs with significant TF-1 agonist activity (>40% growth) generally had little cross-reactivity with murine EPOR (see Figure 1 A). In addition, we also tested the agonist properties of several native IgG clones screened from library F, which were obtained as described above using human EPOR as the immobilized target. None of these IgGs had any significant effect on cell proliferation ( Figure 1C). This is in stark contrast to the previously described studies of EPOR IgG agonists and clearly demonstrates the novelty and non-obviousness of the current embodiments. Two of the most potent clones (D-Fc-1 and D-Fc-2) were selected for further characterization and are hereinafter referred to as 4636 and 4635, respectively (e.g., see Figure 3 A). These two D-Fc constructs exhibit significant homology in their heavy chain CDR sequences, suggesting that they may bind to EPOR in a similar manner.

[0121] To characterize the mechanism of action of 4636 and 4635, titration ELISA (enzyme-linked immunosorbent assay) was performed. The binding to immobilized hEPOR was plotted against the concentration of the bispecific antibody, and the EC50 values of 4636 and 4635 were calculated from this plot to be 1.5 nM and 0.56 nM, respectively (see Figure 3 B). Single-concentration ELISA was also used to detect the specificity of 4636 and 4635. Two receptors previously shown to bind EPO are EPHB4 and CD131. However, neither of the bispecific antibody-Fcs showed any significant binding to these receptors (see Figure 3 C). Then, an epitope sorting assay was performed using competitive biolayer interferometry (“BLI”) to determine whether 4636 and 4635 bind to a common epitope (see Figure 3 D). Briefly, EPOR was immobilized and used to capture the indicated bispecific antibody-Fc in solution phase at a saturating concentration (pre-loaded tip). After washing away the excess bispecific antibody-Fc, control D-Fc, 4636, or 4635 was re-loaded into the solution phase. If the epitope on EPOR was blocked by the pre-loaded saturating D-Fc, no D-Fc binding would occur in the second round. 4636 and 4635 were observed to block each other, indicating that these two bispecific antibody-Fcs share a similar epitope (see Figure 3 D). Competitive ELISA was used to determine whether this epitope also overlaps with that of EPO. As the concentration of EPO increased, the binding of both 4636 and 4635 to immobilized EPOR was blocked (see Figure 3 E).

[0122] Functional Activity of EPOR-Binding Molecules

[0123] Activation of EPOR stimulates the proliferation of the human erythroid cell line. Therefore, the ability of 4636 and 4635 to stimulate the proliferation of the erythroid UT7 / EPO cell line was tested. A bioluminescence-based cell proliferation assay showed that both 4636 and 4635 are potent inducers of UT7 / EPO cell growth. In fact, they are both more effective than EPO itself (see Figure 4A). Then, Western blotting was used to detect the biochemical effects of 4636 and 4635 in UT7 cells. The functional activity of erythropoiesis-stimulating agent requires EPOR dimerization, which initiates a series of protein phosphorylation events, including phosphorylation of JAK2, STAT3 / 5, ERK, and AKT. These modifications in response to 4636 and 4635 were monitored in UT-7 / EPO cells (see Figure 4 B). The UT-7 / EPO cell line is an EPO-dependent human erythroid cell line that endogenously expresses hEPOR. Different from TF-1 cells, it has normal EPOR expression. Both 4636 and 4635 induced phosphorylation of EPOR downstream effectors, very similar to EPO (see Figure 4 B). EPO induced phosphorylation of typical targets JAK2, STAT3, STAT5, ERK, and AKT in a dose-dependent manner. Importantly, both 4636 and 4635 induced phosphorylation of all 5 signaling molecules in a manner very similar to EPO.

[0124] Affinity Maturation of EPOR-Binding Molecules

[0125] According to the strategy described herein, a phage antibody display affinity maturation library was developed to design other embodiments of the present invention. Four libraries were made - one heavy chain (HC) library and one light chain (LC) library each for 4636 and 4635. The exact positions within the complementarity-determining regions (CDRs) and the randomization scheme are as shown in Figure 5 A to Figure 5 D. After 5 rounds of selection against immobilized hEPOR-ECD, sequencing of the binding phage clones revealed unique sequences of the affinity maturation clones ( Figure 6 A to Figure 6 D). Phage ELISA was performed to confirm target binding compared to an immunized Fc control (see Figure 6 A to Figure 6 D). Plates were coated with EPOR-His or Fc, then phage clones expressing each diabody were added to the wells and incubated for an additional hour at room temperature. Samples were washed and then incubated with an anti-M13 secondary antibody. Color development was performed using a TMB substrate. OD450 was measured using a plate reader. All tested phage clones showed significant and specific binding to EPOR-His. Figure 6 A to Figure 6D schematically shows the frequency and distribution of amino acids at random positions. This analysis indicates that LC positions either have a strong preference for the parental amino acids (CDR-L3) or are under little selective pressure due to the random distribution of amino acids (CDR-L1 and CDR-L2). Similarly, HC positions also tend to have a strong preference for the parental amino acids, except for position 39 in CDR-H1, which favors the substitution of isoleucine by methionine.

[0126] Based on the amino acid distribution, 7 clones were selected from the HC selection for further screening to maintain the parental LC positions. All 7 clones from the HC selection had methionine at position 39 of CDR-H1 and different amino acids at position 30. These 7 clones were cloned and purified as proteins in the D-Fc form. Titration ELISA found that in a preferred embodiment, the affinity-matured derived clone 14949D-Fc (see Figure 6 A) had an EC50 very similar to 4636 ( Figure 7 A and Figure 7 B). Cell proliferation assays using UT7 / EPO cells also found that clone 14949 was more effective (higher maximum effect) than the parental 4636 and EPO, and was approximately 5-fold more potent (lower EC50) ( Figure 7 C and 7D). Overall, the functional data indicate that 4636, 4635, and their affinity-matured derived clones bind to hEPOR with high affinity but do not bind to other receptors that interact with EPO, and furthermore, they compete with each other and with EPO for receptor binding. A 4636 affinity-matured derived clone or the affinity-matured clone 14949 exhibits better or desired functional properties.

[0127] EPOR Conformation in the Affinity-Maturation-Derived Clone 14949 Fab Complex

[0128] To explore the structural basis for the binding of the embodiments of the present invention to EPOR, a construct (identified as the affinity-matured derived clone 14949 of the preferred embodiment of the present invention) was cloned, expressed, and purified as Fab and crystallized with the hEPOR-ECD complex ( Figure 8 A and Figure 8 B). Human EPO binds to its cognate receptor through two sites - a high-affinity site 1 on one receptor and a low-affinity site 2 on an adjacent receptor. The binding of these sites induces the formation of an asymmetric dimer signal complex ( Figure 8 A). Based on this, to understand how 14949 activates EPOR signaling, the 14949-Fab / EPOR structure was refined to accommodate the 18A linker in the lowest energy state. Then EPO was superimposed to reveal how the high-affinity site 1 remains accessible when site 2 is blocked.Figure 8 B). This explains the early data indicating that EPO competes for binding with the current embodiments of the present invention (e.g., see Figure 3 E).

[0129] As Figure 8 shown in C, the model of the preferred embodiment was compared with the known EPOR structure. This improved model is very similar to the EPO - EPOR model and the asymmetric dimer formed using an antagonistic peptide (Reference 4). Thus, dimerization itself may not explain the agonistic properties of the preferred embodiment 14949, suggesting a separate and distinct mechanism of action.

[0130] As Figure 8 shown in D, the asymmetric dimer formed by 14949 is different from the previously developed bispecific antibodies that activate EPOR. All previous bispecific antibodies induce EPOR dimerization in a manner different from EPO. The structural similarity of the modeled ternary 14949 - EPOR complex is unique compared to the prior art.

[0131] 14949 Fab Complementary Determining Regions and hEPOR Epitopes

[0132] Careful examination of the 14949Fab - EPOR complex can reveal the precise residues that constitute the complementarity - determining regions in the Fab and the hEPOR epitope ( Figure 9 A). In the complementarity - determining regions, both the VL domain and the VH domain make multiple contacts ( Figure 9 B and Figure 9 C). These positions are all within the CDRs. In the VL domain, the CDR positions of these interactions for 14949 are: Ala38 in CDR - L1; Tyr55 and Ser56 in CDR - L2; and Ser07, Ser108, and Ser114 in CDR - L3. In the VH domain, the interacting residues include: Ser36, Tyr37, and Tyr38 in CDR - H1; Pro58, Tyr61, Tyr62, Tyr64, and Tyr66 in CDR - H2; and Arg106, His107, Gly108, Tyr109, and Gly113 in CDR - H3.

[0133] The hEPOR residues that make up the epitope ( Figure 9 D and Figure 9 E) include Gln82, Glu84, Asp85, Glu86, Pro87, Trp88, Leu90, Pro119, Glu121, Arg123, Thr125, Ser128, Gly129, Pro131, His134, and Val136. As shown previously ( Figure 3 E and Figure 8B), the site 2 position including Leu83 to Lys89 overlaps with the 14949Fab binding. In addition, except for two binding residues (Gln82 and Val136), the other residues in M. fascicularis EPOR are the same. In contrast, several non-conservative substitutions were found in mouse EPOR, namely at positions 85, 87, 88, 90 and 123. This may explain the lack of binding of the current embodiment to mouse EPOR ( Figure 1 A).

[0134] Comparison of the Epitopes Recognized by 14949 Fab with the Prior Art

[0135] By directly comparing the epitope recognized by 14949Fab with all prior art starting from the native binding site of hEPO (Reference 7)( Figure 10 A), the novelty and non-obviousness of the present invention can be best highlighted. hEPO binds to its homologous receptor through high-affinity and low-affinity binding sites. Two residues (Glu84 and Asp85) in the high-affinity site 1 are also the residues that bind to 14949Fab. Glu86 is involved in the binding of site 1 and site 2 and also interacts with 14949Fab. Compared with hEPO, the vast majority of the 14949Fab epitope is unique, which emphasizes that its activation mechanism is non-natural.

[0136] Next, the 14949Fab binding epitope was compared with the previously identified antibody hEPOR agonist ABT-007 (Reference 2)( Figure 10 B). The overlap between these two interfaces is larger than that of the hEPO interface( Figure 10 A). Eleven residues on hEPOR are involved in the binding to 14949Fab and ABT-007. Only 3 residues specifically bind to 14949Fab and do not bind to ABT-007 - Ser87, Pro87 and Leu90( Figure 10 B, medium gray residues). However, the mechanism of ABT-007 targeted binding is significantly different from that of 14949Fab, because its epitope is much larger than that of 14949Fab. This is not fully shown in this structure, but is summarized in Figure 10 E.

[0137] Previous studies have identified three bispecific antibodies with moderate agonist activity - 305 (4Y5V), 310 (4Y5X) and 330 (4Y5Y) (Reference 5). Therefore, these epitopes were compared with the epitope of 14949Fab( Figure 10 C). Among the 22 residues of the bispecific antibody - 305 epitope, only 3 residues are the same as those of 14949, namely Glu84, Asp85 and Glu86(Figure 10 C, left panel). For diabody-310, 19 residues form this epitope, but only 2 (Glu84 and Asp85) are identical to those in 14949Fab ( Figure 10 C, middle panel). Similarly, only 3 out of 14 epitope residues in diabody-330 are also present in the 14949Fab epitope ( Figure 10 C, right panel). The differences in 14949Fab binding compared to these past diabodies are not surprising since these past diabodies were all first derived from the IgG format, while 14949 was designed from a diabody library. There is little similarity between the potential molecular mechanism of 14949-mediated EPOR activation and the way these diabodies function.

[0138] Comparison of the prior art scFv-Fc proteins with the present invention further reveals the uniqueness of this embodiment (Reference 8) ( Figure 10 D). Three scFv-Fc's were identified as having high agonistic activity towards hEPOR - scFv-Fc-10, -29, and -15. Although the structures of these molecules complexed with hEPOR have never been resolved, mutagenesis studies have been used to map the interface. The epitope was defined as any hEPOR residue that, when mutated to alanine, results in a >50% reduction in binding (Reference 8). These positions were compared to the 14949Fab epitope ( Figure 9 D and Figure 9 E). In most cases, the interacting residues of all 3 scFv-Fc proteins are different from those of 14949Fab. In fact, scFv-Fc-10, -29, and -15 have only 3, 1, and 2 overlapping contact residues, respectively ( Figure 10 D and Figure 10 E).

[0139] Overall, Figure 10 E summarizes the comparison of 14949Fab with all prior art. It shows that the VL and VH domains of 14949 contact its hEPOR target in a very unique way that has never been described before. It is non-natural as it is not observed in hEPO either. Thus, the mechanism of action of the current embodiment is novel and non-obvious.

[0140] Characterization of Different Bispecific Antibody Formats

[0141] Additional experiments demonstrate how the embodiments of the present invention function with different modes of expression (e.g., as Figure 11as shown in A). Each form of EPOR agonist has its unique characteristics. For example, bispecific antibody-Fc has an extended serum half-life due to the attachment of the Fc fragment; the intermolecular bispecific antibody (or diabody) is significantly smaller and more similar in size to the natural ligand EPO; while the intramolecular bispecific antibody with a (GGGGS)3 linker enables the formation of a bivalent dimer within one fragment. The Fc format has been tested previously, and the latter format's binding to human EPOR and induction of UT7 / EPO cell proliferation were subsequently tested by titration ELISA ( Figure 11 B and Figure 11 C). Whether the embodiments of the present invention are expressed as bispecific antibody-Fc, between bispecific antibodies, or within covalently linked bispecific antibodies, they all have similar target binding and agonistic effects.

[0142] Additional Affinity Maturation of 14949

[0143] Following the strategy described herein, a phage antibody display affinity maturation library was developed to design additional embodiments of 14949. Two sublibraries - AP229 and AP230 - were established. Each sublibrary consisted of an HC library and an LC library. The exact positions within the complementarity-determining regions (CDRs) and the randomization scheme are as shown in Figure 12 A to Figure 12 D. After 5 rounds of selection against immobilized hEPOR-ECD, the binding phage clones were used in phage ELISA, where the antigen was first pre-saturated with 100 nM 14949D-Fc protein. After washing 5 times with 1X PBS to remove the excess 14949D-Fc protein, the phage ELISA procedure was continued, using an anti-M13 secondary antibody and a TMB substrate for color development. Figure 13 The phage clone sequences with significantly reduced binding to 14949 are shown, and these sequences were subsequently cloned and expressed as bispecific antibody-Fc fusion proteins. The transient production yields (mg / L) using HEK293expi cells are also shown ( Figure 13 ). The proteins with higher yields were selected for further experiments. Despite the high yields, 19432 could not be further developed due to the introduction of an N-glycosylation site in CDR-H2.

[0144] Characterization of 14949 Family Clones

[0145] The high-yield clones shown in Figure 13 were subjected to functional analysis. Two molecules (19113 and 19429) performed better than the parental 14949 bispecific antibody-Fc, with higher maximum proliferation responses in UT7 / EPO cells ( Figure 14 A). Non-specific ELISA was performed using a representative set of antigens ( Figure 14B). All subclones except 19434 showed excellent specificity and very low binding to these unrelated antigens. In particular, 19113 and 19429 had the best specificity, with even negligible binding to KLH ( Figure 14 B). Trastuzumab was used as a control to determine the performance of the clinical-grade molecules in this assay. 15033 was the negative control and 6606 was the highly cross-reactive control with high background binding to multiple antigens. Then the binding affinities of 19113 and 19429 to hEPOR were determined using BLI. Figure 14 C). Compared with the parental 14949, the bispecific antibody-Fc proteins of 19113 and 19429 bound with similar dissociation constants, indicating that their improved hEPOR activation might be related to receptor geometry rather than binding strength. This is consistent with recent findings that agonism is not always related to affinity (Reference 9).

[0146] The Format Is Critical for the Agonistic Effect of the Lead Molecule

[0147] The lead molecules 19113 and 19429 bispecific antibody-Fc molecules were cloned and expressed as IgG1 proteins and functionally tested in the UT7 / EPO cell proliferation assay. Figure 15 ). Different from the 19429 D-Fc form, as IgG1, neither 19113 nor 19429 had much agonistic activity. The previously identified ABT-007 agonistic IgG also did not show much functional agonism. This experiment clearly demonstrated that the engineered VL / VH domains cannot function like classical antibodies and provided evidence for the claim that the current implementation is a novel agonist acting through a non-obvious mechanism of action.

[0148] Other Variants of 19429

[0149] The affinity maturation randomization scheme left several invariant positions that might affect the agonistic function. Based on our structural analysis, these positions generally do not have binding affinity. For example, Arg35 in CDR-H1 of 14949 was never mutated. Figure 13 ). These individual positions were mutated to hydrophilic residues and then the activity was tested in the UT7 / EPO cell proliferation assay. Figure 16 A and Figure 16 B). Overall, the hydrophilic substitutions at these positions were well tolerated. The only exception was the change in CDR-L3 of 19429, where two variants (L3DSxF and L3DS) showed significantly reduced hEPOR activity in this assay.

[0150] Testing the Activity of Higher-Order Species of 19113 and 19429

[0151] The 19429-H1 D2 / L3Q3 double variant was used to perform a more detailed analysis of the activity profiles of various higher-order substances present in the protein A purification material ( Figure 17 A and Figure 17 B). The mixture was separated by size-exclusion chromatography (SEC) and consisted of three peaks, most likely corresponding to three substances with different molecular weights ( Figure 17 A). Using the UT7 / EPO cell proliferation assay, peak 3 was the major substance, most likely a bispecific antibody-Fc monomer, but had the lowest activity ( Figure 17 B). Peaks 1 and 2 corresponded to the molecular weights of the dimer and tetramer, respectively, and had higher activities. These data indicate that valency is a key determinant for activating biological functions.

[0152] The 19113-H1E2 variant was also tested. SEC identified three different substances with different molecular weights, corresponding to the tetramer (peak 1), dimer (peak 2), and monomer (peak 3), respectively ( Figure 18 A). Functional analysis using the UT7 / EPO cell proliferation assay showed that the monomeric substance had the lowest activity ( Figure 18 B). These results indicate that the design of agonists must consider the valency of the target and will affect process development.

[0153] In summary, the functional and structural features of the present invention highlight a new mechanism for activating EPOR and stimulating erythropoiesis. This mechanism involves inducing an asymmetric receptor geometry, which, although very similar to the penultimate form of the native EPO ligand, is still unique because it involves a discontinuous, non-natural, and non-obvious receptor binding mechanism that has not been found in biology or all previous technologies.

[0154] References

[0155] 1. Persson, H., Ye, W., Wernimont, A., Adams, J. J., Koide, A., Koide, S., Lam, R. & Sidhu, S. S. CDR-H3 diversity is not required for antigen recognition by synthetic antibodies. J Mol Biol 425, 803-11 (2013).

[0156] 2. Liu, Z., Stoll, V. S., Devries, P. J., Jakob, C. G., Xie, N., Simmer, R. L., Lacy, S. E., Egan, D. A., Harlan, J. E., Lesniewski, R. R. & Reilly, E. B. A potent erythropoietin-mimicking human antibody interacts through a novel binding site. Blood 110, 2408-13 (2007).

[0157] 3. Goncalves, F., Lacout, C., Feger, F., Cohen-Solal, K., Guichard, J., Cramer, E., Vainchenker, W. & Dumenil, D. Inhibition of erythroid differentiation and induction of megakaryocytic differentiation by thrombopoietin are regulated by two different mechanisms in TPO-dependent UT-7 / c-mpl and TF-1 / c-mpl cell lines. Leukemia 12, 1355-66 (1998).

[0158] 4. Livnah, O., Stura, E. A., Johnson, D. L., Middleton, S. A., Mulcahy, L. S., Wrighton, N. C., Dower, W. J., Jolliffe, L. K. & Wilson, I. A. Functional mimicry of a protein hormone by a peptide agonist: the EPO receptor complex at 2.8 A. Science 273, 464-71 (1996).

[0159] 5. Moraga, I., Wernig, G., Wilmes, S., Gryshkova, V., Richter, C. P., Hong, W. J., Sinha, R., Guo, F., Fabionar, H., Wehrman, T. S., Krutzik, P., Demharter, S., Pio, I., Weissman, I. L., Minary, P., Majeti, R., Constantinescu, S. N., Piehler, J. & Garcia, K. C. Tuning Cytokine Receptor Signaling by Re-orienting Dimer Geometry with Surrogate Ligands. Cell 160, 1196 - 208 (2015).

[0160] 6. Nelson, B. & Sidhu, S. S. Synthetic antibody libraries. Methods Mol Biol 899, 27 - 41 (2012).

[0161] 7. Syed, R., Reid, S., Li, C. et al. Efficiency of signalling through cytokine receptors depends critically on receptor orientation. Nature 395, 511 - 516 (1998).

[0162] 8. Lim, A. R., Ketchem, R. R., Borges, L. et al. Diversity of Antibody Epitopes Can Induce Signaling through the Erythropoietin Receptor. Biochemistry 49, 18, 3797 - 3804 (2010). 9. Yu, X., Orr, C. M., Chan, H. T. C. et al. Reducing affinity as a strategy to boost immunomodulatory antibody agonism. Nature 614, 539 - 547 (2023).

Claims

1. A selective activation region of human erythropoietin receptor (hEPOR), said selective activation region providing selective activation of the erythropoietic-specific effect of hEPOR, said selective activation region comprising a first epitope, said first epitope comprising residues 82, 84 to 88 and 90 of hEPOR, wherein, The numbering refers to SEQ ID NO:

29.

2. The selective activation region according to claim 1, further comprising a second epitope, said second epitope comprising residues 119, 121, 123, 125, 128, 129, 131, 134 and 136 of hEPOR.

3. The selective activation region according to claim 1, comprising residues 82, 84 to 88, 90, 119, 121, 123, 125, 128, 129, 131, 134 and 136 of hEPOR.

4. The selective activation region according to claim 2, wherein, The first epitope comprises QEDEPWL (SEQ ID NO: 1).

5. The selective activation region according to claim 3, wherein, The second epitope comprises PERTSGPHV (SEQ ID NO: 2).

6. An antibody or an antigen-binding portion thereof that binds to the selective activation region of human erythropoietin receptor (hEPOR), said selective activation region providing selective activation of the erythropoietic-specific effect of hEPOR, said selective activation region being the selective activation region according to any one of claims 1 to 5.

7. An antibody or an antigen-binding portion thereof that binds to human erythropoietin receptor (hEPOR), said antibody or antigen-binding portion comprising a CDR-L1 region having a continuous amino acid sequence X1X2X3X4X5, wherein: X1 is S, D or T; X2 is V or an aliphatic amino acid; X3 and X4 are D, E, G, H, K, N, Q, R or S; and X5 is A or an aliphatic amino acid.

8. The antibody according to claim 7, wherein, In the CDR-L1 region: X1 is S or D; X2 is V; X3 is S or D; X4 is S; and X5 is A.

9. The antibody or antigen-binding portion according to claim 7, further comprising a CDR-L2 having a continuous amino acid sequence X1X2X3X4X5X6X7, wherein: X1 is S or T; X2 is A, D or an aliphatic amino acid; X3 and X4 are D, E, G, H, K, N, Q, R or S; X5 is L, D or an aliphatic amino acid; X6 is Y or a polar amino acid; and X7 is S, D or T.

10. The antibody according to claim 9, wherein, In the CDR-L2 region: X1 is S; X2 is A or D; X3 and X4 are D or S; X5 is L or D; X6 is Y; and X7 is S or D.

11. The antibody or antigen-binding portion thereof according to claim 7, further comprising a CDR-L3 region having a continuous amino acid sequence X1X2X3X4X5X6, wherein: X1 is S, F, T, A, I or P; X2 is S, P, T, C, A, F, Y, G, R or D; X3 is Y, R, P, S, D, R, H, F, N, I, G, P, E, Q or T; X4 is S, F, A, G, V, Y, P, T or N; X5 is L, P or an aliphatic amino acid; and X6 is I, F or a hydrophobic amino acid.

12. The antibody according to claim 11, wherein, In the CDR-L3 region: X1 is S; X2 is S; X3 is D, H, N, E, Y or Q; X4 is S or F; X5 is L; and X6 is I or F.

13. The antibody or antigen-binding portion according to claim 7, further comprising a CDR-H1 region having a continuous amino acid sequence X1X2X3X4X5X6, wherein: X1 is L, F or an aliphatic amino acid; X2 is Y, S, N, G, D, H, R, F, T, D, P, Q, K or I; X3 is S, A, F, Y, R, N, G, T, D or H; X4 is Y, F, H, S, E or N; X5 is Y, A, F, V, L, G, P, T, S or an aliphatic or aromatic amino acid; and X6 is I, M or a hydrophobic amino acid.

14. The antibody according to claim 13, wherein, In the CDR-H1 region: X1 is L; X2 is R, D, T, Q, K, S, Y or H; X3 is S; X4 is Y; X5 is Y; and X6 is M.

15. The antibody or antigen-binding portion according to claim 7, further comprising a CDR-H2 region having a continuous amino acid sequence X1X2X3X4X5X6X7X8X9X10, wherein: X1 is S, Y or T; X2 is I or an aliphatic amino acid; X3 is S, Y, A or a polar amino acid; X4 is P or an aliphatic amino acid; X5 is Y, H, F or a polar amino acid; X6 is Y, S, H or a polar amino acid; X7 is S, T, D or G; X8 is Y, F or a polar amino acid; X9 is T, D or S amino acid; and X10 is Y, S or a polar amino acid.

16. The antibody according to claim 15, wherein, In the CDR-H2 region: X1 is S; X2 is I; X3 is S or A; X4 is P; X5 is Y or H; X6 is Y or H; X7 is S, D or G; X8 is Y; X9 is T, D or S amino acid; and X10 is Y.

17. The antibody or its antigen-binding portion according to claim 7, further comprising a CDR-H3 region having a continuous amino acid sequence X1X2X3X4X5X6, wherein: X1 is H, R or N; X2 is G, A, V or S; X3 is Y, F or H; X4 is G, S, I, V, A, T or an aliphatic amino acid; X5 is A, G or an aliphatic amino acid; and X6 is M, L or a hydrophobic amino acid.

18. The antibody according to claim 17, wherein, In the CDR-H2 region: X1 is H; X2 is G; X3 is Y; X4 is G or S; X5 is A; and X6 is L or M.

19. The antibody or antigen-binding portion according to claim 7, wherein, The CDR-L1 comprises the contiguous amino acid sequence SVSSA (SEQ ID NO:3).

20. The antibody or antigen-binding portion according to claim 9, wherein, The CDR-L2 comprises the contiguous amino acid sequence SASSLYS (SEQ ID NO:4).

21. The antibody or antigen-binding portion according to claim 11, wherein, The CDR-L3 comprises the contiguous amino acid sequence SSYSLI (SEQ ID NO:5), or the contiguous amino acid sequence AYWPI (SEQ ID NO:6), or the contiguous amino acid sequence SSYSLF (SEQ ID NO:24), or the contiguous amino acid sequence SSDSLF (SEQ ID NO:25), or the contiguous amino acid sequence SSNFLI (SEQ ID NO:30), or the contiguous amino acid sequence SSQFLI (SEQ ID NO:36).

22. The antibody or antigen-binding portion according to claim 13, wherein, The CDR-H1 comprises the contiguous amino acid sequence LYSYYI (SEQ ID NO:7), or the contiguous amino acid sequence LRSYYM (SEQ ID NO:38) or the contiguous amino acid sequence LSSYYI (SEQ ID NO:8), or the contiguous amino acid sequence LESYYI (SEQ ID NO:37), or the contiguous amino acid sequence LDSYYI (SEQ ID NO:35), or the contiguous amino acid sequence LESYYM (SEQ ID NO:34).

23. The antibody or antigen-binding portion according to claim 15, wherein, The CDR-H2 comprises the contiguous amino acid sequence SISPYYSYTY (SEQ ID NO:9), or the contiguous amino acid sequence SISPHYGYTY (SEQ ID NO:26), or the contiguous amino acid sequence SIAPYHGYTY (SEQ ID NO:31).

24. The antibody or antigen-binding portion according to claim 17, wherein, The CDR-H3 comprises the contiguous amino acid sequence HGYGAM (SEQ ID NO:10), or the contiguous amino acid sequence HSYAAL (SEQ ID NO:11), or the contiguous amino acid sequence HGFGAM (SEQ ID NO:27), or the contiguous amino acid sequence HGYSAM (SEQ ID NO:28), or the contiguous amino acid sequence HGYGAL (SEQ ID NO:32).

25. The antibody or antigen-binding portion according to any one of claims 6 to 24, wherein, CDR-L1 includes SEQ ID NO:3, CDR-L2 includes SEQ ID NO:4, CDR-L3 includes SEQ ID NO:30, CDR-H1 includes SEQ ID NO:38, CDR-H2 includes SEQ ID NO:31, CDR-H3 includes SEQ ID NO:

32.

26. The antibody or antigen-binding portion according to any one of claims 6 to 24, wherein, CDR-L1 includes SEQ ID NO:3, CDR-L2 includes SEQ ID NO:4, CDR-L3 includes SEQ ID NO:25, CDR-H1 includes SEQ ID NO:38, CDR-H2 includes SEQ ID NO:26, CDR-H3 includes SEQ ID NO:

28.

27. The antibody or antigen-binding portion according to any one of claims 6 to 24, wherein, CDR-L1 comprises SEQ ID NO:3, CDR-L2 comprises SEQ ID NO:4, CDR-L3 comprises SEQ ID NO:30, CDR-H1 comprises SEQ ID NO:35, CDR-H2 comprises SEQ ID NO:31, and CDR-H3 comprises SEQ ID NO:

32.

28. The antibody or antigen-binding portion according to any one of claims 6 to 24, wherein, CDR-L1 comprises SEQ ID NO:3, CDR-L2 comprises SEQ ID NO:4, CDR-L3 comprises SEQ ID NO:36, CDR-H1 comprises SEQ ID NO:38, CDR-H2 comprises SEQ ID NO:31, and CDR-H3 comprises SEQ ID NO:

32.

29. The antibody or antigen-binding portion according to any one of claims 6 to 24, wherein, CDR-L1 comprises SEQ ID NO:3, CDR-L2 comprises SEQ ID NO:4, CDR-L3 comprises SEQ ID NO:5, CDR-H1 comprises SEQ ID NO:7, CDR-H2 comprises SEQ ID NO:9, and CDR-H3 comprises SEQ ID NO:

10.

30. The antibody or antigen-binding portion according to any one of claims 6 to 24, wherein, CDR-L1 comprises SEQ ID NO:3, CDR-L2 comprises SEQ ID NO:4, CDR-L3 comprises SEQ ID NO:6, CDR-H1 comprises SEQ ID NO:8, CDR-H2 comprises SEQ ID NO:9, and CDR-H3 comprises SEQ ID NO:

11.

31. The antibody or antigen-binding portion according to any one of claims 6 to 24, wherein, CDR-L1 comprises SEQ ID NO:3, CDR-L2 comprises SEQ ID NO:4, CDR-L3 comprises SEQ ID NO:5, CDR-H1 comprises SEQ ID NO:38, CDR-H2 comprises SEQ ID NO:9, and CDR-H3 comprises SEQ ID NO:

10.

32. An antibody or antigen-binding portion according to any one of claims 6 to 24, wherein, CDR-L1 comprises SEQ ID NO:3, CDR-L2 comprises SEQ ID NO:4, CDR-L3 comprises SEQ ID NO:25, CDR-H1 comprises SEQ ID NO:37, CDR-H2 comprises SEQ ID NO:26, and CDR-H3 comprises SEQ ID NO:

28.

33. An antibody or antigen-binding portion according to any one of claims 6 to 32, wherein, The antibody or antigen-binding portion is a diabody, Fab, F(ab)2, tetravalent body or multivalent body.

34. An antibody or antigen-binding portion thereof according to any one of claims 6 to 33, which is used in a method for treating kidney disease or anemia.

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