Synthetic antibody agonists of the erythropoietin receptor

Abstract

The present invention relates to the use of erythropoietin receptor (EPOR) binding sites and EPOR agonists and antagonists in the diagnosis and treatment of diseases, particularly anemia resulting from renal disease and cancer treatment, or genetic syndromes. More specifically, the present invention relates to synthetic EPOR antibody constructs, particularly diabodies.

Description

[Technical Field]

[0001] The present invention relates to erythropoietin receptor (EPOR) binding sites and EPOR agonists and antagonists for the diagnosis and treatment of diseases, particularly anemia caused by kidney disease and cancer treatment, or genetic syndromes. More specifically, the present invention relates to synthetic EPOR antibody constructs, particularly diabodies. These EPOR agonists offer new treatment options for patients with chronic kidney disease, treatment-related anemia, and hereditary EPO deficiency. [Background technology]

[0002] background Human erythropoietin (EPO) is a growth factor that promotes red blood cell production. It binds to the human EPO receptor (hEPOR) on erythroid progenitor cells and activates the intracellular signaling cascade involved in red blood cell production. EPO is naturally produced by peritubular cells in the kidney in response to hypoxia. Patients with chronic kidney disease (CKD) often have reduced EPO production, resulting in anemia due to reduced red blood cells. Therefore, frequent administration of recombinant EPO or other erythropoiesis-stimulating agents (ESAs) is required to maintain normal red blood cell production. The most common ESAs are epoetin alfa and darbepoetin alfa, administered 2–3 times weekly and every 1–2 weeks, respectively.

[0003] Erythropoiesis begins with the differentiation of hematopoietic stem and progenitor cells (HSPCs) into the erythroid lineage. Once HSPCs differentiate into erythroid progenitors, they begin to express EPOR and become responsive to circulating EPO. EPO promotes further proliferation and ultimately maturation into differentiated erythrocytes. hEPO binds to hEPOR and induces a conformational change in the receptor via high-affinity binding site (site 1) and low-affinity binding site (site 2), activating downstream signaling cascades. In the absence of ligand, the inactive hEPOR is thought to be expressed on the cell surface as a preformed dimer. hEPO binding proceeds via asymmetric binding, first via the interaction of high-affinity hEPO site 1 and then the low-affinity hEPO site 2. This stimulates reorientation of the hEPOR monomer within the dimeric complex, resulting in activation of the JAK2 receptor-associated kinase.

[0004] In addition to CKD, anemia is a common complication affecting approximately 40% of cancer patients and 90% of those undergoing chemotherapy. Recombinant hEPO is an effective treatment for cancer-associated anemia; however, concerns about disease recurrence and adverse effects on patient survival limit its use in many oncology patients. hEPO exerts its harmful effects in cancer patients, at least in part, through non-erythroid signaling pathways, such as the ephrin B4 receptor (EPHB4) and the IL-3R common β subunit (CD131). hEPO may also promote cancer growth by increasing oxygen delivery to hypoxic regions within tumors. The pleiotropic effects of EPO highlight the need and potential for the development of erythropoiesis-specific therapies that stimulate EPO, as well as methods to selectively inhibit cancer-promoting EPO signaling mechanisms.

[0005] Agonists and antagonists of EPOR that can solve the above problems and be useful in the diagnosis and treatment of diseases, particularly anemia associated with cancer and other diseases, have yet to be discovered. Summary of the Invention

[0006] The present invention provides novel epitopes on hEPOR that provide selective activation of the erythropoiesis-specific activity of hEPOR without side effects such as activation of other potentially tumorigenic cell surface receptors, such as EPHB4 and CD131.

[0007] The present invention also provides antibodies and polypeptide sequences that can selectively bind to human EPOR with similarly high affinity, but with little affinity to mouse EPOR (see, e.g., Figures 1A, 2, and 9), which 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 or treatment-related anemia, and inherited anemia.

[0009] One aspect of the present invention relates to highly selective hePOR agonists, which in preferred embodiments include synthetic binding molecules, such as antibodies, diabodies, and higher order molecules such as tetravalent antibodies. In a more preferred embodiment of the present invention, synthetic diabodies are provided that act as hePOR agonists.

[0010] One aspect of the present invention relates to highly selective hePOR antagonists, which in preferred embodiments include synthetic binding molecules such as antibodies. In a more preferred embodiment of the present invention, synthetic antibodies that act as hePOR antagonists are provided.

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

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

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

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

[0015] According to another embodiment, the antibody further comprises a CDR-L1 having the consecutive 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 the consecutive 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 an L, D, or aliphatic amino acid; and 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 the consecutive 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 an L, P, or 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 the consecutive 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 an L, P, or 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 the consecutive amino acid sequence X1X2X3X4X5X6, wherein: X1 is an L or 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 the consecutive 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 the consecutive 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 a T, D, or 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 the consecutive 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; and 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 the consecutive 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; and 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 the consecutive 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 the consecutive 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 the consecutive 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 the consecutive 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 the consecutive 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 a T, D, or S amino acid; and X10 is Y.

[0029] According to another embodiment, the antibody further comprises a CDR-H3 having the consecutive 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] In one embodiment, an antibody is provided that comprises (a) a CDR-L1 described herein; (b) a CDR-L2 described herein; and (c) a CDR-L3 described herein. In one embodiment, the antibody of the present invention comprises (a) a CDR-H1 described herein; (b) a CDR-H2 described herein; and (c) a CDR-H3 described herein.

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

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

[0033] According to yet another embodiment of the present invention, there is provided a higher order, such as tetravalent, molecule comprising: (a) a CDR-L1 as described herein; (b) a CDR-L2 as described herein; (c) a CDR-L3 as described herein; (d) a CDR-H1 as described herein; (e) a CDR-H2 as described herein; and (e) a CDR-H3 as described herein. According to yet another embodiment of the present invention, the diabodies of the present invention comprise a linker that facilitates diabody formation, preferably an 18A (±2A) amino acid linker, more preferably a linker having any combination of amino acids between 5 and 15 amino acids, even more preferably comprising 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 (SEQ ID NO: 17), NKGGGGAKV (SEQ ID NO: 18), IKGSSRDDI (SEQ ID NO: 19), MKHAGRGGV (SEQ ID NO: 20), SKGGGGGEV (SEQ ID NO: 21), MKHAGRGGV (SEQ ID NO: 22), LECSDCSGI (SEQ ID NO: 23), and even more preferably a linker having the sequence GGGGG (SEQ ID NO: 12).

[0034] In one embodiment, an antibody of the invention comprises a CDR-L1 comprising the contiguous amino acid sequence SVSSA (SEQ ID NO: 3).

[0035] In one embodiment, an antibody of the invention comprises a CDR-L2 comprising the consecutive amino acid sequence SASSLYS (SEQ ID NO: 4).

[0036] In one embodiment, an antibody of the present invention comprises a CDR-L3 comprising the consecutive amino acid sequence SSYSLI (SEQ ID NO: 5). In one embodiment, an antibody of the present invention comprises a CDR-L3 comprising the consecutive amino acid sequence AYWPI (SEQ ID NO: 6). According to one embodiment, an antibody of the present invention comprises a CDR-L3 comprising the consecutive amino acid sequence SSYSLF (SEQ ID NO: 24). According to one embodiment, an antibody of the present invention comprises a CDR-L3 comprising the consecutive amino acid sequence SSDSLF (SEQ ID NO: 25). According to one embodiment, an antibody of the present invention comprises a CDR-L3 comprising the consecutive amino acid sequence SSNFLI (SEQ ID NO: 30) or the consecutive amino acid sequence SSQFLI (SEQ ID NO: 36).

[0037] According to one embodiment, an antibody of the invention comprises a CDR-H1 comprising the consecutive amino acid sequence LYSYYI (SEQ ID NO: 7). According to one embodiment, an antibody of the invention comprises a CDR-H1 comprising the consecutive amino acid sequence LSSYYI (SEQ ID NO: 8), LESYYM (SEQ ID NO: 34), LRSYYM (SEQ ID NO: 38), LESYYI (SEQ ID NO: 37), or LDSYYI (SEQ ID NO: 35).

[0038] According to one embodiment, an antibody of the invention comprises a CDR-H2 comprising the consecutive amino acid sequence SISPYYSYTY (SEQ ID NO: 9). According to one embodiment, an antibody of the invention comprises a CDR-H2 comprising the consecutive amino acid sequence SISPHYGYTY (SEQ ID NO: 26) or the consecutive amino acid sequence SIAPYHGYTY (SEQ ID NO: 31).

[0039] In one embodiment, an antibody of the present invention comprises a CDR-H3 comprising the consecutive amino acid sequence HGYGAM (SEQ ID NO: 10). In one embodiment, an antibody of the present invention comprises a CDR-H3 comprising the consecutive amino acid sequence HSYAAL (SEQ ID NO: 11). In one embodiment, an antibody of the present invention comprises a CDR-H3 comprising the consecutive amino acid sequence HGFGAM (SEQ ID NO: 27). In one embodiment, an antibody of the present invention comprises a CDR-H3 comprising the consecutive amino acid sequence HGYSAM (SEQ ID NO: 28) or the consecutive amino acid sequence HGYGAL (SEQ ID NO: 32).

[0040] In yet another embodiment of the invention, the antibody further comprises CDR-L1, CDR-L2, and CDR-L3 of the antibody of any one of Figures 6A-6E, or Figure 13, or Figure 16.

[0041] According to yet another embodiment, the antibody or antigen-binding portion comprises CDR-L1 comprising SEQ ID NO: 3, CDR-L2 comprising SEQ ID NO: 4, CDR-L3 comprising SEQ ID NO: 30, CDR-H1 comprising SEQ ID NO: 38, CDR-H2 comprising SEQ ID NO: 31, and CDR-H3 comprising SEQ ID NO: 32, preferably wherein the antibody is Ab19429.

[0042] According to yet another embodiment, the antibody or antigen-binding portion comprises CDR-L1 comprising SEQ ID NO: 3, CDR-L2 comprising SEQ ID NO: 4, CDR-L3 comprising SEQ ID NO: 25, CDR-H1 comprising SEQ ID NO: 38, CDR-H2 comprising SEQ ID NO: 26, and CDR-H3 comprising SEQ ID NO: 27. Preferably, the antibody comprising SEQ ID NO: 28 is Ab19113.

[0043] According to yet another embodiment, the antibody or antigen-binding portion comprises CDR-L1 comprising SEQ ID NO:3, CDR-L2 comprising SEQ ID NO:4, CDR-L3 comprising SEQ ID NO:30, CDR-H1 comprising SEQ ID NO:35, CDR-H2 comprising SEQ ID NO:31, and CDR-H3 comprising SEQ ID NO:32, preferably said antibody is Ab19429 H1 D2.

[0044] According to yet another embodiment, the antibody or antigen-binding portion comprises CDR-L1 comprising SEQ ID NO:3, CDR-L2 comprising SEQ ID NO:4, CDR-L3 comprising SEQ ID NO:36, CDR-H1 comprising SEQ ID NO:38, CDR-H2 comprising SEQ ID NO:31, and CDR-H3 comprising SEQ ID NO:32, preferably the antibody is Ab19429 L3Q3. According to yet another embodiment, the antibody or antigen-binding portion comprises CDR-L1 comprising SEQ ID NO:3, CDR-L2 comprising SEQ ID NO:4, CDR-L3 comprising SEQ ID NO:5, CDR-H1 comprising SEQ ID NO:7, CDR-H2 comprising SEQ ID NO:9, and CDR-H3 comprising SEQ ID NO:10, preferably the antibody is Ab4636.

[0045] According to yet another embodiment, the antibody or antigen-binding portion comprises CDR-L1 comprising SEQ ID NO: 3, CDR-L2 comprising SEQ ID NO: 4, CDR-L3 comprising SEQ ID NO: 6, CDR-H1 comprising SEQ ID NO: 8, CDR-H2 comprising SEQ ID NO: 9, and CDR-H3 comprising SEQ ID NO: 11. Preferably, the antibody is Ab4635.

[0046] According to yet another embodiment, the antibody or antigen-binding portion comprises CDR-L1 comprising SEQ ID NO: 3, CDR-L2 comprising SEQ ID NO: 4, CDR-L3 comprising SEQ ID NO: 5, CDR-H1 comprising SEQ ID NO: 38, CDR-H2 comprising SEQ ID NO: 9, and CDR-H3 comprising SEQ ID NO: 10, preferably wherein the antibody is Ab14949.

[0047] According to yet another embodiment, the antibody or antigen-binding portion comprises CDR-L1 comprising SEQ ID NO: 3, CDR-L2 comprising SEQ ID NO: 4, CDR-L3 comprising SEQ ID NO: 25, CDR-H1 comprising SEQ ID NO: 37, CDR-H2 comprising SEQ ID NO: 26, and CDR-H3 comprising SEQ ID NO: 28, preferably said antibody is Ab19113 H1 E2.

[0048] According to yet another embodiment, CDR-L1 is located at about residues 28-38, CDR-L2 is located at about residues 56-65, and CDR-L3 is located at about residues 107-116. According to another embodiment, CDR-H1 is located at about residues 30-39, CDR-H2 is located at about residues 55-66, and CDR-H3 is located at about residues 107-116.

[0049] A further embodiment of the present invention relates to anti-hEPOR antibodies that selectively bind to human and monkey EPORs but not to EPORs from other non-human mammalian species. Such antibodies with high species-specific binding affinity are particularly useful in preclinical studies and diagnostic and therapeutic applications.

[0050] Also contemplated are synthetic antibody variants that have improved binding affinity for human and monkey EPORs compared to EPORs of other non-human species.

[0051] The present invention contemplates various forms of antibodies, diabodies, and other binding molecules, as well as variants thereof. For example, antibody variants may be full-length antibodies (e.g., those having a human immunoglobulin constant region), or antibody fragments (e.g., Fab or F(ab')2) or diabodies. Furthermore, the compounds of the present invention may be labeled with a detectable label, immobilized on a solid phase, and / or conjugated to a heterologous compound (e.g., a cytotoxic agent).

[0052] Diagnostic and therapeutic uses of the compounds of the present invention are contemplated. As one diagnostic use, the present invention provides a method for determining the presence of a protein of interest. The method involves exposing a sample suspected of containing the protein of interest to a compound of the present invention and determining binding of the compound to the sample. For this use, the present invention may include a kit containing a compound of the present invention and instructions for use in detecting the protein.

[0053] The present invention also provides a composition comprising a compound of the present invention and a pharmaceutically acceptable carrier or diluent. The therapeutic composition is sterile and can be lyophilized. The use of the compound of the present invention in the manufacture of a medicament for treating the indications described herein is also contemplated.

[0054] The present invention further provides a method of treating a mammal, comprising administering to the mammal an effective amount of an embodiment of the present invention. The mammal treated in this method may be a non-human mammal, such as a primate suitable for collecting preclinical data, or a rodent (e.g., a mouse, rat, or rabbit). The non-human mammal may be healthy (e.g., in a toxicity study) or may be suffering from a disease that is being treated with the subject composition or compound. In one embodiment, the mammal is suffering from or at risk of developing a disease. In a specific embodiment, the disease is renal disease, cancer-related or treatment-related anemia, and inherited anemia syndrome. The amount of the composition or compound administered is a therapeutically effective amount for treating the disease. In a dose-escalation study, various doses of the compound or composition can 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 useful for treating renal disease, cancer-related or treatment-related anemia, and inherited anemia is a diabody. Thus, the compounds of the present invention can be used in the manufacture of a medicament for the treatment of kidney disease, cancer-related or treatment-related anemia, and hereditary anemia. [Brief explanation of the drawings]

[0055] These and other objects, features, and advantages of the present invention will become apparent from the following description taken in conjunction with the accompanying drawings. This patent or patent application document contains at least one color drawing. Copies of this patent or patent application publication with color drawing(s) will be provided by the Patent and Trademark Office upon request and payment of the necessary fee.

[0056] [Figure 1]Figures 1A-1C illustrate embodiments of the present invention. A. Naive binding molecule constructs derived from the diabody phage library described herein exhibit binding activity to human EPOR and mouse EPOR and demonstrate activity in a TF-1 proliferation assay. Naive binding molecule constructs were cloned in a VL-VH diabody-Fc format, expressed in mammalian cells, and purified. ELISAs using immobilized human EPOR (black bars) or mouse EPOR (white bars) were performed with all diabody-Fc proteins. The ability of the diabody-Fc proteins to induce proliferation of the human erythroid TF-1 cell line was also measured, normalized to EPO (silver bars). B. Schematic diagram showing the diabody format used for each construct, showing the variable heavy and light chains, binding, and constant heavy chains. C. Naive human EPOR binding molecule constructs obtained from selections using Library F as described above were expressed and purified as IgG as previously described (References 1 and 2). These IgGs (solid lines) were tested for their ability to induce TF-1 cell proliferation in comparison with EPO (dotted line) at various concentrations.

[0057] [Figure 2-1] Figures 2A and 2B show the binding of the native diabody-Fc protein to TF-1 cells using flow cytometry. As a negative control, goat anti-human Alexa488-labeled secondary antibody alone was used. [Figure 2-2] Same as above.

[0058] [Figure 3]Figures 3A-3E show the characteristics of preferred embodiments of the present invention. A. CDR amino acid sequences of two preferred embodiments (4636 and 4635) according to the IMGT nomenclature. B. Titration ELISA of the two preferred embodiments binding to immobilized human EPOR. EC50 values ​​calculated from the two curves are 1.5 and 0.56 for 4636 and 4635, respectively. C. ELISA was also performed to examine the specificity of 4636 and 4635 for two receptors previously shown to bind EPO (EPHB4 and CD131). D. Epitope binding using competitive biolayer interferometry (BLI) was performed by first immobilizing human EPOR and using it to capture saturating (200 nM) D-Fc in solution phase. After washing away uncaptured D-Fc, a second competing D-Fc was added, and the binding capacity to EPOR was measured and normalized. E. Competitive phage ELISA was performed by measuring binding of the indicated phage D-Fc to immobilized EPOR in the presence of the indicated EPO concentrations.

[0059] [Figure 4] Figures 4A and 4B show the functional activity of preferred embodiments of the present invention. A. Inventive embodiments (4636 and 4635) stimulated proliferation of the human erythroid UT-7 / EPO cell line (Reference 3). B. Inventive embodiments (4636 and 4635) induced phosphorylation of the downstream effectors of EPO, JAK2, STAT3, STAT5, ERK, and AKT, in a dose-dependent manner (D-Fc used at 1 nM, 10 nM, and 100 nM, and EPO used at 1 1 / 4 L / mL, 10 1 / 4 L / mL, and 100 1 U / mL).

[0060] [Figure 5-1] 5A-5D show the randomization scheme of the heavy and light chains of 4636 and 4635 in the affinity maturation phage library. [Figure 5-2] Same as above. [Figure 5-3] Same as above. [Figure 5-4] Same as above.

[0061] [Figure 6-1] Figures 6A-6D show the amino acid CDR sequences of the heavy and light chain affinity-matured clones 4636 (Figures 6A and 6B) and 4635 (Figures 6C and 6D) and their phage ELISA binding data. [Figure 6-2] Same as above. [Figure 6-3] Same as above. [Figure 6-4] Same as above.

[0062] [Figure 7] Figures 7A-7D show preferred embodiments obtained from 4636 affinity maturation screening. A. Binding of embodiments compared to 4636 parent D-Fc protein. B. EC50 values ​​calculated from the curves in Figure 7A. C. Stimulation of UT7 / EPO cell proliferation by preferred embodiments compared to parent 4636. D. EC50 values ​​calculated from the curves in Figure 7C.

[0063] [Figure 8-1] Figures 8A-8D show the structural features of a preferred embodiment of 14949 in the Fab format bound to EPOR. A. Structure of one EPO molecule bound to two asymmetric EPORs via sites 1 and 2 to induce activation (1 EER). B. Reconstruction of the 14949-Fab / EPOR ternary structure using Molecule Operating Environment (MOE) software to fit an 18 Å diabody linker that connects the VH and VL domains in the most energetically favorable manner. The CH1 domain is not shown. EPO is overlaid to show that site 2 is blocked and site 1 is unblocked. C. The 14949-Fab / EPOR ternary structure is compared to the ternary structure formed by EPO and a previously identified EPOR peptide antagonist (1 EBA), which induces receptor dimerization without activation (Reference 4). D. Structure of EPOR in complex with a previously discovered diabody is shown compared to the EPO-EPOR structure (Reference 5). [Figure 8-2] Same as above.

[0064] [Figure 9-1] Figures 9A to 9E show the 14949 paratope and its corresponding hEPOR epitope. A. Ribbon diagram showing the interaction interface between hEPOR (dark gray) and binding residues annotated on the 14949 heavy (light gray) and light (gray) chains. B. Open-book view of the paratope, showing interacting residues (dark gray) in both the heavy (left panel) and light (right panel) chains. C. Sequence of the entire CDR region (underlined) of 14949 with interacting residues (gray shaded) shown in B. All amino acid positions are numbered according to the IMGT nomenclature. D. Open-book view of the hEPOR epitope showing interacting residues (gray). E. Sequence alignment of hEPOR compared to the sequences of M. fascicularis and M. musculus. Residues in Site 1 and Site 2 that bind EPO are indicated by bold (*), shaded (*), or both. The 14949 binding residues identical to hePOR are shown in light grey, and non-conserved residues in dark grey. [Figure 9-2] Same as above. [Figure 9-3] Same as above. [Figure 9-4] Same as above.

[0065] [Figure 10-1]Figures 10A-10E compare the binding of the inventive molecule 14949 to that of EPO, prior art ABT-007, prior art diabodies 305, 310, and 330, and scFv-Fc-10, -29, and -15. A. hEPOR residues that interact with hEPO (light gray) are compared to residues of the 14949 epitope in this structure (medium gray). Both high-affinity residues (left panel) and low-affinity residues (right panel) are shown. Residues common to both sites are shown in dark gray. B. The prior art ABT-007 epitope is compared to the 14949 epitope, with specific binding residues shown in light gray and medium gray, respectively. Dark gray indicates hEPOR residues that interact with both ABT-007 and the 14949 Fab. C. Comparison of the 14949 epitope (medium gray) with three previously identified diabodies, 305 (4Y5V), 310 (4Y5X), and 330 (4Y5Y) (light gray). Residues that interact with 14949 and each of 305, 310, and 330 are shown in dark gray. D. The 14949 epitope is also compared to the epitopes of three prior art scFv-Fc agonist molecules based on mutagenesis screening. Residues that, when mutated to alanine, reduce scFv-Fc binding to the hEPOR that constitutes the 14949 epitope by less than 50% are shown in dark gray. Residues that only affect scFv-Fc binding (≦50%) and are not part of the 14949 epitope are shown in light gray, while amino acids specific to the 14949 Fab are shown in medium gray. A diagram summarizing all structural comparisons of EA~D. [Figure 10-2] Same as above. [Figure 10-3] Same as above. [Figure 10-4] Same as above. [Figure 10-5] Same as above.

[0066] [Figure 11]Figures 11A-11C show different modalities of preferred 14949 embodiments and their functional properties. A. Schematic of different modalities, including a diabody-Fc format in which VL and VH are linked to CH2 and CH3 domains. Also shown are inter- and intra-diabody formats, the latter with a (GGGGS)s covalent linker. B. Titration ELISA experiments showing similar binding across different formats. C. Proliferation of UT7 / EPO cells was measured at various protein doses to determine whether format affected agonist activity.

[0067] [Figure 12-1] 12A-12D show randomization schemes using 14949 as a template. A. CDR-L scheme of sublibrary AP229. B. CDR-H scheme of sublibrary AP229. C. CDR-L scheme of sublibrary AP230. D. CDR-H scheme of sublibrary AP230. [Figure 12-2] Same as above.

[0068] [Figure 13] Figure 13 shows the sequences of affinity-matured clones that bound to hEPOR in phage ELISA and whose binding was inhibited by saturating 14949 D-Fc protein. Positions randomized in affinity maturation libraries AP229 and AP230 are shown in gray. Production yields are also shown.

[0069] [Figure 14-1]Figures 14A-14C show the characteristics of affinity-matured clones (excluding 19432, which has a potential N-glycosylation site) that showed higher yields than the parental strain 14949. A. To examine the activity of these clones, a UT7 / EPO cell proliferation assay was performed. EPO and 15033 D-Fc were used as positive and negative controls, respectively. B. A nonspecific ELISA was performed using a representative antigen panel to examine the nonspecific binding of high-value clones. C. Affinity measurements using BLI compared the dissociation constants (KD) of the parental strain 14949 with those of 19429 and 19113. [Figure 14-2] Same as above.

[0070] [Figure 15] Figure 15 shows the results of a UT7 / EPO cell proliferation assay in which the agonist activity of affinity-matured 19113 and 19429 VL-VH constructs was measured and compared in both diabody-Fc and IgG formats. The activity of ABT-007 in IgG1 format was also tested in this assay. hEPO and 15033 D-Fc were used as positive and negative controls, respectively.

[0071] [Figure 16-1] Figures 16A and 16B,B' show the analysis of mutants at individual positions that were not previously randomized in the AP229 and AP230 sublibraries. A. Table showing the individual mutations tested. B,B'. Collection of UT7 / EPO cell proliferation assays showing the activity of the mutants shown in A. EPO and 15033 D-Fc were included in each experiment as positive and negative controls, respectively. [Figure 16-2] Same as above. [Figure 16-3] Same as above.

[0072] [Figure 17]Figures 17A and 17B show the results of Protein A purification of 19429-H1 D2 / L3Q3 supernatant followed by fractionation and analysis of various species based on molecular weight. A. Size-exclusion chromatogram showing peaks eluted from the column. There are three major peaks, designated 1, 2, and 3. B. UT7 / EPO cell proliferation assays were performed using material from peaks 1, 2, and 3 to measure the agonist activity of each peak. EPO and 15033 D-Fc were used as controls.

[0073] [Figure 18] Figures 18A and 18B show the results of Protein A purification of 19113-H1 E2 supernatant followed by fractionation and analysis of various molecular species based on molecular weight. A. Size-exclusion chromatogram showing peaks eluted from the column. Trastuzumab was used as a control. There were three major peaks belonging to 19113-H1 E2, designated 1, 2, and 3, respectively. B. Material from peaks 1, 2, and 3 was used in a UT7 / EPO cell proliferation assay to measure the agonist activity of each peak using EPO and 15033 D-Fc as controls.

[0074] Detailed Description of the Invention In this disclosure, a number of terms and abbreviations are used. Definitions of these terms and abbreviations are provided below.

[0075] As used herein, those skilled in the art will generally understand that the term "erythropoietin" or its abbreviation "EPO" refers to the protein erythropoietin and, when used with reference to a nucleic acid, refers to a nucleic acid encoding EPO. As used herein, those skilled in the art will generally understand that the term "erythropoietin receptor" or its abbreviation "EPOR" refers to the EPO receptor and, when used with reference to a nucleic acid, refers to a nucleic acid encoding EPOR.

[0076] In this specification, those skilled in the art will generally understand the term "comprising" to generally mean the presence of the features, integers, steps, or components recited in the claim, but not excluding the presence or addition of one or more other features, integers, steps, components, or groups thereof.

[0077] As used herein, the term "treatment" generally refers to an approach for obtaining beneficial or desired results, as generally understood by those skilled in the art. Beneficial or desired results include, but are not limited to, prevention or prophylaxis, alleviation or amelioration of one or more symptoms or conditions, reduction in the extent of disease, stabilization of disease (i.e., a state in which the disease is not worsening), prevention of the spread of disease, delay or slowing of disease progression, alleviation or mitigation of the disease state, and remission (partial or complete) (whether detectable or undetectable). "Treatment" can also mean prolonging survival compared to expected survival if not receiving treatment.

[0078] As used herein, the term "therapeutically effective amount" is generally understood by those skilled in the art to mean an amount sufficient to effectively treat a subject in need of treatment when administered to the subject. In the context of embodiments of the present invention, a therapeutically effective amount includes, but is not limited to, an amount that eliminates or reduces the effects of a disease in a subject, such as a tumor burden.

[0079] As used herein, those skilled in the art will generally understand the term "amino acid sequence" to refer to the amino acid sequence of a naturally occurring or non-naturally occurring protein molecule. The use of "amino acid sequence" and similar terms, such as "polypeptide" and "protein," is not intended to limit the amino acid sequence to the complete, naturally occurring amino acid sequence associated with the described protein molecule. An amino acid sequence may be referred to as having an amino (N)-terminus and a carboxyl-terminus. Individual amino acids in a peptide or polypeptide are referred to as "residues," and such residues are numbered in increasing order, beginning with the N-terminus and proceeding toward the C-terminus. The amino acid located proximal to the N-terminus is generally referred to as the N-terminal amino acid, and the amino acid located proximal to the C-terminus is generally referred to as the C-terminal amino acid. Those skilled in the art will understand that whether an amino acid residue is the N-terminal or C-terminal amino acid residue may vary depending on the protein.

[0080] As used herein, those skilled in the art will generally understand that the term "aliphatic amino acid" may include any of the amino acids alanine, glycine, isoleucine, leucine, proline, valine, or methionine, and the term "polar amino acid" may include any of the amino acids serine, threonine, cysteine, asparagine, glutamine, or tyrosine. The term "hydrophobic amino acid" may include any of the amino acids glycine, alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, and tryptophan.

[0081] As used herein, those skilled in the art will generally understand the terms "nucleic acid molecule encoding," "DNA sequence encoding," "RNA sequence encoding," "mRNA sequence encoding," "oligonucleotide having a nucleotide sequence encoding a gene," "polynucleotide having a nucleotide sequence encoding a gene," "DNA encoding," "RNA encoding," and similar terms to refer to the order or sequence of nucleotides in a single- or double-stranded nucleic acid comprising the coding region of a gene, i.e., the nucleic acid sequence encoding a gene product. The order of these nucleotides determines the order of amino acids in the polypeptide chain. The coding region may be present in the form of cDNA, genomic DNA, or RNA. The oligonucleotide or polynucleotide may be single-stranded (e.g., the sense strand) or double-stranded (e.g., the antisense and sense strands). Appropriate regulatory elements (e.g., enhancers / promoters, splice junctions, polyadenylation signals, etc.) may be positioned adjacent to the coding region of a gene if necessary to allow proper initiation of transcription and / or correct processing of the primary RNA transcript. Alternatively, the coding region utilized in an expression vector may include endogenous enhancers / promoters, splice junctions, intervening sequences, polyadenylation signals, etc., or a combination of both endogenous and exogenous regulatory elements. Those skilled in the art will understand that nucleic acid molecules are said to have "5' ends" and "3' ends" because mononucleotides are linked via phosphodiester bonds, and oligonucleotides or polynucleotides are linked such that the 5' phosphate of one mononucleotide's pentose ring is unidirectionally attached to the 3' oxygen of the adjacent mononucleotide. Thus, an end of an oligonucleotide or polynucleotide is referred to as the "5' end" if its 5' phosphate is not attached to the 3' oxygen of the preceding mononucleotide's pentose ring, and as the "3' end" if its 3' oxygen is not attached to the 5' phosphate of the succeeding mononucleotide's pentose ring. As used herein, a nucleic acid sequence may also be referred to as having a 5' end and a 3' end, even if it is internal to a larger oligonucleotide or polynucleotide.In either linear or circular nucleic acid molecules, individual elements are referred to as "upstream" or 5' of the "downstream" or 3' element. Because DNA molecules are typically in a double-helical structure, DNA molecules are said to have a "sense" strand and an "antisense" strand. The sense and antisense strands are said to be reverse complementary because the 3' end of the sense strand can anneal to the 5' end of the antisense strand, and the 5' end of the antisense strand can anneal to the 3' end of the sense strand. The "sense" strand of a DNA molecule is typically copied into messenger RNA (mRNA) during transcription. Because the mRNA produced during transcription has the same sequence as the sense strand through transcription of the antisense strand, the final protein can be generated based on the sense strand of the DNA molecule. The term "antisense strand" refers to the nucleic acid strand complementary to the "sense" strand. The antisense strand is sometimes designated (-) (i.e., "negative"), and the sense strand is sometimes designated (+) (i.e., "positive").

[0082] As used herein, the term "homology" refers to the degree of complementarity. There can be partial or complete homology (i.e., identity). When applied to polypeptides, the term "substantial homology" means that two peptide sequences optimally aligned, such as by the GAP or BESTFIT programs using default gap weights, share at least 80% sequence identity, preferably at least 90% sequence identity, and more preferably at least 95% or more sequence identity (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 with chemically similar side chains. For example, the group of amino acids with aliphatic side chains can include glycine, alanine, valine, leucine, and isoleucine. The group of amino acids with aliphatic hydroxyl side chains can include serine and threonine. The group of amino acids with amide-containing side chains can include asparagine and glutamine. The group of amino acids having aromatic side chains may include phenylalanine, tyrosine, and tryptophan, the group of amino acids having basic side chains may include lysine, arginine, and histidine, and the group of amino acids having sulfur-containing side chains may include cysteine ​​and methionine.

[0083] As used herein, the term "fragment," when used in reference to a single-chain amino acid sequence, refers to a polypeptide that may lack an amino (N)-terminal portion and / or a carboxy (C)-terminal portion compared to the native protein, but the remaining amino acid sequence of the fragment is identical to that of the native protein. Those skilled in the art will appreciate that the term "fragment" can also refer to a portion of a multi-chain protein molecule (e.g., an antibody fragment).

[0084] As used herein, when applied to an object, the term "naturally occurring" or "natural" refers to the fact that the object can exist in nature. For example, a polypeptide or polynucleotide sequence that can be isolated from a natural source, exists in an unmodified organism (including viruses), and exists in nature is naturally occurring. Those skilled in the art will understand that the term "synthetic" refers to a compound that does not exist in nature.

[0085] As used herein, the term "target" refers to a structure, such as a nucleic acid or protein molecule, that is to be identified, detected, characterized, or amplified. Thus, a "target" is sought to be separated from other structures.

[0086] The term "isolated," when used with respect to a nucleic acid or peptide, as in "isolated oligonucleotide," "isolated polynucleotide," or "isolated polypeptide," refers to a nucleic acid or amino acid sequence that has been identified and separated from at least one contaminant normally associated with it in the natural source. An isolated compound exists in a form or setting that is different from that found in nature. In contrast, a non-isolated compound, such as a nucleic acid or amino acid sequence, is found in the state it exists in nature. For example, a particular DNA sequence (e.g., a gene) is found in close proximity to neighboring genes on the chromosome of a host cell. An RNA sequence, such as a particular mRNA sequence encoding a particular protein, exists in a cell mixed with many other mRNAs that encode many other proteins.

[0087] As used herein, the term "portion" when used in reference to a nucleotide sequence or amino acid sequence refers to a fragment of that sequence. As used herein, the term "purified" or "to purify" refers to the removal of contaminants from a sample. For example, an EPOR agonist is purified by removing contaminating non-immunoglobulin proteins, but also by removing immunoglobulins that do not bind to EPOR. Removal of non-immunoglobulin proteins and / or removal of immunoglobulins that do not bind to EPOR results in an increase in the proportion of EPOR agonist in the sample.

[0088] As used herein, the term "recombinant protein" or "recombinant polypeptide" refers to a protein molecule that is expressed from a recombinant DNA molecule.

[0089] In the context of the present invention, numerous techniques well known in the art can be used to detect antibody binding, including, but not limited to, RIA (radioimmunoassay), ELISA (enzyme-linked immunosorbent assay), "sandwich" immunoassay, immunoradiometry, 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.

[0090] 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 separated by electrophoresis on an acrylamide gel, and then transferred from the gel to a solid support such as nitrocellulose or a nylon membrane. The immobilized proteins are then exposed to an antibody reactive with the antigen of interest. Antibody binding can be detected by a variety of methods, including the use of radiolabeled antibodies, enzyme-linked antibodies, and the like.

[0091] As used herein, the term "antigenic determinant" refers to the portion of an antigen that contacts an antibody (i.e., epitope). When a host animal is immunized with a protein or protein fragment, many regions of the protein may induce the production of antibodies that specifically bind to particular regions or three-dimensional structures on the protein. These regions or structures are called antigenic determinants. Antigenic determinants may compete with the complete antigen (i.e., the "immunogen" used to elicit an immune response) for binding to an antibody. Those skilled in the art will understand that a "paratope" (also called an antigen-binding site) is the portion of an antibody that recognizes and binds to an epitope of an antigen. Each paratope is composed of six complementarity-determining regions, three from each of the light and heavy chains. Identical paratopes are present at the end of each arm of a Y-shaped antibody.

[0092] The term "conformational epitope" refers to an epitope in which non-contiguous amino acids form a three-dimensional conformation. In a conformational epitope, the points of interaction occur across amino acid residues that are separated from one another on the protein. In one embodiment, the epitope is one described in the Examples herein.

[0093] As used herein, the term "sample" is used in the broadest sense. Samples suspected of containing nucleic acid or amino acid sequences include cells, chromosomes isolated from cells (e.g., metaphase chromosome spreads), 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 include cells, tissue fragments, extracts containing one or more proteins, etc.

[0094] As used herein, the term "response," when used in reference to an assay or other result, refers to the production of a detectable signal (e.g., accumulation of a reporter molecule, increase in ion concentration, accumulation of a detectable chemical product (e.g., antibody)).

[0095] As used herein, the terms "agonist" and "agonistic" refer to or describe a molecule that can directly or indirectly initiate, activate, stimulate, or induce one or more aspects of a response (e.g., a physiological response) or other biological activity upon binding to a receptor. In a preferred embodiment, a compound of the present invention, such as a diabody, can include an agonist of EPO in that it mimics one or more properties of EPO. As used herein, the terms "antagonist" and "antagonistic" refer to or describe a molecule that can directly or indirectly affect or inhibit one or more aspects of a physiological response or other biological activity upon binding to a receptor. Antagonist is the opposite of agonist. In another preferred embodiment of the present invention, a compound of the present invention, such as a complete antibody, can include an antagonist of EPO in that it inhibits the action of EPO.

[0096] As used herein, the term "antibody" or "Ab" is used in the broadest sense and specifically encompasses single anti-EPOR monoclonal antibodies (including agonistic, antagonistic, neutralizing, or blocking antibodies) and anti-EPOR antibody compositions with polyepitopic specificity. As used herein, "antibody" also includes 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'), or Fv), and synthetic diabodies, so long as they exhibit any of the desired agonistic or antagonistic properties described herein. Various procedures known in the art can be used to generate such antibodies against a particular antigen, or derivatives, fragments, analogs, homologs, or homologs thereof.

[0097] Antibodies are typically proteins or polypeptides that exhibit binding specificity to a specific antigen. Natural antibodies are usually heterotetrameric glycoproteins consisting of two identical light chains (L chains) and two identical heavy chains (H chains). Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, although the number of disulfide bonds varies among heavy chains of an immunoglobulin isotype. Each heavy and light chain has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (VH) at one end followed by multiple constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end. The constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. Specific amino acid residues are believed to form an interface between the light and heavy chain variable domains. The light chains of antibodies from all vertebrate species can be classified into two distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains. Depending on the amino acid sequence of their heavy-chain constant domains, immunoglobulins can be divided into different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which are further divided into subclasses (isotypes), such as IgG-1, IgG-2, IgG-3, IgG-4, IgA-1, and IgA-2. The heavy-chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0098] As used herein, the term "antibody fragment" refers to a portion of an intact antibody, typically constituting the antigen-binding or variable region of the intact 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.

[0099] The term "diabody" refers to a small antibody fragment having two antigen-binding sites, each of which comprises a heavy-chain variable domain (VH) linked to a light-chain variable domain (VL) in the same polypeptide chain (VH and VL). In diabodies, each VL domain is linked to one VH domain in a single-chain Fv (scFv) fragment by a short linker. The use of a short linker (e.g., in a preferred embodiment of the present invention, 6-15 amino acids, i.e., 18 Å) to allow pairing between the two domains on the same chain forces these domains to pair with complementary domains on another chain to form two antigen-binding sites. Diabodies are described in further detail, for example, in EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0100] As used herein, the term "linker" refers to a linking sequence that facilitates diabody formation, preferably an 18 Å (±2 Å) amino acid linker, more preferably a linker having any combination of 5 to 15 amino acids, even more preferably having the sequence GGGGG, IKGGGGGEV, LKVLSRGVV, ISARAGSLV, SKSRAGGEV, LKGGRGGKV, NKGGGGAKV, IKGSSRDDI, MKHAGRGGV, SKGGGGGEV, MKHAGRGGV, LECSDCSGI, and even more preferably having the sequence GGGGG.

[0101] As used herein, the term "variable domain" refers to a specific portion of an antibody whose sequence differs among antibodies and is responsible for each antibody's binding and specificity for a particular antigen. However, this variability is not typically evenly distributed throughout the variable domain of an antibody. In both light and heavy chain variable domains, it is typically concentrated in three segments called complementarity-determining regions ("CDRs"), "hypervariable regions," or "hypervariable domains." The more highly conserved portions of the variable domain are called framework regions ("FRs"). Naturally occurring heavy and light chain variable domains each consist of four FR regions, most of which adopt a beta-sheet configuration and are connected by three CDRs. The CDRs form loops that connect and, in some cases, form part of the beta-sheet structure. The CDRs of each chain are held in close proximity by the FR regions and, together with the CDRs of the other chain, contribute to the formation of the antibody's antigen-binding site. The constant domains are not directly involved in antibody-antigen binding but exert various effector functions, such as antibody participation in antibody-dependent cellular cytotoxicity.

[0102] As used herein, the term "monoclonal antibody" or "mAb" refers to an antibody obtained from a population of substantially homogeneous antibodies; that is, the individual antibodies comprising the population are identical except for possible minor naturally occurring mutations. Monoclonal antibodies are highly specific, targeting a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies targeting different antigenic determinants (epitopes), each monoclonal antibody targets a single antigenic determinant on the antigen.

[0103] The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method.

[0104] Those skilled in the art will understand that modifications of the antibodies of the present invention are contemplated herein. The antibodies of the present invention can be modified by conjugating, tagging, or labeling them with any known diagnostic or therapeutic agent, including, but not limited to, cytotoxic agents (e.g., immunotoxin conjugates), prodrugs, drugs (e.g., pharmacologically active substances), or other effector molecules effective in treating disease, as well as known reporter molecules, using methods known in the art. Such modified antibodies (also referred to as immunochemical derivatives thereof) 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, in which 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 a substance that inhibits or inhibits the function of cells and / or causes cell destruction, and include, but are not limited to, radioactive isotopes, chemotherapeutic agents, and toxins, such as small molecule 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 agent that has less cytotoxicity to target cells compared to the pharmaceutically active agent and can be activated or converted to a more pharmaceutically active agent.

[0105] Those skilled in the art will appreciate that the antibodies of the present invention can also be produced by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567, which is incorporated herein by reference. DNA encoding the monoclonal antibodies of the present invention can be readily isolated and sequenced using conventional methods, such as using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of mouse antibodies. The isolated DNA is then inserted into an expression vector and introduced into host cells, such as monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not produce immunoglobulin proteins, to synthesize monoclonal antibodies in the recombinant host cells. The DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous mouse sequences (U.S. Pat. No. 4,816,567; Morrison, Nature 368, 812-13 (1994)), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Such a non-immunoglobulin polypeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the variable domains of one antigen-binding site of an antibody of the invention to create a chimeric bivalent antibody.

[0106] Those skilled in the art will understand that "phage display" refers to a technique for generating and selecting novel proteins that bind to ligands such as antigens. Using phage display technology, it is possible to generate large libraries of protein variants and rapidly select sequences that bind to target antigens with high affinity. Methods for generating peptide libraries and screening these libraries are disclosed in numerous patents (e.g., U.S. Pat. Nos. 5,723,286, 5,432,018, 5,580,717, 5,427,908, and 5,498,530). It goes without saying that antibody phage display libraries can also be generated. (Smith et al., Science (1985), 228:1315; Skerra and Pluckthun, Science (1988), 240:1038) Antibody or antigen-binding polypeptide libraries have been generated using a variety of methods, including modifying single genes by inserting random DNA sequences and cloning families of related genes. Methods for displaying antibodies or antigen-binding fragments using phage display are 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 screened for the expression of antibodies or antigen-binding proteins with desired properties. Phage display technology offers several advantages over traditional hybridoma and recombinant methods for preparing antibodies with desired properties. This technology allows for the development of large libraries of antibodies with diverse sequences in a shorter time and without the use of animals. Preparation of hybridomas and humanized antibodies often requires several months of preparation time. Furthermore, because immunization is not required, phage antibody libraries can be generated even against toxic or weakly antigenic antigens (Hogenboom, Immunotechniques (1988), 4:1-20).Commercially available phage antibody libraries are also available, such as those developed by Cambridge Antibody Technology and Morphosys (Vaughan et al. (1996) Nature Biotech 14:309; Knappik et al. (1999) J. Mol. Biol. 296:57).

[0107] Generating a diverse antibody or antigen-binding protein library is crucial for isolating high-affinity antibodies. Libraries with limited CDR diversity have been generated using a variety of approaches. See, for example, Tomlinson, Nature Biotech. (2000), 18:989-994. The CDR3 region is of interest because it is often implicated in antigen binding. The CDR3 region on the heavy chain varies widely in size, sequence, and structural conformation. Another approach has been to randomize the CDR regions of the variable heavy and light chains to generate diversity by using all 20 amino acids at each position. It was thought that using all 20 amino acids would result in greater sequence diversity in mutant antibodies, increasing the probability of identifying novel antibodies. (Barbas, PNAS 91:3809 (1994); Yelton, DE, J. Immunology, 155:1994 (1995); Jackson, JR, J. Immunology, 154:3310 (1995) and Hawkins, RE, J. Mol. Biology, 226:889 (1992))

[0108] Once a binding molecule with the desired binding and functional activity has been identified, in a preferred embodiment of the present invention, binding variants are generated, preferably by introducing one or more amino acid substitutions (e.g., substitutions) in one or more hypervariable or CDR regions of the binding molecule. One method is to utilize affinity maturation using phage display (Hawkins et al. J. Mol. Biol. 254:889-896 (1992) and Lowman et al. Biochemistry 30(45):10832-10837 (1991)). In this technique, multiple hypervariable region sites are mutated to generate possible amino acid substitutions at each site. The antibody variants thus generated are then displayed and screened for their biological activity (e.g., binding affinity). Those skilled in the art will appreciate that this process is commonly referred to as affinity maturation.

[0109] Those skilled in the art will 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 manner conventional for administering such substances, using standard pharmaceutical formulation chemistry and methodology readily available to those skilled in the art. Those skilled in the art will also understand that such pharmaceutical compositions may contain one or more excipients, carriers, stabilizers, or other pharmaceutically inactive compounds (e.g., wetting agents, emulsifiers, pH buffering agents, etc.). Pharmaceutically acceptable salts may also be included. A detailed discussion of pharmaceutically acceptable excipients, vehicles, and auxiliary substances is provided in Remington's Pharmaceutical Sciences (Mack Pub. Co. NJ 1991), which is incorporated herein by reference. Such pharmaceutical compositions can be prepared as injectable or oral preparations. The antibodies of the present invention can be administered by injection, including, but not limited to, intramuscular, intravenous, subcutaneous, peritoneal, transdermal, or nasal injections. The therapeutically effective amount will vary depending on body weight, and the timing and duration of administration will be determined by the specific clinical study protocol.

[0110] The following description and the embodiments described therein exemplify specific embodiments of the principles and aspects of the present invention. These examples are provided to explain the principles and aspects of the present invention, but are not intended to limit the invention. In this specification, like reference numerals refer to like parts throughout the specification and drawings.

[0111] The present invention is directed to the diagnosis and treatment of major disease- or cancer-associated anemia through selective activation or signaling of EPOR without adversely affecting disease recurrence (e.g., cancer, kidney disease) and patient survival. More preferably, embodiments of the present invention are directed to specific synthetic binding molecules, including antibodies, diabodies, and the like, as therapeutic agents for diseases, including cancer-associated anemia. Even more preferably, the present invention is directed to methods of activating the EPOR activation site upon binding with an agonist of the present invention. The present invention is applicable to many disease states, including, but not limited to, cancer-associated anemia and kidney disease, and can be used alone or in combination with other therapeutic agents.

[0112] The two antigen-binding sites of synthetic agonists (e.g., antibodies or diabodies) enable EPOR dimerization, which is crucial for EPOR phosphorylation and activation of downstream signaling pathways leading to erythropoiesis. EPOR agonists demonstrated potent erythropoietic stimulation in vitro and long-lasting activity in vivo. These EPOR agonists may represent a novel therapeutic option for patients with chronic kidney disease or treatment-related anemia. DETAILED DESCRIPTION OF THE INVENTION

[0113] Example Production, preparation and characterization of EPOR-binding molecules To select for EPOR agonists, a diabody library was constructed by modifying a phagemid vector encoding a human framework scFv6. In a preferred embodiment, diabodies can be generated in which the C-terminus of the variable light (VL) domain is linked to the N-terminus of the heavy chain variable light (VH) domain via a linker, preferably a Gly5 linker (e.g., GGGGG), and linked to the M13 gene 3 minor coat protein via a modified IgG hinge sequence. In a preferred embodiment, four of the six CDRs (the three heavy chain CDRs and CDR-L3) were diversified as described above for library F1, but the length diversity of CDR-H3 was reduced. This library contained 4.2 x 109 unique clones and was used for selection as described above. 1 .

[0114] The diabody phage library described herein was used for binding selection with the recombinant extracellular domain (ECD) of human EPOR (hEPOR-ECD). Phage antibodies that specifically bound to the antigen (e.g., ECD) but not to a control protein by ELISA were PCR-amplified and sequenced to identify unique sequence clones. Sequencing of individual binding clones identified 14 unique clones that bound to human and mouse EPOR in vitro as purified diabody-Fc (fragment crystallizable region) proteins (D-Fc) (see Figures 1A and 1B). The binding of the Fc region is thought to extend the half-life of diabodies in serum. These 14 D-Fc clones were also shown to bind to EPOR expressed on the cell surface by flow cytometry analysis using human erythroid TF-1 cells (Figures 2A and 2B). However, only seven of the 14 D-Fc clones (D-Fc-1 through D-Fc-7) stimulated TF-1 cell proliferation, as expected for EPOR activation (see Figure 1A). Interestingly, all D-Fc clones exhibiting significant TF-1 agonist activity (proliferation rates ≤ 40%) generally had little cross-reactivity with mouse EPOR (see Figure 1A). Furthermore, we tested the agonist properties of several naive IgG clones obtained from Library F selection using human EPOR as an immobilized target, as previously described. None of these IgG clones had a significant effect on cell proliferation (Figure 1C). This is in stark contrast to previous studies describing EPOR IgG agonists, clearly distinguishing the current embodiment as novel and non-obvious. 2 The two most potent clones (D-Fc-1 and D-Fc-2) were selected for further characterization and are hereafter designated 4636 and 4635, respectively (see, e.g., Figure 3A). These two D-Fc constructs showed high homology in the heavy chain CDR sequences, suggesting that they likely bind to EPOR in a similar manner.

[0115] To characterize the mechanism of action of 4636 and 4635, we performed titration ELISA (enzyme-linked immunosorbent assay). Binding to immobilized hEPOR was plotted against diabody concentration, and these graphs were used to calculate EC50 values ​​of 1.5 nM and 0.56 nM for 4636 and 4635, respectively (see Figure 3B). Single-concentration ELISA was also used to examine the specificity of 4636 and 4635. Two receptors previously shown to bind to EPO are EPHB4 and CD131. However, neither diabody-Fc showed significant binding to these receptors (see Figure 3C). Next, we performed an epitope binning assay using competitive biolayer interferometry (BLI) to determine whether 4636 and 4635 bind to a common epitope (see Figure 3D). Briefly, EPOR was immobilized and captured the designated diabody-Fc in solution phase at saturating concentrations (preloaded chip). After washing away excess diabody-Fc, control D-Fc, 4636, or 4635 was loaded again in the solution phase. If the epitope on EPOR was blocked by saturating D-Fc preloading, D-Fc did not bind in this second round. 4636 and 4635 were observed to inhibit each other, suggesting that these two diabody-Fc share a similar epitope (Figure 3D). To confirm whether this epitope also overlaps with EPO, a competitive ELISA was used. Binding of 4636 and 4635 to immobilized EPOR was inhibited with increasing EPO concentrations (Figure 3E).

[0116] Functional activity of EPOR-binding molecules Activation of EPOR induces proliferation of human erythroid cell lines. Therefore, we tested the ability of 4636 and 4635 to stimulate proliferation of the erythroid UT7 / EPO cell line. In a bioluminescence-based cell proliferation assay, both 4636 and 4635 were potent inducers of UT7 / EPO cell proliferation. In fact, both were more effective than EPO itself (Figure 4A). Next, we used Western blotting to examine the biochemical effects of 4636 and 4635 in UT7 cells. The functional activity of erythropoiesis-stimulating factors requires the dimerization of EPOR, which initiates a series of protein phosphorylation cascades, including the phosphorylation of JAK2, STAT3 / 5, ERK, and AKT. These changes were observed in UT-7 / EPO cells in response to 4636 and 4635 (Figure 4B). The UT-7 / EPO cell line is an EPO-dependent human erythroid cell line that endogenously expresses hEPOR. Unlike TF-1 cells 3, these cells exhibit normal EPOR expression. Both 4636 and 4635 induced phosphorylation of EPOR downstream effectors in a manner very similar to that of EPO (see Figure 4B). EPO induced phosphorylation of the canonical targets JAK2, STAT3, STAT5, ERK, and AKT in a dose-dependent manner. Importantly, both 4636 and 4635 induced phosphorylation of all five signaling molecules in a manner very similar to that of EPO.

[0117] Affinity maturation of EPOR-binding molecules Following the strategy described herein, phage antibody display affinity maturation libraries were developed to design further embodiments of the present invention. Four libraries were generated: one heavy chain (HC) library and one light chain (LC) library for each of 4636 and 4635. The exact locations within the complementarity-determining regions (CDRs) and the randomization scheme are shown in Figures 5A-5D. After five rounds of selection against immobilized hEPOR-ECD, sequencing of the binding phage clones revealed unique sequences of the affinity-matured clones (Figures 6A-6D). Phage ELISA was performed to confirm target binding compared with an immunoimmobilized Fc control (see Figures 6A-6D). After coating plates with EPOR-His or Fc, phage clones expressing each diabody were added to the wells and incubated for an additional hour at room temperature. Samples were washed and incubated with anti-M13 secondary antibody. TMB substrate was used for color development. OD450 was measured using a plate reader. All phage clones tested showed significant and specific binding to EPOR-His. The frequency and distribution of amino acids at randomized positions are shown schematically in Figures 6A-6D. This analysis indicated that LC positions either had a strong preference for the parent amino acid (CDR-L3) or had little selective pressure given the random distribution of amino acids (CDR-L1 and CDR-L2). Similarly, HC positions also tended to have a strong preference for the parent amino acid, with the exception of position 39 in CDR-H1, where a substitution of isoleucine with methionine was favored.

[0118] Based on the amino acid distribution, seven clones that maintained the LC position of the parent strain were selected from the HC-selected strain for further screening. All seven clones derived from the HC-selected strain had methionine at position 39 of CDR-H1 and various amino acids at position 30. These seven clones were cloned in the D-Fc format and purified as proteins. Using a titration ELISA, the preferred embodiment, affinity-matured clone 14949 D-Fc (see Figure 6A), showed a very similar EC50 to that of 4636 (Figures 7A and 7B). In a cell proliferation assay using UT7 / EPO cells, clone 14949 was found to be more effective (higher maximal effect) and approximately five-fold more potent (lower EC50) than the parent strain 4636 and EPO (Figures 7C and 7D). Functional data showed that 4636, 4635, and their affinity-matured clones bound with high affinity to hEPOR but not to other receptors that interact with EPO; moreover, they competed with each other and with EPO for receptor binding. The 4636 affinity-matured clone, or one of the affinity-matured clones, 14949, exhibited favorable or desirable functional properties.

[0119] Arrangement of EPOR complexed with affinity-matured clone 14949 Fab To explore the structural basis for EPOR binding by embodiments of the present invention, we cloned, expressed, purified, and crystallized one construct, identified as affinity-matured clone 14949, a preferred embodiment of the present invention, in complex with hEPOR-ECD (Figures 8A and 8B). Human EPO binds to its cognate receptor through two sites: high-affinity site 1 located on one receptor and low-affinity site 2 on the adjacent receptor. Binding to these sites induces the formation of an asymmetric dimeric signaling complex (Figure 8A). Based on this, to understand how 14949 activates EPOR signaling, we refined the 14949-Fab / EPOR structure to account for the 18A linker in its lowest energy state. Next, superimposition of EPO revealed that high-affinity site 1 is accessible, while site 2 is blocked (Figure 8B). This explains previous data (see, e.g., Figure 3E) that EPO competes for binding with current embodiments of the present invention.

[0120] As shown in Figure 8C, a model of the preferred embodiment is compared to the known EPOR structure. This improved model shows striking similarities not only to the EPO-EPOR model, but also to the asymmetric dimer formed with antagonist peptides (Reference 4). Therefore, dimer formation itself may not explain the agonistic properties of the preferred embodiment 14949, suggesting a separate and distinct mechanism of action.

[0121] As shown in Figure 8D, the asymmetric dimer formed by 14949 is distinct from previously developed diabodies that activate EPOR5. All previous diabodies induce EPOR dimerization in a manner distinct from that of EPO. The structural similarity of the modeled 14949-EPOR trimeric complex is unique compared to prior art.

[0122] 14949 Fab paratope and hEPOR epitope Closer inspection of the 14949 Fab-EPOR complex reveals the precise residues that make up the paratope of the Fab and hEPOR epitopes (Figure 9A). Within the paratope, both the VL and VH domains form multiple contacts (Figures 9B and 9C). All of these positions are within the CDRs. In the VL domain, the interacting CDR positions of 14949 are Ala38 in CDR-L1, Tyr55 and Ser56 in CDR-L2, and Ser107, Ser108, ​​and Ser114 in CDR-L3. In the VH domain, the interacting residues are Ser36, Tyr37, and Tyr38 in CDR-H1. In CDR-H2, they are Pro58, Tyr61, Tyr62, Tyr64, and Tyr66, and in CDR-H3, they are Arg106, His107, Gly108, Tyr109, and Gly113.

[0123] The hEPOR residues that make up the epitope (Figures 9D and 9E) include Gln82, Glu84, Asp85, Glu86, Pro87, Trp88, Leu90, Pro119, Glu121, Arg123, Thr125, Ser128, Gly129, Pro131, His134, and Val136. As previously shown (Figures 3E and 8B), the site 2 position, encompassing Leu83 through Lys89, overlaps with 14949 Fab binding. Furthermore, all but two of these binding residues (Gln82 and Val136) match those of M. fascicularis EPOR. In contrast, mouse EPOR contains several nonconservative substitutions at positions 85, 87, 88, 90, and 123. This may explain the lack of binding to mouse EPOR and the lack of binding of embodiments of the present invention to mouse EPOR (Figure 1A).

[0124] Comparison of epitopes recognized by 14949 Fab with those of the prior art The novelty and non-obviousness of this invention are best revealed by a direct comparison of the epitope recognized by the 14949 Fab with all prior art, including the native binding site of hEPO (Reference 7) (Figure 10A). hEPO binds to its homologous receptor through high- and low-affinity binding sites. Two residues in high-affinity Site 1 (Glu84 and Asp85) are also found to bind to the 14949 Fab. Glu86 is involved in binding to both Site 1 and Site 2 and also interacts with the 14949 Fab. The majority of the 14949 Fab epitope is unique compared to hEPO, highlighting the non-natural mechanism of its activation.

[0125] Next, we compare the 14949 Fab binding epitope with that of ABT-007, a previously identified antibody hEPOR agonist (ref. 2) (Figure 10B). The overlap between these two interfaces is greater than that of the hEPO interface (Figure 10A). Eleven residues on hEPOR are involved in binding to both 14949 Fab and ABT-007. Only three residues, Ser87, Pro87, and Leu90, specifically bind to 14949 Fab but not ABT-007 (Figure 10B, medium gray residues). However, the target-binding mechanism of ABT-007 is significantly different from that of 14949 Fab, as the epitope of ABT-007 is much larger than that of 14949 Fab. While this is not fully demonstrated in this structure, it is summarized in Figure 10E.

[0126] Previous studies have identified three diabodies, 305 (4Y5V), 310 (4Y5X), and 330 (4Y5Y), which exhibited moderate agonist activity (Ref. 5). Therefore, we compared these epitopes with the epitope of the 14949 Fab (Fig. 10C). Of the 22 residues in the diabody 305 epitope, only three are shared with the 14949 Fab: Glu84, Asp85, and Glu86 (Fig. 10C, left panel). For diabody 310, 19 residues form this epitope, but only two are shared with the 14949 Fab: Glu84 and Asp85 (Fig. 10C, center panel). Similarly, of the 14 residues in diabody 330, only three are also found in the 14949 Fab epitope (Fig. 10C, right panel). This discrepancy between the binding of the 14949 Fab and these previous diabodies is not surprising given that these previous diabodies were all initially derived from the IgG modality, whereas 14949 was engineered from a diabody library. There are few similarities between the molecular mechanisms underlying 14949-mediated EPOR activation and how these diabodies function.

[0127] Comparison of the present invention with prior art scFv-Fc proteins further highlights the uniqueness of this embodiment (Reference 8) (Figure 10D). Three scFv-Fcs were confirmed to exhibit high agonistic activity toward hEPOR: scFv-Fc-10, -29, and -15. Although the structures of these molecules in complex with hEPOR have not been elucidated, their interfaces were mapped using mutagenesis studies. Epitopes were defined as hEPOR residues that, when mutated to alanine, reduce binding by less than 50% (Reference 8). These positions were compared with the 14949 Fab epitope (Figures 9D and 9E). The majority of interacting residues in the three scFv-Fc proteins differed from those in the 14949 Fab. Indeed, scFv-Fc-10, -29, and -15 had only three, one, and two overlapping contact residues, respectively (Figures 10D and 10E).

[0128] A comparison of 14949 Fab with all prior art is summarized in Figure 10E. Figure 10E shows that the VL and VH domains of 14949 contact the hEPOR target in a highly unique manner that has not been previously reported. This is not observed in hEPO and is therefore non-natural. Therefore, the mechanism of action of this embodiment is novel and non-obvious.

[0129] Characterization of different diabody modalities Additional experiments demonstrate how embodiments of the present invention are functionally active when expressed in different modalities (e.g., as shown in Figure 11A). Each modality of EPOR agonist has its own unique properties. For example, diabody-Fc enhances serum half-life through the addition of an Fc fragment. Intermolecular diabodies (or diabodies) are significantly smaller than, and more similar in size to, the natural ligand, EPO. Additionally, intramolecular diabodies with a (GGGGS)3 linker allow for the formation of bivalent dimers within a single fragment. The Fc modality has already been tested, and the latter modality was tested for binding to human EPOR using a titration ELISA and a UT7 / EPO cell proliferation induction assay (Figures 11B and 11C). Whether expressed as diabody-Fc, interdiabody, or covalently linked intradiabody, embodiments of the present invention have similar target binding and agonistic effects.

[0130] Further affinity maturation of 14949 Following the strategy described herein, a phage antibody display affinity maturation library was developed to design further embodiments of 14949. Two sublibraries, AP229 and AP230, were created. Each sublibrary consisted of one HC library and one LC library. The exact locations within the complementarity-determining regions (CDRs) and the randomization scheme are shown in Figures 12A-12D. After five rounds of selection against immobilized hEPOR-ECD, binding phage clones were used for phage ELISA. The antigen was first presaturated with 100 nM 14949 D-Fc protein. Excess 14949 D-Fc protein was washed five times with 1X PBS, and then the phage ELISA procedure was continued using an anti-M13 secondary antibody and TMB substrate for color development. The sequences of phage clones whose binding was significantly reduced by 14949 are shown in Figure 13. These clones were then cloned and expressed as diabody-Fc fusion proteins. The transient production yields (mg / L) using HEK293expi cells are also shown (Figure 13). Proteins that showed higher yields were used in subsequent experiments. Although 19432 showed a high yield, it was not subjected to further experiments because an N-glycosylation site had been introduced into CDR-H2.

[0131] Characterization of 14949 family clones Functional analysis was performed on the high-yielding clones shown in Figure 13. Two molecules (19113 and 19429) performed better than the parent clone, 14949 diabody-Fc, demonstrating higher maximal proliferative responses in UT7 / EPO cells (Figure 14A). A nonspecific ELISA was performed using a representative antigen panel (Figure 14B). All daughter clones, except 19434, showed significantly reduced binding to these unrelated antigens, demonstrating excellent specificity. In particular, 19113 and 19429 showed minimal binding to KLH, demonstrating the best specificity (Figure 14B). To assess how clinical-grade molecules perform in this assay, trastuzumab was used as a control. 15033 was a negative control, and 6606 was a highly cross-reactive control that showed high background binding to various antigens. Next, the binding affinities of 19113 and 19429 to hEPOR were measured using BLI (Figure 14C). Compared with the parent protein 14949, the diabody-Fc proteins 19113 and 19429 bound with similar dissociation constants, suggesting that the enhanced activation of hEPOR is likely related to receptor geometry rather than binding strength, which is consistent with recent findings that agonism and affinity do not necessarily correlate (Ref. 9).

[0132] Modality is essential for lead molecule agonism The lead diabody-Fc molecules, 19113 and 19429, were cloned, expressed as IgG1 proteins, and functionally tested in a UT7 / EPO cell proliferation assay (Figure 15). Unlike the 19429 D-Fc format, as IgG1s, both 19113 and 19429 exhibited little to no agonist activity. The previously identified ABT-007 agonist IgG also exhibited little to no functional agonist activity. This experiment clearly demonstrates that Applicants' engineered VLA / H domains do not function like classical antibodies, providing evidence supporting the claim that the current embodiments are novel agonists that exert their effects through a non-obvious mechanism of action.

[0133] 19429 additional variants The affinity maturation randomization scheme left several positions that could potentially affect agonist function unaltered. These positions are generally non-binding based on our structural analysis. For example, Arg35 in CDR-H1 of 14949 was not mutated (Figure 13). These individual positions were mutated to hydrophilic residues and activity was examined in the UT7 / EPO cell proliferation assay (Figures 16A and 16B). In general, hydrophilic substitutions at these positions were very well tolerated. The only exception was mutations in CDR-L3 of 19429, of which two mutants, L3DSxF and L3DS, significantly reduced hEPOR activation in this assay.

[0134] Activity test of higher molecular species of 19113 and 19429 The 19429-H1 D2 / L3Q3 double mutant was used to more closely analyze the activity profile of the various higher molecular species present in the Protein A purified product (Figures 17A and 17B). Size-exclusion chromatography (SEC) separation of this mixture revealed three peaks likely corresponding to three distinct molecular species with different molecular weights (Figure 17A). Using the UT7 / EPO cell proliferation assay, peak 3, the major molecular species and likely the diabody-Fc monomer, had the lowest activity (Figure 17B). Peaks 1 and 2, corresponding to the molecular weights of the dimer and tetramer, respectively, exhibited much higher activity. This data indicates that valency is an important determinant of biological function as an agonist.

[0135] Similar experiments were performed using the 19113-H1 E2 mutant. SEC identified three distinct species with different molecular weights, corresponding to a tetramer (peak 1), a dimer (peak 2), and a monomer (peak 3) (Figure 18A). Functional analysis using a UT7 / EPO cell proliferation assay revealed that the monomer species exhibited the lowest activity (Figure 18B). These results suggest that target valency must be considered in agonist design, which has implications for process development.

[0136] The combined functional and structural features of the present invention highlight a novel mechanism of EPOR activation and erythropoiesis stimulation that, while highly similar to the penultimate form of the natural EPO ligand, is unique in that it involves a discontinuous, non-natural, and non-obvious receptor binding mechanism not seen in biology or any prior art, which involves inducing an asymmetric receptor geometry.

[0137] reference JPEG2025540009000002.jpg152164

Claims

1. A selective activation region of human erythropoietin receptor (hEPOR) that provides selective activation of an erythropoiesis-specific function of hEPOR, said selective activation region comprising a first epitope, said first epitope comprising residues 82, 84-88, and 90 of hEPOR, the numbering of which is relative to SEQ ID NO:

29.

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

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

4. The selective activating region of claim 2 , wherein the first epitope comprises QEDEPWL (SEQ ID NO: 1).

5. The selectively activating region of claim 3 , wherein the second epitope comprises PERTSGPHV (SEQ ID NO: 2).

6. An antibody or antigen-binding portion thereof that binds to a selective activation region of human erythropoietin receptor (hEPOR) that provides selective activation of the erythropoiesis-specific activity of hEPOR, wherein the activation region is one of those described in any one of claims 1 to 5.

7. 1. An antibody or antigen-binding portion thereof that binds to the human erythropoietin receptor (hEPOR), comprising a CDR-L1 region having the consecutive 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; An antibody or antigen-binding portion thereof.

8. 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, The antibody described in claim 7.

9. and further comprising a CDR-L2 having the consecutive 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 an L, D or aliphatic amino acid; X6 is Y or a polar amino acid; and X7 is S, D or T; The antibody or antigen-binding portion of claim 7.

10. 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; The antibody described in claim 9.

11. and further comprising a CDR-L3 having the consecutive 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 an L, P, or aliphatic amino acid; and X6 is I, F, or a hydrophobic amino acid; The antibody or antigen-binding portion of claim 7.

12. In the CDR-L3, 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; 12. The antibody or antigen-binding portion of claim 11.

13. and further having a CDR-H1 having the consecutive 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; The antibody or antigen-binding portion of claim 7.

14. 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, The antibody described in claim 13.

15. and further comprising a CDR-H2 having the consecutive 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 a T, D, or S amino acid; and X10 is Y, S or a polar amino acid; The antibody or antigen-binding portion of claim 7.

16. 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 a T, D, or S amino acid; and X is Y; The antibody described in claim 15.

17. and further comprising a CDR-H3 having the consecutive 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; and X5 is A, G, or an aliphatic amino acid; and X6 is M, L or a hydrophobic amino acid; The antibody or antigen-binding portion of claim 7.

18. 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; The antibody described in claim 17.

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

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

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

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

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

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

25. 25. The antibody or antigen-binding portion of 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: 38, CDR-H2 comprises SEQ ID NO: 31, and CDR-H3 comprises SEQ ID NO:

32.

26. 25. The antibody or antigen-binding portion of 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: 38, CDR-H2 comprises SEQ ID NO: 26, and CDR-H3 comprises SEQ ID NO:

28.

27. 25. The antibody or antigen-binding portion of 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. 25. The antibody or antigen-binding portion of 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. 25. The antibody or antigen-binding portion of 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. 25. The antibody or antigen-binding portion of 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. 25. The antibody or antigen-binding portion of 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. 25. The antibody or antigen-binding portion of 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. 33. The antibody or antigen-binding portion thereof of any one of claims 6 to 32, which is a diabody, Fab, F(ab)2, tetravalent antibody, or multivalent antibody.

34. The antibody or antigen-binding portion thereof according to any one of claims 6 to 33 for use in a method for treating kidney disease or anemia.

Images