Antibodies specifically recognizing granulocyte-macrophage colony-stimulating factor receptor alpha and uses thereof

Abstract

The present application relates to antibodies or antigen-binding fragments that specifically recognize granulocyte-macrophage colony-stimulating factor receptor (GM-CSFRa), and methods of making and uses thereof.

Description

[0001] Submit sequence list

[0002] The contents of the sequence listing file (GM-CSFR-20220729SEQ.xml, record date: 2022.07.29, size: 290KB) submitted below are incorporated herein by reference in their entirety. Technical Field

[0003] This invention relates to antibodies that specifically recognize granulocyte-macrophage colony-stimulating factor receptor α (GM-CSFRα), their preparation methods and uses, including methods for treating autoimmune diseases, inflammation and cancer. Background Technology

[0004] Granulocyte-macrophage colony-stimulating factor (GM-CSF), also known as colony-stimulating factor 2 (CSF2), is a type I pro-inflammatory cytokine that plays a role in exacerbating inflammatory, respiratory, and autoimmune diseases. The GM-CSF receptor is a member of the hematopoietic receptor superfamily and is a heterodimer composed of α and β subunits. GM-CSF can bind to the α subunit alone with relatively low affinity (Kd 1-5 nM), but does not bind to the β subunit alone at all. However, when the α and β subunits are present simultaneously, a high-affinity ligand-receptor complex is formed (Kd ≈ 100 pM). Therefore, neutralizing the binding of GM-CSF to GM-CSFRα has become a therapeutic approach for GM-CSFRα-mediated diseases and symptoms. Patent application WO2007110631 discloses the anti-human GM-CSFRα antibody Mavrilimumab (Mab, used as a control antibody in the embodiments section of this patent).

[0005] All publications, patents, patent applications, and disclosed information in published patent applications mentioned herein are incorporated herein by reference in their entirety. Invention Overview

[0006] On one hand, the present invention relates to an isolated anti-GM-CSFRα antibody capable of specifically binding to an epitope of human GM-CSFRα, wherein the epitope comprises one, two, three, four, five, or six amino acid residues selected from the group consisting of Val50, Glu59, Lys194, Lys195, Arg283, and Ile284 of human GM-CSFRα. In some embodiments, the epitope further comprises the following amino acid residues: (i) Val51, Thr63, and Ile196; (ii) Leu191 and Ile196; or (iii) Arg49, Val51, Asn57, and Ser61. In some embodiments, the Kd value for binding of the isolated anti-GM-CSFRα antibody to human GM-CSFRα is 0.1 pM to 1 nM.

[0007] In some embodiments, any of the isolated anti-GM-CSFRα antibodies described above, the isolated anti-GM-CSFRα antibody comprising a heavy chain variable domain (V H ), the V H Contains: a heavy chain complementarity-determining region (HC-CDR)1 containing the sequence X1LX2X3H (SEQ ID NO:76), wherein X1 is E, N, G, D, M, S, P, F, Y, A, V, K, W, R, or C, X2 is S, C, or P, and X3 is I or M; an HC-CDR2 containing the sequence GFDX1X2X3X4EX5X6YAQKX7QG (SEQ ID NO:77), wherein X1 is P, G, T, S, or V, X2 is E, D, G, or A, X3 is D, G, I, W, S, or V, X4 is G, E, D, or H, X5 is T or A, X6 is N or I, and X7 is S or F; and a region containing the sequence GRYX1X2X3X4X5X6YGFDY (SEQ ID NO:77). HC-CDR3 of NO:78, wherein X1 is C, T, S, I, A or V; X2 is S, G, E, F, W, H, I, V, N, Y, T or R; X3 is T, H, L, F, P, I, S, Y, K, A, D, V, N or G; X4 is D, A, M, Y, F, S, T, G or W; X5 is T, S, F, Q, A, N, L, E, I, G or M; X6 is C, T, N, S or A; and a light chain variable domain (V L ), the V LContains: an LC-CDR1 containing the sequence RAX1X2X3VX4X5X6LA (SEQ ID NO:293), wherein X1 is S, L, N, A, K, R, I, Q, G, T, H, M, or C; X2 is Q, Y, P, A, I, F, T, R, V, L, E, S, or C; X3 is S, H, W, L, R, K, T, P, I, F, V, E, A, or Q; X4 is S, L, W, M, A, Y, K, R, G, T, E, V, N, F, or C; X5 is S, T, R, A, H, Q, P, M, L, or G; and X6 is Y, L, or F; and a LC-CDR1 containing the sequence X1X2X3LA (SEQ ID NO:293), wherein X1 is S, L, N, A, Q, P, M, L, or G; and X6 is Y, L, or F. LC-CDR2 (SEQ ID NO:294), wherein X1 is G or T, X2 is A, G, R, H, K, S, T, M or F, and X3 is S, A, W, R, L, T, Q, F, Y, H or N; and LC-CDR3 comprising the sequence QQYX1X2X3PX4T (SEQ ID NO:79), wherein X1 is N, D, S, R, A, T, L, Y, Q, W or G, X2 is N, D, E, T, Y, G, A, M, F, S, I or L, X3 is W, S, P, V, G or R, and X4 is P, Y, H, S, F, N, D, V or G. In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains: an HC-CDR1 containing the sequence ELX1X2H (SEQ ID NO: 295), wherein X1 is S, C, or P, and X2 is I or M; an HC-CDR2 containing the sequence GFDX1X2X3X4EX5X6YAQKX7QG (SEQ ID NO: 77), wherein X1 is P, G, T, S, or V, X2 is E, D, G, or A, X3 is D, G, I, W, S, or V, X4 is G, E, D, or H, X5 is T or A, X6 is N or I, and X7 is S or F; and a HC-CDR2 containing the sequence GRYX1X2X3X4X5X6YGFDY (SEQ ID NO: 295). NO:78) of HC-CDR3, wherein X1 is C, T, S, I, A or V; X2 is S, G, E, F, W, H, I, V, N, Y, T or R; X3 is T, H, L, F, P, I, S, Y, K, A, D, V, N or G; X4 is D, A, M, Y, F, S, T, G or W; X5 is T, S, F, Q, A, N, L, E, I, G or M; X6 is C, T, N, S or A; and V L The V LIt comprises: an LC-CDR1 containing the sequence RASQSVSSYLA (SEQ ID NO:51); an LC-CDR2 containing the sequence GASSRAT (SEQ ID NO:52); and an LC-CDR3 containing the sequence QQYX1X2X3PX4T (SEQ ID NO:79), wherein X1 is N, D, S, R, A, T, L, Y, Q, W, or G; X2 is N, D, E, T, Y, G, A, M, F, S, I, or L; X3 is W, S, P, V, G, or R; and X4 is P, Y, H, S, F, N, D, V, or G.

[0008] In some embodiments, an isolated anti-GM-CSFRα antibody is involved, comprising V H The V H Comprising: an HC-CDR1 comprising any amino acid sequence of SEQ ID NOs:1-4 or a variant thereof, the variant comprising up to 3 amino acid substitutions; an HC-CDR2 comprising any amino acid sequence of SEQ ID NOs:5-16 or a variant thereof, the variant comprising up to 3 amino acid substitutions; and an HC-CDR3 comprising any amino acid sequence of SEQ ID NOs:17-50 or a variant thereof, the variant comprising up to 3 amino acid substitutions; and V L The V L It comprises an LC-CDR1 containing an amino acid sequence SEQ ID NO:51 or a variant thereof, the variant containing up to 3 amino acid substitutions; an LC-CDR2 containing an amino acid sequence SEQ ID NO:52 or a variant thereof, the variant containing up to 3 amino acid substitutions; and an LC-CDR3 containing any of the amino acid sequences in SEQ ID NOs:53-75 or a variant thereof, the variant containing up to 3 amino acid substitutions.

[0009] In some embodiments, an isolated anti-GM-CSFRα antibody is involved, comprising V H It contains V having any of the amino acid sequences in SEQ ID NOs:80-121 H HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V having any of the amino acid sequences in SEQ ID NOs:122-144. L LC-CDR1, LC-CDR2 and LC-CDR3.

[0010] In some embodiments, an isolated anti-GM-CSFRα antibody is involved, comprising: (i) V H The V HContains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:5, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:17, or a variant containing up to 5 amino acid substitutions in the HC-CDRs; and V L The V L Contains: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:54, or a variant containing up to 5 amino acid substitutions in the LC-CDRs; (ii) V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:8, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:22, or a variant comprising up to 5 amino acid substitutions in all HC-CDRs; and V L The V L Contains: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:56, or a variant containing up to 5 amino acid substitutions in all LC-CDRs; (iii) V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:7, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:23, or a variant containing up to 5 amino acid substitutions in all HC-CDRs; and V L The V L Contains: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:57, or a variant containing up to 5 amino acid substitutions in all LC-CDRs; (iv)V H The V HContains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:6, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:27, or a variant comprising up to 5 amino acid substitutions in all HC-CDRs; and V L The V L Contains: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:57, or a variant containing up to 5 amino acid substitutions in all LC-CDRs; (v)V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:7, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:35, or a variant comprising up to 5 amino acid substitutions in all HC-CDRs; and V L The V L Contains: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:53, or a variant containing up to 5 amino acid substitutions in all LC-CDRs; (vi)V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:7, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:37, or a variant containing up to 5 amino acid substitutions in all HC-CDRs; and V L The V L Contains: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:57, or a variant containing up to 5 amino acid substitutions in all LC-CDRs; (vii)V H The V HContains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:3, an HC-CDR2 containing the amino acid sequence SEQ ID NO:6, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:39, or a variant containing up to 5 amino acid substitutions in all HC-CDRs; and V L The V L Contains: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:54, or a variant containing up to 5 amino acid substitutions in all LC-CDRs; (viii)V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:7, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:35, or a variant comprising up to 5 amino acid substitutions in all HC-CDRs; and V L The V L Contains: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:57, or a variant containing up to 5 amino acid substitutions in all LC-CDRs; (ix)V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:7, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:50, or a variant containing up to 5 amino acid substitutions in all HC-CDRs; and V L The V L It includes: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:57, or a variant containing up to 5 amino acid substitutions in all LC-CDRs.

[0011] In some embodiments, any of the isolated anti-GM-CSFRα antibodies described above, the isolated anti-GM-CSFRα antibody comprises amino acid residues: (i) V H31-bit E, H, N, G, D, M, S, P, F, Y, A, V, K, W, R, or C; and / or (ii) V L 26-bit S, L, N, A, K, R, I, Q, G, T, H, M or C; and / or (iii) V L The 27-bit Q, Y, P, A, I, F, T, R, V, L, E, S, or C; and / or (iv) V L The 28-bit S, H, W, L, R, K, T, P, I, F, V, E, A, or Q; and / or (v)V L 30 bits of S, L, W, M, A, Y, K, R, G, T, E, V, N, F or C; and / or (vi)V L 31-bit S, T, R, A, H, Q, P, M, L or G; and / or (vii)V L 32-bit Y, L, or F; and / or (viii) V L 50-bit G or T; and / or (ix)V L 51-bit A, G, R, H, K, S, T, M, F, N, or V; and / or (x)V L 52-bit S, A, W, R, L, T, Q, F, Y, H or N; and / or (xi)V L 92-bit D, A, Q, or W; and / or (xii)V L 93-bit N, D, E, T, Y, G, A, M, F, S, I or L; and / or (xiii) V H 28-bit T, H, V, E, P, L, M, S, W, C, A, G, N, or K; and / or (xiv)V H The 30-bit T, P, D, E, Y, W, V, M, N, L, Q, G, S, A, K, or R, where the numbering is defined according to the EU Kabat numbering definition.

[0012] In some embodiments, any of the isolated anti-GM-CSFRα antibodies described above, the isolated anti-GM-CSFRα antibody comprises: V H It contains any amino acid sequence from SEQ ID NOs:80-121 and 246-287, or contains V that has at least 90% sequence homology with any amino acid sequence from SEQ ID NOs:80-121 and 246-287. H Variant; and V L It contains any amino acid sequence of SEQ ID NOs:122-144, 150-245, and 288-289, or contains V with at least 90% sequence homology to any amino acid sequence of SEQ ID NOs:122-144, 150-245, and 288-289.L Variant. In some embodiments, the isolated anti-GM-CSFRα antibody comprises: (i) V containing SEQ ID NO:80 H and V containing SEQ ID NO:123 L (ii) V containing SEQ ID NO:85 H and V containing SEQ ID NO:125 L (iii) V containing SEQ ID NO:86 H and V containing SEQ ID NO:126 L (iv) V containing SEQ ID NO:91 H and V containing SEQ ID NO:126 L (v) V containing SEQ ID NO:99 H and V containing SEQ ID NO:122 L (vi) V containing SEQ ID NO:101 H and containing SEQ ID NO:126; (vii) containing SEQ ID NO:103 V H and V containing SEQ ID NO:123 L (viii) V containing SEQ ID NO: 99 H and V containing SEQ ID NO:126 L ;(ix) contains SEQ ID NO:121 and V contains SEQ ID NO:126 L (x) V containing SEQ ID NO:250 H and V containing SEQ ID NO:241 L ;(xi) contains V of SEQ ID NO:250 H and V containing SEQ ID NO:193 L ;(xii) contains V of SEQ ID NO:248 H and V containing SEQ ID NO:188 L (xiii) V containing SEQ ID NO:248 H and V containing SEQ ID NO:193 L ;(xiv) contains V containing SEQ ID NO:250 H and V containing SEQ ID NO:288 L ;(xv) contains V of SEQ ID NO:250 H and V containing SEQ ID NO:188 L;(xvi) contains V of SEQ ID NO:250 H and V containing SEQ ID NO:236 L ; or (xvii) contains V of SEQ ID NO:91 H and V containing SEQ IDNO:288 L .

[0013] In some embodiments, an isolated anti-GM-CSFRα antibody is involved, which competitively binds to GM-CSFRα with any of the isolated anti-GM-CSFRα antibodies described above. In some embodiments, an isolated anti-GM-CSFRα antibody is involved, which specifically binds to the same epitope as any of the isolated anti-GM-CSFRα antibodies described above.

[0014] In some embodiments, the isolated anti-GM-CSFRα antibody, as described above, comprises an Fc fragment. In some embodiments, the isolated anti-GM-CSFRα antibody is a full-length IgG antibody. In some embodiments, the isolated anti-GM-CSFRα antibody is a full-length IgG1 or IgG4 antibody. In some embodiments, the isolated anti-GM-CSFRα antibody is chimeric, human, or humanized. In some embodiments, the isolated anti-GM-CSFRα antibody is an antigen-binding fragment selected from the group consisting of Fab, Fab', F(ab)'2, Fab'-SH, single-chain antibody (scFv), Fv fragment, dAb, Fd, nanobodies, double-chain antibodies, and linear antibodies.

[0015] In some embodiments, the invention relates to an isolated nucleic acid molecule encoding any of the anti-GM-CSFRα antibodies described above. In some embodiments, the invention relates to a vector comprising any of the nucleic acid molecules described above. In some embodiments, the invention relates to a host cell comprising any of the anti-GM-CSFRα antibodies described above, any of the nucleic acid molecules described above, or any of the vectors described above. In some embodiments, the invention relates to a method for preparing an anti-GM-CSFRα antibody, comprising: a) culturing any of the host cells that effectively express an anti-GM-CSFRα antibody; and b) obtaining the expressed anti-GM-CSFRα antibody from the host cells.

[0016] In some embodiments, a method of treating a desired individual disease or condition includes administering an effective amount of any of the anti-GM-CSFRα antibodies described above to the individual. In some embodiments, the disease or condition is an inflammatory, respiratory, or autoimmune disease or condition. In some embodiments, the disease or condition is selected from the group consisting of rheumatoid arthritis, asthma, chronic obstructive pulmonary disease, allergic reactions, multiple sclerosis, myeloid leukemia, and atherosclerosis.

[0017] It also relates to pharmaceutical compositions, kits, and manufactured products containing any of the anti-GM-CSFRα antibodies described above. Attached Figure Description

[0018] Figure 1A-1C The figure shows the binding affinity of an exemplary anti-GM-CSFRα antibody to human GM-CSFRα, as analyzed by ELISA. Figure 1A The diagram shows the binding curves of T119, E9, E16, E27, E29, E30, E35, E36, E54 and E34 with human GM-CSFRα. Figure 1B The figure shows the binding curves of T119, E108, E105, E113, E87, E85, E39, E40, EII55, E200a and EII81 with human GM-CSFRα. Figure 1C The diagram shows the binding curves of T119, E61, E83, E88, E90, E84, E172, E164, E1, and E31 with human GM-CSFRα.

[0019] Figure 2 The binding affinity of E35, E200a, T119, E87, and E108 to cynomolgus monkey GM-CSFRα is shown by ELISA analysis.

[0020] Figure 3 The diagram shows the binding affinity comparison between E35, E87, and E108 with IL3RA, IL5RA, GM-CSFR, and GM-CSFRα, respectively.

[0021] Figure 4 The image shows a comparison of the binding affinity of E35-IgG4 to WIL2S cells expressing GM-CSFRα and to control WIL2S cells not expressing GM-CSFRα, as analyzed by FACS.

[0022] Figures 5A-5D The results of the competitive binding assay are shown. The ability of the lead antibody T119 and the optimized lead antibody to block the binding of GM-CSF to GM-CSFRα was determined by competitive ELISA. Figure 5AThe results of the competitive binding experiments for T119, E01, E09, E194, E27, E29, E34, E35, E40 and E30 are shown. Figure 5B The results of the competitive binding experiments for T119, E83, E87, EII81, E85, E54, EII55, E31, E105 and E84 are shown. Figure 5C The results of the competitive binding experiments for T119, E164, E172, E108, E16, E36, E61, E88 and E39 are shown. Figure 5D The results of the competitive binding experiments for T119, E90, EII33, E200a, E94, E113 and EII52 are shown.

[0023] Figure 6A and 6B The figures show the melting temperature curves and aggregation temperature curves of anti-GM-CSFRα antibodies Mab-IgG1, T119-IgG1, E35-IgG1, and E35b-IgG1 analyzed by UNCLE. Figure 6A The figure shows the melting temperature curve of the antibody. Figure 6B The figure shows the antibody aggregation temperature curve.

[0024] Figure 7 The results of TF-1 cell proliferation experiments using the lead antibody T119 and the lead optimized antibody are shown.

[0025] Figure 8 The results of human granulocyte deformability assays using E35, E108, and E87b antibodies are shown.

[0026] Figure 9 The results of the E35 antibody assay on cynomolgus monkey granulocytes are shown.

[0027] Figure 10 The results of granulocyte survival assays using antibodies E35, E108, and E87b are shown.

[0028] Figure 11 The results of the CD11b expression experiment are shown for E35, E87b and the control antibody Mab.

[0029] Figure 12A and 12B The results of the TNFα release experiment are shown for E35, E87b, and Mab. Figure 12A The results show the experimental results of TNFα release of E35, E87b compared to Mab, measured by the Human Macrophage / Microglia Panel. Figure 12BThe results show the experimental results of TNFα release by E35, E87b compared with Mab, as measured by ELISA.

[0030] Figure 13 The results show the experimental results of IL-1β release of E35, E87b, and Mab.

[0031] Figure 14A and 14B The results of pharmacokinetic analysis of Mab and E35 in rats, measured by ELISA, are shown. Figure 14A The figures show the serum antibody concentrations after intravenous injection of 2 mg / kg Mab or E35, respectively. Figure 14B The pharmacokinetic results are shown for intravenous injection of 20 mg / kg Mab or E35, respectively.

[0032] Figure 15 The results of pharmacokinetic analysis of Mab and E35 in cynomolgus monkeys, measured by ELISA, are shown.

[0033] Figures 16A-16D The image shown is a FACS diagram of in vivo granulocyte deformability analysis after administration of Mab-IgG4 or E35-IgG4 to cynomolgus monkeys. Figure 16A The image shown is a FACS diagram of granulocytes before antibody administration. Figure 16B The image shown is a FACS diagram of granulocyte deformability analysis 14 days after antibody administration. Figure 16C The image shown is a FACS diagram of granulocyte deformability analysis 21 days after antibody administration. Figure 16D The results show the in vivo granulocyte deformation trend analysis from before antibody administration to 21 days after antibody administration.

[0034] Figure 17A-17G The results show the inhibitory effect of E35-IgG4 on GM-CSF-induced inflammatory cell proliferation. Figure 17A The results show the inhibitory effect of E35-IgG4 on GM-CSF-induced leukocyte proliferation. Figure 17B The results show the inhibitory effect of E35-IgG4 on GM-CSF-induced neutrophil proliferation. Figure 17C The results show the inhibitory effect of E35-IgG4 on GM-CSF-induced lymphocyte proliferation. Figure 17D The results show the inhibitory effect of E35-IgG4 on GM-CSF-induced basophil proliferation. Figure 17E The results show the inhibitory effect of E35-IgG4 on GM-CSF-induced eosinophil proliferation. Figure 17F The results show the inhibitory effect of E35-IgG4 on GM-CSF-induced monocyte proliferation. Figure 17GThe results show the inhibitory effect of E35-IgG4 on GM-CSF-induced erythrocyte proliferation.

[0035] Figures 18A-18C The binding affinity of E35-IgG4, E87b-IgG4, and T119-IgG4 to wild-type GMRαh and GMRαh containing exemplary amino acid residue mutations, as measured by ELISA, is shown. Figure 18A The figure shows the binding affinity of E35-IgG4 to wild-type GMRαh and mutant GMRαh. Figure 18B The figure shows the binding affinity of E87b-IgG4 to wild-type GMRαh and mutant GMRαh. Figure 18C The figure shows the binding affinity of T119-IgG4 to wild-type GMRαh and mutant GMRαh.

[0036] Figures 19A-19B The image shows the sequence alignment of the variable region of the anti-GM-CSFRα antibody, with the complementarity-determining region already marked. Figure 19A The image shows a sequence alignment diagram of the heavy chain variable field sequence. Figure 19B The image shows a sequence alignment diagram of the light chain variable field sequence.

[0037] Figure 20 The figure shows the numbers of amino acid residues 16-296 in GM-CSFRα.

[0038] Detailed description of the invention

[0039] This invention relates, in one aspect, to an anti-GM-CSFRα antibody molecule. Through a combination of screening a natural scFv phage library, affinity maturation, and appropriately designed biochemical and biological experiments, we have identified a highly efficient antibody molecule capable of binding to human GM-CSFRα and inhibiting the action of human GM-CSF on its receptor. The results presented herein demonstrate that our antibody binds to different regions or epitopes of GM-CSFRα compared to the known anti-GM-CSFRα antibody Mavrilimumab, and surprisingly, in various biological experiments, our antibody has proven to be even more effective than Mavrilimumab.

[0040] The anti-GM-CSFRα antibodies involved in this invention include, for example, full-length anti-GM-CSFRα antibodies, anti-GM-CSFRα single-chain antibodies (scFvs), anti-GM-CSFRα Fc fusion proteins, multispecific (e.g., bispecific) anti-GM-CSFRα antibodies, anti-GM-CSFRα immunoconjugates, and the like.

[0041] On the one hand, it involves anti-GM-CSFRα antibodies that can specifically bind to epitopes on human GM-CSFRα, which contain the amino acid residues Val50, Glu59, Lys194, Lys195, Arg283, and Ile284 of human GM-CSFRα.

[0042] On the other hand, the present invention relates to anti-GM-CSFRα antibodies, wherein the anti-GM-CSFRα antibody comprises a heavy chain variable domain (V H ), the V H Contains: an HC-CDR1 containing the sequence X1LX2X3H (SEQ ID NO:76), wherein X1 is E, N, G, D, M, S, P, F, Y, A, V, K, W, R, or C, X2 is S, C, or P, and X3 is I or M; an HC-CDR2 containing the sequence GFDX1X2X3X4EX5X6YAQKX7QG (SEQ ID NO:77), wherein X1 is P, G, T, S, or V, X2 is E, D, G, or A, X3 is D, G, I, W, S, or V, X4 is G, E, D, or H, X5 is T or A, X6 is N or I, and X7 is S or F; and a HC-CDR2 containing the sequence GRYX1X2X3X4X5X6YGFDY (SEQ ID NO:77). HC-CDR3 (IDNO:78), wherein X1 is C, T, S, I, A or V; X2 is S, G, E, F, W, H, I, V, N, Y, T or R; X3 is T, H, L, F, P, I, S, Y, K, A, D, V, N or G; X4 is D, A, M, Y, F, S, T, G or W; X5 is T, S, F, Q, A, N, L, E, I, G or M; X6 is C, T, N, S or A; and a light chain variable domain (V L ), the V LContains: an LC-CDR1 containing the sequence RAX1X2X3VX4X5X6LA (SEQ ID NO:293), wherein X1 is S, L, N, A, K, R, I, Q, G, T, H, M, or C; X2 is Q, Y, P, A, I, F, T, R, V, L, E, S, or C; X3 is S, H, W, L, R, K, T, P, I, F, V, E, A, or Q; X4 is S, L, W, M, A, Y, K, R, G, T, E, V, N, F, or C; X5 is S, T, R, A, H, Q, P, M, L, or G; and X6 is Y, L, or F; and a LC-CDR1 containing the sequence X1X2X3LA (SEQ ID NO:293), wherein X1 is S, L, N, A, Q, P, M, L, or G; and X6 is Y, L, or F. LC-CDR2 (NO:294), wherein X1 is G or T, X2 is A, G, R, H, K, S, T, M or F, and X3 is S, A, W, R, L, T, Q, F, Y, H or N; and LC-CDR3 containing the sequence QQYX1X2X3PX4T (SEQ ID NO:79), wherein X1 is N, D, S, R, A, T, L, Y, Q, W or G, X2 is N, D, E, T, Y, G, A, M, F, S, I or L, X3 is W, S, P, V, G or R, and X4 is P, Y, H, S, F, N, D, V or G.

[0043] It also relates to nucleic acids encoding anti-GM-CSFRα antibodies, compositions containing anti-GM-CSFRα antibodies, and methods for preparing and using anti-GM-CSFRα antibodies.

[0044] definition

[0045] As described herein, "treatment" or "treatment" is a method of obtaining a beneficial or desired outcome, including clinical outcomes. For the purposes of this application, the beneficial or desired clinical outcomes include, but are not limited to, one or more of the following: relief of one or more symptoms caused by a disease, reduction of disease severity, stabilization of the disease (e.g., prevention or delay of disease progression), prevention or delay of disease progression (e.g., metastasis), prevention or delay of disease recurrence, delay or slowing of disease progression, improvement of disease status, relief of the disease (partial or complete), reduction of the dosage of one or more other medications required to treat the disease, delay of disease progression, improvement or enhancement of quality of life, weight gain, and / or prolongation of survival. Simultaneously, "treatment" also includes a reduction in disease pathological outcomes (e.g., tumor volume in the case of cancer). The methods of this application consider any one or more aspects of these treatments.

[0046] The term "antibody" includes both full-length antibodies and their antigen-binding fragments. Full-length antibodies consist of two heavy chains and two light chains. Variable regions in both the light and heavy chains are responsible for antigen binding. The variable regions in both chains typically include three hypervariable loops, known as complementarity-determining regions (CDRs) (light chain (LC) CDRs include LC-CDR1, LC-CDR2, and LC-CDR3; heavy chain (HC) CDRs include HC-CDR1, HC-CDR2, and HC-CDR3). The CDR boundaries of the antibodies or antigen-binding fragments disclosed herein can be defined or identified according to the Kabat, Chothia, or Al-Lazikani conventions (Al-Lazikani 1997; Chothia 1985; Chothia 1987; Chothia 1989; Kabat 1987; Kabat 1991). Three CDR regions of the heavy or light chain are inserted between flanking segments called framework regions (FRs), which are more conserved than the CDR regions and form a scaffold supporting the hypervariable loop. The constant regions of the heavy and light chains do not participate in antigen binding but exhibit a variety of effector functions. Antibodies are classified based on the amino acid sequence of their heavy chain constant regions. The five major classes or isotypes of antibodies are IgA, IgD, IgE, IgG, and IgM, characterized by having α, δ, ε, γ, and μ-type heavy chains, respectively. Several major antibody classes are subclassed into subclasses such as IgG1 (γ1 heavy chain), IgG2 (γ2 heavy chain), IgG3 (γ3 heavy chain), IgG4 (γ4 heavy chain), IgA1 (α1 heavy chain n), or IgA2 (α2 heavy chain).

[0047] As described herein, the term "antigen-binding fragment" refers to an antibody fragment, including, for example, double-chain antibodies, Fab, Fab', F(ab')2, Fv fragments, disulfide-stabilized Fv fragments (dsFv), (dsFv)2, bispecific dsFv (dsFv-dsFv'), disulfide-stabilized double-chain antibodies (ds double-chain antibodies), single-chain antibodies (scFv), scFv dimers (bivalent double-chain antibodies), multispecific antibodies composed of antibody fragments containing one or more CDRs, single-domain antibodies, nanobodies, domain antibodies, bivalent domain antibodies, or any other antibody fragment capable of binding to an antigen but not containing a complete antibody structure. The antigen-binding fragment is capable of binding the same antigen to a parent antibody or a parent antibody fragment (such as a parental scFv). In some embodiments, the antigen-binding fragment may include one or more CDRs from a specific human antibody grafted onto a frame region from one or more different human antibodies.

[0048] As described herein, the term "epitope" refers to a specific atom or amino acid group on an antigen that an antibody or antibody portion binds to. If two antibodies or antibody portions exhibit competitive binding to an antigen, they may bind to the same epitope on the antigen.

[0049] As described herein, when the first antibody inhibits the binding of the second antibody to the GM-CSFRα target by at least 50% (e.g., at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) at an equimolar concentration, the first antibody “competitively” binds to the GM-CSFRα target with the second antibody, and vice versa. PCT Publication WO 03 / 48731 describes a high-throughput antibody “epitope classification” method based on cross-competition.

[0050] As described herein, the terms "specifically bind," "specifically recognize," or "specific to" refer to measurable and reproducible interactions, such as antibody binding to a target that confirms the presence of that target in a heterogeneous group of molecules, including biomolecules. For example, an antibody's ability to specifically recognize a target (which may be an epitope) means that the antibody binds to that target with higher affinity, stronger binding, and / or more persistent binding compared to binding to other targets. In some embodiments, an antibody that specifically recognizes an antigen reacts with one or more antigenic determinants of the antigen with a binding affinity at least 10 times greater than its binding affinity to other targets.

[0051] As described herein, an “isolated” anti-GM-CSFRα antibody is an anti-GM-CSFRα antibody that (1) is not related to a naturally occurring protein, (2) does not contain other proteins of the same origin, (3) is expressed by cells of a different species, or (4) does not exist in nature.

[0052] As described herein, the term “isolated nucleic acid” refers to a nucleic acid of genomic, cDNA, or synthetic origin, or some combination thereof. Depending on its origin, the “isolated nucleic acid” (1) is not related to all or part of the polynucleotides in “isolated nucleic acids” found in nature, (2) can be operatively linked to polynucleotides that are not linked to it in their natural state, or (3) does not exist in nature as part of a longer sequence.

[0053] As described in this article, the term “CDR” or “complementarity-determining region” refers to a discontinuous antigen-binding site found within the variable domain of heavy and light chain polypeptides. In the literature, Kabat et al., J. Biol. Chem. 252: 6609-6616 (1977); Kabatet al., USDept. of Health and Human Services, "Sequences of proteins of immunological interest" (1991); Chothia et al., J. Mol. Biol. 196: 901-917 (1987); Al-Lazikani B. et al. al., J. Mol. Biol., 273: 927-948 (1997); MacCallum et al., J. Mol. Biol. 262: 732-745 (1996); Abhinandan and Martin, Mol. Immunol., 45: 3832-3839 (2008); Lefranc MPet These specific regions have been described in al., Dev. Comp. Immunol., 27:55-77 (2003); and Honegger and Plückthun, J. Mol. Biol., 309:657-670 (2001), where these definitions include overlap or subsets of amino acid residues when compared relative to each other. However, each definition of a CDR for an antibody or grafted antibody or variant thereof mentioned in this application is meant to be within the scope of the terminology defined and used herein. The amino acid residues containing the CDRs defined by the foregoing references are listed in Table 1 for comparison. Algorithms and interfaces for CDR prediction are known in the art, including, for example, those described in Abhinandan and Martin, Mol. Immunol., 45:3832-3839 (2008); Ehrenmann F. et al., Nucleic Acids Res., 38:D301-D307 (2010); and Adolf-Bryfogle J. et al., Nucleic Acids Res., 43:D432-D438 (2015). The entire contents of the references cited in this paragraph are incorporated herein by reference for the purposes of this application and one or more claims that may be included herein.

[0054] Table 1: CDR Definition

[0055]

[0056] 1 The amino acid residue numbering follows the nomenclature method described in Kabat et al. above.

[0057] 2 The amino acid residue numbering follows the nomenclature method described in Chothia et al. above.

[0058] 3 The amino acid residue numbering follows the nomenclature method described in MacCallum et al. above.

[0059] 4 The amino acid residue numbering follows the nomenclature method described in Lefranc et al. above.

[0060] 5 The amino acid residue numbering follows the nomenclature method described in Honegger and Plückthun.

[0061] The term "chimeric antibody" refers to an antibody whose heavy chain and / or light chain portion is identical or homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular antibody species or subclass, while the remaining portion of this chain is identical or homologous to the corresponding sequence in an antibody from another genus or belonging to another antibody species or subclass, as well as fragments of such antibodies, provided they possess the biological activity described in this application (see US Patent No. 4,816,567; and Morrison et al., Proc. Natl. Acad. Sci. USA, 81:6851-6855 (1984)).

[0062] "Fv" is the smallest antibody fragment containing a complete antigen recognition and binding site. This fragment is a dimer composed of a heavy chain variable domain and a light chain variable domain linked tightly non-covalently. The folding of these two domains derives six hypervariable rings (three rings from each of the light and heavy chains), which contribute amino acid residues for antigen binding and confer the antibody's specificity for antigen binding. However, even a single variable domain (or half of the Fv fragment, containing only three CDRs, which are antigen-specific) can recognize and bind to antigens, although its affinity is lower than that of the complete binding site.

[0063] "Single-chain Fv", also abbreviated as "sFv" or "scFv", is a type of Fv containing V molecules linked together to form a single polypeptide chain. H and V L Antibody fragments of antibody domains. In some embodiments, the scFv polypeptide further includes V H and V LThe connecting polypeptide between the domains allows scFv to form an ideal structure for antigen binding. For an overview of scFv, see Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0064] The term "diabodies" refers to antibodies that are formed in V... H and V L Between domains, short linkers (e.g., 5–10 residues) are used to construct scFv fragments (see above paragraph), creating small antibody fragments. This allows the variable domains to pair between chains rather than within chains, resulting in a bivalent fragment, i.e., a fragment with two antigen-binding sites. Bispecific double-chain antibodies are heterodimers of two “crossed” scFv fragments, where the V0 of the two antibodies... H and V L The domains are located on different polypeptide chains. Double-chain antibodies have been fully described in EP 404,097; WO 93 / 11161; Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0065] The “humanized” form of non-human (e.g., rodent) antibodies is a chimeric antibody, which includes a minimal sequence from the non-human antibody. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies), where the hypervariable region (HVR) residues of the receptor antibody are replaced by hypervariable region residues from non-human species such as mice, rats, rabbits, or non-human mammals that possess desirable antibody specificity, affinity, and performance (donor antibody). In some cases, residues in the human immunoglobulin framework region are replaced by corresponding non-human residues. Additionally, humanized antibodies may include residues not found in either the receptor or donor antibody. These modifications can further improve antibody performance. Typically, humanized antibodies contain substantially all, at least one, usually two, variable domains, where all or substantially all of the hypervariable loops correspond to the hypervariable loops of the non-human immunoglobulin, and all or substantially all of the framework regions are human immunoglobulin sequences. Humanized antibodies may also optionally include at least a portion of the immunoglobulin constant region (Fc), typically the constant region of a human immunoglobulin. For details, please refer to Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992).

[0066] The "percentage of amino acid sequence identity (%)" or "homology" of the peptide and antibody sequences identified in this paper is defined as the percentage of identical amino acid residues in the candidate sequence and the peptide sequence to be compared, with any conserved substitutions of amino acid residues considered as part of the sequence identity after sequence alignment. The percentage of amino acid sequence identity can be determined by a variety of alignment methods within the scope of the art, for example, using publicly available computer software such as BLAST, BLAST-2, ALIGN, Megalign (DNASTAR), or MUSCLE software. Those skilled in the art can determine suitable parameters for measuring alignment, including any algorithm required to achieve maximum alignment across the full length of the compared sequences. However, for the purposes of this paper, the percentage of amino acid sequence identity values ​​were generated using the sequence alignment computer program MUSCLE (Edgar, RC, Nucleic Acids Research 32(5):1792-1797, 2004; Edgar, RC, BMC Bioinformatics 5(1):113, 2004).

[0067] The term "Fc receptor" or "FcR" is used to describe receptors that bind to the Fc region of an antibody. In some embodiments, the FcR described in this application is an FcR that binds to an IgG antibody (a type of γ receptor) and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternative splicing forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), which have similar amino acid sequences and differ primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine activation motif (ITAM) in its cytoplasmic domain. The inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine inhibition motif (ITIM) in its cytoplasmic domain (see M.in.). Annu.Rev.Immunol.15:203-234 (1997)). The term also includes allotypes, such as FcγRIIIA allotypes: FcγRIIIA-Phe158, FcγRIIIA-Val158, FcγRIIA-R131, and / or FcγRIIA-H131. FcRs are described in Ravetch and Kinet, Annu.Rev.Immunol 9:457-92 (1991) and Capel et al., Immunomethods 4:25-34 (1994); and de Haas et al., J.Lab.Clin.Med.126:330-41 (1995). In this application, the term FcR covers other types of FcRs, including those to be identified in the future. The term FcR also includes the neonatal receptor FcRn, which is responsible for transferring maternal IgGs to the newborn (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

[0068] The term "FcRn" refers to the neonatal Fc receptor (FcRn). FcRn is structurally similar to the major histocompatibility complex (MHC), consisting of an α-chain non-covalently bound to β2-microglobulin. The various functions of the neonatal Fc receptor FcRn are described in Ghetie and Ward (2000) Annu. Rev. Immunol. 18, 739-766. FcRn plays an important role in the passive transport of immunoglobulin IgGs from the mother to the newborn and in regulating serum IgG levels. As a rescue receptor, FcRn can bind intact both intracellularly and intercellularly, transporting endocytized IgG and rescuing it from the default degradation pathway.

[0069] The "CH1 domain" of the human IgG Fc region usually refers to the region extending from amino acid 118 to amino acid 215 (EU numbering system).

[0070] The "hinge region" is typically defined as extending from Glu at position 216 to Pro at position 230 of human IgG1 (Burton, Molec. Immunol. 22:161-206 (1985)). By placing the first and last cysteine ​​residues that form the inter-heavy chain disulfide bond at the same positions as IgG1, it is possible to align the hinge regions of other IgG isotypes with the IgG1 sequence.

[0071] The "CH2 domain" of the human IgG Fc region typically extends from amino acid 231 to amino acid 340. The unique feature of the CH2 domain is that it does not pair tightly with another region. Instead, two N-terminal branched sugar chains are inserted between the two CH2 domains of the intact native IgG molecule. It is speculated that the sugars can provide substitutions for domain-to-domain pairing, helping to maintain the stability of the CH2 domain. (Burton, Molec Immunol. 22:161-206 (1985)).

[0072] The “CH3” domain includes the extension from the C-terminal residue to the CH2 domain within the Fc region (from amino acid 341 to the C-terminus of the antibody sequence, typically amino acid 446 or 447 of IgG).

[0073] "Functional Fc fragments" possess the "effective functions" inherent in the natural Fc region sequence. Typical effector functions include C1q binding; complement-dependent cytotoxicity (CDC); Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and downregulation of cell surface receptors (such as B cell receptors; BCR). These effector functions typically require the Fc region to bind to a binding domain (such as the antibody variable region) and can be evaluated using a variety of experimental methods known in the art.

[0074] Antibodies with IgG Fc variants exhibiting "altered" FcR binding affinity or ADCC activity show enhanced or weakened FcR binding and / or ADCC activity compared to the parent peptide or peptides containing the native Fc sequence. Fc variants exhibiting "enhanced" FcR binding have a higher affinity (e.g., lower apparent K) with at least one FcR compared to the parent peptide or peptides containing the native IgG Fc sequence. d or IC 50(Value). In some embodiments, the binding affinity is approximately 3-fold stronger than that of the parent peptide, for example, 5, 10, 25, 50, 60, 100, 150, 200, or even up to 500-fold stronger, or any one of 25% to 1000% stronger. Fc variants exhibiting “reduced binding” to FcRs have at least one lower affinity (e.g., higher apparent Kc) compared to the parent peptide. d or IC 50 (Value). Compared with the parent peptide, its binding ability is reduced by 40% or more.

[0075] Antibody-dependent cell-mediated cytotoxicity (ADCC) is a form of cytotoxicity in which secreted Ig binds to Fc receptors (FcRs) present on certain cytotoxic cells, such as natural killer (NK) cells, neutrophils, and macrophages. This allows these cytotoxic effector cells to specifically bind to target cells carrying antigens and subsequently kill the target cells using cytotoxins. Antibodies "arm" the cytotoxic cells and are essential for this killing. Among the main cell types mediating ADCC, NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcRs on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). The ADCC activity of target molecules can be assessed by in vitro ADCC experiments, described in U.S. Patent Nos. 5,500,362 or 5,821,337. Effector cells suitable for such experiments include peripheral blood mononuclear cells (PBMCs) and natural killer cells (NK cells). Alternatively, or additionally, the ADCC activity of the target molecule can also be assessed in vivo, as described, for example, in animal models disclosed in Clynes et al. PNAS (USA) 95:652-656 (1998).

[0076] Peptides containing Fc region variants exhibit "enhanced ADCC activity" or are more effective at mediating ADCC effects in the presence of human effector cells compared to peptides containing wild-type IgG Fc or parental peptides. These peptides, when experimentally present in substantially the same quantity as those containing wild-type IgG Fc (or parental peptides), mediate ADCC more effectively both in vitro and in vivo. Such variants are typically identified using any in vitro ADCC assay known in the art, such as experiments or methods for identifying ADCC activity, for example in animal models. In some embodiments, such variants mediate ADCC more effectively than wild-type Fc (or parental peptides), by approximately 5 to 100-fold, for example, approximately 25 to approximately 50-fold.

[0077] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (a subclass with a suitable structure) that binds to a homologous antigen. To assess complement activation, CDC assays can be performed, as described in Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996). Peptide variants with altered Fc region amino acid sequences and increased or decreased C1q binding capacity are described in U.S. Patent Nos. 6,194,551B1 and WO99 / 51642. The contents of these patent publications are expressly incorporated herein by reference. See also Idusogie et al. J. Immunol. 164:4178-4184 (2000).

[0078] Unless otherwise stated, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences in their degenerate form that encode the same amino acid sequence. Nucleotide sequences encoding proteins or RNA may also include introns; for example, nucleotide sequences encoding proteins may contain introns in certain forms.

[0079] The term "operationally ligated" refers to a functional link between a regulatory sequence and a heterologous nucleotide sequence, thereby enabling the latter to be expressed. For example, the first and second nucleotide sequences are operably ligated when they are in a functional relationship. Similarly, if a promoter affects the transcription or expression of a coding sequence, the promoter and coding sequence are operably ligated. Typically, operably ligated DNA sequences are contiguous, and, if necessary, two protein-coding regions can be ligated within the same reading frame.

[0080] "Homology" refers to the sequence similarity or identity between two polypeptides or two nucleic acid molecules. If the same position in two compared sequences is the same base or amino acid monomer subunit, for example, if both DNA molecules have adenine at the same position, then the two DNA molecules are homologous at that position. The percentage of homology between two sequences is a function calculated by multiplying the number of matching or homologous positions in the two sequences to the total number of positions, and then multiplying by 100. For example, if 6 out of 10 positions in two sequences are matching or homologous, then the two sequences are 60% homologous. For instance, the DNA sequences ATTGCC and TATGGC have 50% homology. Generally, when comparing two sequences, the goal is to obtain the maximum homology.

[0081] The “effective amount” of the anti-GM-CSFRα antibody or composition disclosed herein refers to an amount sufficient to achieve a specific purpose. The “effective amount” can be determined empirically and by known methods associated with the stated purpose.

[0082] The term "therapeutic effective dose" refers to the dosage at which the anti-GM-CSFRα antibody or its composition disclosed herein can effectively treat an individual's disease or symptoms. For example, in the case of cancer, a therapeutic effective dose of the anti-GM-CSFRα antibody or its composition means the ability to reduce the number of cancer cells; reduce the size or weight of the tumor; inhibit (i.e., to some extent slow down or preferably stop) the invasion of tumor cells into surrounding organs; inhibit (i.e., to some extent slow down or preferably stop) tumor metastasis; inhibit tumor growth to some extent; and / or alleviate one or more cancer-related symptoms to some extent. The anti-GM-CSFRα antibody or its composition disclosed herein can, to some extent, prevent and / or kill existing tumor cells; this can be cytoseptic or cytotoxic. In some embodiments, a therapeutic effective dose means the dosage at which the patient's survival is prolonged. In some embodiments, a therapeutic effective dose means the dosage at which the patient's progression-free survival is improved.

[0083] As used herein, "pharmaceuticalally acceptable" or "pharmacologically compatible" means a material that is biologically inactive or otherwise free from undesirable properties, such as the ability to be incorporated into a pharmaceutical composition given to a patient without causing significant adverse biological reactions, or that does not interact harmfully with any other components contained in the composition. Pharmaceutically acceptable carriers or excipients preferably meet the required standards for toxicological or manufacturing testing and / or are included in the inactive ingredient guidelines prepared by the U.S. Food and Drug Administration.

[0084] The embodiments of the application described herein should be understood to include embodiments that are “composed of” and / or “substantially composed of”.

[0085] In this article, “about” is used to refer to a numerical value or parameter, including (and descriptions) variations for that value or parameter itself. For example, a description involving “about X” includes a description of “X”.

[0086] As used in this article, mentioning "not" a value or parameter usually means and describes something other than a certain value or parameter. For example, "this method cannot be used to treat type X cancer" means that this method is generally used to treat other types of cancer besides type X.

[0087] Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “the” used herein and in the claims include plural objects.

[0088] Anti-GM-CSFRα antibody

[0089] On one hand, this application relates to anti-GM-CSFRα antibodies that specifically bind to GM-CSFRα. These anti-GM-CSFRα antibodies include, but are not limited to, humanized antibodies, chimeric antibodies, mouse antibodies, human antibodies, and antibody molecules comprising heavy chain and / or light chain CDRs as described herein. On the other hand, this application relates to isolated antibodies that bind to GM-CSFRα. Anticipated anti-GM-CSFRα antibodies include, for example, full-length anti-GM-CSFRα antibodies (such as full-length IgG1 or IgG4), anti-GM-CSFRα single-chain antibodies, anti-GM-CSFRα Fc fusion proteins, multispecific (e.g., bispecific) anti-GM-CSFRα antibodies, anti-GM-CSFRα immunoconjugates, and the like. In some embodiments, the anti-GM-CSFRα antibody is a full-length antibody (such as full-length IgG1 or IgG4) or its antigen-binding fragment that specifically binds to GM-CSFRα. In some embodiments, the anti-GM-CSFRα antibody is Fab, Fab', F(ab)'2, Fab'-SH, a single-chain antibody (scFv), an Fv fragment, dAb, Fd, a nanobody, a double-chain antibody, or a linear antibody. In some embodiments, an antibody that specifically binds to GM-CSFRα means that the antibody's affinity for GM-CSFRα is at least 10 times greater than its affinity for binding to non-target antibodies (including, for example, 10, 10). 2 10 3 10 4 10 5 10 6 , or 10 7 (Multiple times). In some embodiments, non-target refers to an antigen that is not GM-CSFRα. Binding affinity can be determined by methods known in the art, such as ELISA, fluorescence-activated cell sorting (FACS) analysis, or radioimmunoprecipitation (RIA). K d The value can be determined by methods known in the art, such as surface plasmon resonance (SPR) or biolayer interferometry (BLI).

[0090] While this document discusses anti-GM-CSFRα antibodies containing human sequences (e.g., human heavy and light chain variable domains containing human CDR sequences), non-human anti-GM-CSFRα antibodies are also considered. In some embodiments, a non-human anti-GM-CSFRα antibody comprises the human CDR sequence and non-human frame region sequence of the anti-GM-CSFRα antibody described herein. In some embodiments, the non-human frame region sequence comprises any sequence that can be used to generate heavy and / or light chain variable domains using one or more human CDR sequences as described herein, including, for example, mammals such as mice, rats, rabbits, pigs, cattle (e.g., bulls, buffalo), deer, sheep, goats, chickens, cats, dogs, ferrets, primates (e.g., apes, macaques), etc. In some embodiments, a non-human anti-GM-CSFRα antibody comprises an anti-GM-CSFRα antibody produced by transplanting one or more human CDR sequences described herein into a non-human frame region (e.g., a mouse or chicken frame region sequence).

[0091] The complete amino acid sequence of the exemplary human GM-CSFRα contains or consists of the amino acid sequence shown in SEQ ID NO: 148.

[0092] 1 MLLLVTSLLL CELPHPAFLL IPEKSDLRTV APASSLNVRF DSRTMNLSWD CQENTTFSKC

[0093] 61 FLTDKKNRVV EPRLSNNECS CTFREICLHE GVTFEVHVNT SQRGFQQKLL YPNSGREGTA

[0094] 121 AQNFSCFIYN ADLMNCTWAR GPTAPRDVQY FLYIRNSKRR REIRCPYYIQ DSGTHVGCHL

[0095] 181 DNLSGLTSRN YFLVNGTSRE IGIQFFDSLL DTKKIERFNP PSNVTVRCNT THCLVRWKQP

[0096] 241 RTYQKLSYLD FQYQLDVHRK NTQPGTENLL INVSGDLENR YNFPSSEPRA KHSVKIRAAD

[0097] 301 VRILNWSSWS EAIEFGSDDG NLGSVYIYVL LIVGTLVCGI VLGLFKRFL RIQRLFPPVP

[0098] 361 QIKDKLNDNH EVEDEIIWEE FTPEEGKGYR EEVLTVKEIT

[0099] An exemplary human GM-CSFRα extracellular region amino acid sequence contains or consists of the amino acid sequence shown in SEQ ID NO: 149.

[0100] 1 MLLLVTSLLL CELPHPAFLL IPEKSDLRTV APASSLNVRF DSRTMNLSWD CQENTTFSKC

[0101] 61 FLTDKKNRVV EPRLSNNECS CTFREICLHE GVTFEVHVNT SQRGFQQKLL YPNSGREGTA

[0102] 121 AQNFSCFIYN ADLMNCTWAR GPTAPRDVQY FLYIRNSKRR REIRCPYYIQ DSGTHVGCHL

[0103] 181 DNLSGLTSRN YFLVNGTSRE IGIQFFDSLL DTKKIERFNP PSNVTVRCNT THCLVRWKQP

[0104] 241 RTYQKLSYLD FQYQLDVHRK NTQPGTENLL INVSGDLENR YNFPSSEPRA KHSVKIRAAD

[0105] 301 VRILNWSSWS EAIEFGSDDG

[0106] In some embodiments, the anti-GM-CSFRα antibody described herein specifically recognizes an epitope within human GM-CSFRα. In some embodiments, the anti-GM-CSFRα antibody cross-reacts with GM-CSFRα from species other than humans. In some embodiments, the anti-GM-CSFRα antibody is completely specific to human GM-CSFRα and does not exhibit cross-reactivity with other non-human species or types.

[0107] In some embodiments, the anti-GM-CSFRα antibody described herein specifically binds to a linear epitope within human GM-CSFRα. In some embodiments, the anti-GM-CSFRα antibody described herein specifically binds to a non-linear epitope within GM-CSFRα. In some embodiments, the anti-GM-CSFRα antibody described herein specifically binds to an epitope on human GM-CSFRα, the epitope comprising one, two, three, four, five, or six amino acid residues selected from the group consisting of Val50, Glu59, Lys194, Lys195, Arg283, and Ile284 of human GM-CSFRα. In some embodiments, the anti-GM-CSFRα antibody described herein specifically binds to an epitope on human GM-CSFRα, the epitope comprising Val50, Glu59, Lys194, Lys195, Arg283, and Ile284 of human GM-CSFRα. In some embodiments, the anti-GM-CSFRα antibody described herein specifically binds to an epitope on human GM-CSFRα, said epitope comprising one, two, three, four, five, six, seven, eight, or nine amino acid residues from the group consisting of Val50, Glu59, Lys194, Lys195, Arg283, Ile284, Val51, Thr63, and Ile196. In some embodiments, the anti-GM-CSFRα antibody described herein specifically binds to an epitope on human GM-CSFRα, said epitope comprising amino acid residues Val50, Glu59, Lys194, Lys195, Arg283, Ile284, Val51, Thr63, and Ile196. In some embodiments, the anti-GM-CSFRα antibody described herein specifically binds to an epitope on human GM-CSFRα, said epitope comprising 1, 2, 3, 4, 5, 6, 7, or 8 amino acid residues selected from the group consisting of Val50, Glu59, Lys194, Lys195, Arg283, Ile284, Leu191, and Ile196. In some embodiments, the anti-GM-CSFRα antibody described herein is capable of specifically binding to an epitope of human GM-CSFRα, said epitope comprising amino acid residues at positions Val50, Glu59, Lys194, Lys195, Arg283, Ile284, Leu191, and Ile196 of human GM-CSFRα. In some embodiments, the anti-GM-CSFRα antibody described herein specifically binds to an epitope on human GM-CSFRα, said epitope comprising 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid residues from the group consisting of Val50, Glu59, Lys194, Lys195, Arg283, Ile284, Arg49, Val51, Asn57, and Ser61.In some embodiments, the anti-GM-CSFRα antibody described herein specifically binds to an epitope on human GM-CSFRα, the epitope comprising the amino acid residues Val50, Glu59, Lys194, Lys195, Arg283, Ile284, Arg49, Val51, Asn57, and Ser61.

[0108] In some embodiments, the anti-GM-CSFRα antibody cross-reacts with at least one allelic variant of the GM-CSFRα protein (or a fragment thereof). In some embodiments, the allelic variant has up to 30 amino acid substitutions (e.g., any one of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or 30) compared to the naturally occurring GM-CSFRα protein (or a fragment thereof) (e.g., conserved substitutions). In some embodiments, the anti-GM-CSFRα antibody does not cross-react with any allelic variant of the GM-CSFRα protein (or a fragment thereof).

[0109] In some embodiments, the anti-GM-CSFRα antibody cross-reacts with at least one interspecies variant of the GM-CSFRα protein. In some embodiments, for example, the GM-CSFRα protein (or a fragment thereof) is human GM-CSFRα, and the interspecies variant of the GM-CSFRα protein (or a fragment thereof) is a variant found in cynomolgus monkeys. In some embodiments, the anti-GM-CSFRα antibody does not cross-react with any interspecies variant of the GM-CSFRα protein.

[0110] In some embodiments, any anti-GM-CSFRα antibody as described herein includes an antibody heavy chain constant region and an antibody light chain constant region. In some embodiments, the anti-GM-CSFRα antibody includes an IgG1 type heavy chain constant region. In some embodiments, the anti-GM-CSFRα antibody includes an IgG2 type heavy chain constant region. In some embodiments, the anti-GM-CSFRα antibody includes an IgG3 type heavy chain constant region. In some embodiments, the anti-GM-CSFRα antibody includes an IgG4 type heavy chain constant region. In some embodiments, the heavy chain constant region comprises (including a sequence consisting of or substantially consisting of) the amino acid sequence SEQ ID NO:145. In some embodiments, the heavy chain constant region comprises (including a sequence consisting of or substantially consisting of) the amino acid sequence SEQ ID NO:146. In some embodiments, the anti-GM-CSFRα antibody includes a λ light chain constant region. In some embodiments, the anti-GM-CSFRα antibody includes a κ light chain constant region. In some embodiments, the light chain constant region comprises (including a sequence consisting of or substantially consisting of) the amino acid sequence SEQ ID NO:147. In some embodiments, the anti-GM-CSFRα antibody includes an antibody heavy chain variable domain and an antibody light chain variable domain.

[0111] In some embodiments, the anti-GM-CSFRα antibody includes V H V H Contains: an HC-CDR1 containing the sequence X1LX2X3H (SEQ ID NO:76), wherein X1 is E, N, G, D, M, S, P, F, Y, A, V, K, W, R, or C, X2 is S, C, or P, and X3 is I or M; an HC-CDR2 containing the sequence GFDX1X2X3X4EX5X6YAQKX7QG (SEQ ID NO:77), wherein X1 is P, G, T, S, or V, X2 is E, D, G, or A, X3 is D, G, I, W, S, or V, X4 is G, E, D, or H, X5 is T or A, X6 is N or I, and X7 is S or F; and a HC-CDR2 containing the sequence GRYX1X2X3X4X5X6YGFDY (SEQ ID NO:77). NO:78) of HC-CDR3, wherein X1 is C, T, S, I, A or V; X2 is S, G, E, F, W, H, I, V, N, Y, T or R; X3 is T, H, L, F, P, I, S, Y, K, A, D, V, N or G; X4 is D, A, M, Y, F, S, T, G or W; X5 is T, S, F, Q, A, N, L, E, I, G or M; X6 is C, T, N, S or A; and V L The V LContains: an LC-CDR1 containing the sequence RAX1X2X3VX4X5X6LA (SEQ ID NO:293), wherein X1 is S, L, N, A, K, R, I, Q, G, T, H, M, or C; X2 is Q, Y, P, A, I, F, T, R, V, L, E, S, or C; X3 is S, H, W, L, R, K, T, P, I, F, V, E, A, or Q; X4 is S, L, W, M, A, Y, K, R, G, T, E, V, N, F, or C; X5 is S, T, R, A, H, Q, P, M, L, or G; and X6 is Y, L, or F; and a LC-CDR1 containing the sequence X1X2X3LA (SEQ ID NO:293), wherein X1 is S, L, N, A, Q, P, M, L, or G; and X6 is Y, L, or F. LC-CDR2 (NO:294), wherein X1 is G or T, X2 is A, G, R, H, K, S, T, M or F, and X3 is S, A, W, R, L, T, Q, F, Y, H or N; and LC-CDR3 containing the sequence QQYX1X2X3PX4T (SEQ ID NO:79), wherein X1 is N, D, S, R, A, T, L, Y, Q, W or G, X2 is N, D, E, T, Y, G, A, M, F, S, I or L, X3 is W, S, P, V, G or R, and X4 is P, Y, H, S, F, N, D, V or G.

[0112] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains: an HC-CDR1 containing the sequence ELX1X2H (SEQ ID NO: 295), wherein X1 is S, C, or P, and X2 is I or M; an HC-CDR2 containing the sequence GFDX1X2X3X4EX5X6YAQKX7QG (SEQ ID NO: 77), wherein X1 is P, G, T, S, or V, X2 is E, D, G, or A, X3 is D, G, I, W, S, or V, X4 is G, E, D, or H, X5 is T or A, X6 is N or I, and X7 is S or F; and a HC-CDR2 containing the sequence GRYX1X2X3X4X5X6YGFDY (SEQ ID NO: 295). NO:78) of HC-CDR3, wherein X1 is C, T, S, I, A or V; X2 is S, G, E, F, W, H, I, V, N, Y, T or R; X3 is T, H, L, F, P, I, S, Y, K, A, D, V, N or G; X4 is D, A, M, Y, F, S, T, G or W; X5 is T, S, F, Q, A, N, L, E, I, G or M; X6 is C, T, N, S or A; and V L The V LIt comprises: an LC-CDR1 containing the sequence RASQSVSSYLA (SEQ ID NO:51); an LC-CDR2 containing the sequence GASSRAT (SEQ ID NO:52); and an LC-CDR3 containing the sequence QQYX1X2X3PX4T (SEQ ID NO:79), wherein X1 is N, D, S, R, A, T, L, Y, Q, W, or G; X2 is N, D, E, T, Y, G, A, M, F, S, I, or L; X3 is W, S, P, V, G, or R; and X4 is P, Y, H, S, F, N, D, V, or G.

[0113] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H The HC-CDR1 comprises any amino acid sequence of SEQ ID NOs:1-4 or a variant thereof, the variant comprising up to 3 amino acid substitutions (e.g., any one of 1, 2, or 3); an HC-CDR2 comprises any amino acid sequence of SEQ ID NOs:5-16 or a variant thereof, the variant comprising up to 3 amino acid substitutions (e.g., any one of 1, 2, or 3); and an HC-CDR3 comprises any amino acid sequence of SEQ ID NOs:17-50 or a variant thereof, the variant comprising up to 3 amino acid substitutions (e.g., any one of 1, 2, or 3).

[0114] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H It comprises: an HC-CDR1 containing any amino acid sequence of SEQ ID NOs:1-4, an HC-CDR2 containing any amino acid sequence of SEQ ID NOs:5-16, and an HC-CDR3 containing any amino acid sequence of SEQ ID NOs:17-50.

[0115] In some embodiments, the anti-GM-CSFRα antibody includes V L The V L It comprises: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51 or a variant containing up to 3 amino acid substitutions (e.g., any one of 1, 2, or 3); an LC-CDR2 containing the amino acid sequence SEQ ID NO:52 or a variant containing up to 3 amino acid substitutions (e.g., any one of 1, 2, or 3); and an LC-CDR3 containing any of the amino acid sequences SEQ ID NOs:53-75 or a variant containing up to 3 amino acid substitutions (e.g., any one of 1, 2, or 3).

[0116] In some embodiments, the anti-GM-CSFRα antibody includes V L The V L It comprises: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing any of the amino acid sequences in SEQ ID NOs:53-75.

[0117] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains: an HC-CDR1 comprising any amino acid sequence of SEQ ID NOs:1-4 or a variant comprising up to 3 amino acid substitutions (e.g., any one of 1, 2, or 3); an HC-CDR2 comprising any amino acid sequence of SEQ ID NOs:5-16 or a variant comprising up to 3 amino acid substitutions (e.g., any one of 1, 2, or 3); and an HC-CDR3 comprising any amino acid sequence of SEQ ID NOs:17-50 or a variant comprising up to 3 amino acid substitutions (e.g., any one of 1, 2, or 3); and V L The V L It comprises: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51 or a variant containing up to 3 amino acid substitutions (e.g., any one of 1, 2, or 3); an LC-CDR2 containing the amino acid sequence SEQ ID NO:52 or a variant containing up to 3 amino acid substitutions (e.g., any one of about 1, 2, or 3); and an LC-CDR3 containing any amino acid sequence SEQ ID NOs:53-75 or a variant containing up to 3 amino acid substitutions (e.g., any one of 1, 2, or 3).

[0118] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains: an HC-CDR1 containing any of the amino acid sequences of SEQ ID NOs:1-4, an HC-CDR2 containing any of the amino acid sequences of SEQ ID NOs:5-16, and an HC-CDR3 containing any of the amino acid sequences of SEQ ID NOs:17-50; and V L The V L It comprises: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing any of the amino acid sequences SEQ ID NOs:53-75.

[0119] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:5, an HC-CDR3 containing the amino acid sequence SEQ ID NO:17, or a variant containing up to 5 amino acid substitutions in total in the HC-CDRs; and V L The V L It includes: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:54, or variants containing up to 5 amino acid substitutions in total in the LC-CDRs.

[0120] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:5, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:17; and V L The V L It comprises: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:54.

[0121] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:8, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:22, or variants containing up to 5 amino acid substitutions in total in the HC-CDRs; and V L The V L It includes: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:56, or variants containing up to 5 amino acid substitutions in total in the LC-CDRs.

[0122] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:8, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:22; and V L The V L It comprises: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:56.

[0123] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:7, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:23, or variants containing up to 5 amino acid substitutions in total in the HC-CDRs; and V L The V L It includes: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:57, or variants containing up to 5 amino acid substitutions in total in the LC-CDRs.

[0124] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:7, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:23; and V L The V L It comprises: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:57.

[0125] In some embodiments, the anti-GM-CSFRα antibody includes V H The VH Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:6, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:27, or variants containing up to 5 amino acid substitutions in the HC-CDRs; and V L The V L It comprises: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:57, or variants containing up to 5 amino acid substitutions in the LC-CDRs.

[0126] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:6, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:27; and V L The V L It comprises: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:57.

[0127] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:7, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:35, or variants containing up to 5 amino acid substitutions in the HC-CDRs; and V L The V L It includes: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:53, or variants containing up to 5 amino acid substitutions in the LC-CDRs.

[0128] In some embodiments, the anti-GM-CSFRα antibody includes V H The VH Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:7, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:35; and V L The V L It comprises: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:53.

[0129] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:7, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:37, or variants containing up to 5 amino acid substitutions in the HC-CDRs; and V L The V L It comprises: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:57, or variants containing up to 5 amino acid substitutions in the LC-CDRs.

[0130] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:7, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:37; and V L The V L It comprises: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:57.

[0131] In some embodiments, the anti-GM-CSFRα antibody includes V H The V HContains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:3, an HC-CDR2 containing the amino acid sequence SEQ ID NO:6, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:39, or variants containing up to 5 amino acid substitutions in the HC-CDRs; and V L The V L It includes: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:54, or variants containing up to 5 amino acid substitutions in the LC-CDRs.

[0132] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:3, an HC-CDR2 containing the amino acid sequence SEQ ID NO:6, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:39; and V L The V L It comprises: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:54.

[0133] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:7, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:35, or variants containing up to 5 amino acid substitutions in the HC-CDRs; and V L The V L It comprises: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:57, or variants containing up to 5 amino acid substitutions in the LC-CDRs.

[0134] In some embodiments, the anti-GM-CSFRα antibody includes V H The V HContains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:7, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:35; and V L The V L It comprises: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:57.

[0135] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:7, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:50, or variants containing up to 5 amino acid substitutions in the HC-CDRs; and V L The V L It comprises: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:57, or variants containing up to 5 amino acid substitutions in the LC-CDRs.

[0136] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:7, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:50; and V L The V L It comprises: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:57.

[0137] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Containing amino acid sequences SEQ IDNOs:1-50, or variants containing up to 5 amino acid substitutions; and V LThe V L Contains the amino acid sequence SEQ IDNOs:51-75, or a variant containing up to 5 amino acid substitutions. In some embodiments, the anti-GM-CSFRα antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:1-50; and V L The V L It contains the amino acid sequence SEQ ID NOs:51-75.

[0138] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Containing amino acid sequences SEQ IDNOs:1, 5, and 17, or variants containing up to 5 amino acid substitutions; and V L The V L Contains the amino acid sequences SEQ IDNOs:51, 52, and 54, or a variant containing up to five amino acid substitutions. In some embodiments, the anti-GM-CSFRα antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:1, 5, and 17; and V L The V L It contains the amino acid sequences SEQ ID NOs:51, 52 and 54.

[0139] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Containing amino acid sequences SEQ IDNOs:1, 8, and 22, or variants containing up to 5 amino acid substitutions; and V L The V L Containing amino acid sequences SEQ IDNOs:51, 52, and 56, or variants containing up to five amino acid substitutions. In some embodiments, the anti-GM-CSFRα antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:1, 8, and 22; and V L The V L It contains the amino acid sequences SEQ ID NOs:51, 52 and 56.

[0140] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Containing amino acid sequences SEQ IDNOs:1, 7, and 23, or variants containing up to 5 amino acid substitutions; and V L The VL Contains the amino acid sequences SEQ IDNOs:51, 52, and 57, or a variant containing up to five amino acid substitutions. In some embodiments, the anti-GM-CSFRα antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:1, 7, and 23; and V L The V L It contains the amino acid sequences SEQ ID NOs:51, 52 and 57.

[0141] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Containing amino acid sequences SEQ IDNOs:1, 6, and 27, or variants containing up to 5 amino acid substitutions; and V L The V L Contains any of the amino acid sequences SEQ ID NOs:51, 52, and 57, or a variant containing up to five amino acid substitutions. In some embodiments, the anti-GM-CSFRα antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:1, 6, and 27; and V L The V L It contains amino acid sequences SEQ ID NOs:51, 52 and 57.

[0142] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Containing amino acid sequences SEQ IDNOs:1, 7, and 35, or variants containing up to 5 amino acid substitutions; and V L The V L Contains the amino acid sequences SEQ IDNOs:51, 52, and 53, or a variant containing up to five amino acid substitutions. In some embodiments, the anti-GM-CSFRα antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:1, 7, and 35; and V L The V L It contains the amino acid sequences SEQ ID NOs:51, 52 and 53.

[0143] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Containing amino acid sequences SEQ IDNOs:1, 7, and 37, or variants containing up to 5 amino acid substitutions; and V LThe V L Contains the amino acid sequences SEQ IDNOs:51, 52, and 57, or a variant containing up to five amino acid substitutions. In some embodiments, the anti-GM-CSFRα antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:1, 7, and 37; and V L The V L It contains the amino acid sequences SEQ ID NOs:51, 52 and 57.

[0144] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Containing amino acid sequences SEQ IDNOs:3, 6, and 39, or variants containing up to 5 amino acid substitutions; and V L The V L Contains the amino acid sequences SEQ IDNOs:51, 52, and 54, or a variant containing up to five amino acid substitutions. In some embodiments, the anti-GM-CSFRα antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:3, 6, and 39; and V L The V L It contains the amino acid sequences SEQ ID NOs:51, 52 and 54.

[0145] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Containing amino acid sequences SEQ IDNOs:1, 7, and 35, or variants containing up to 5 amino acid substitutions; and V L The domain V L Contains the amino acid sequences SEQ ID NOs:51, 52, and 57, or a variant containing up to five amino acid substitutions. In some embodiments, the anti-GM-CSFRα antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:1, 7, and 35; V L The V L It contains the amino acid sequences SEQ ID NOs:51, 52 and 57.

[0146] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Variant containing the amino acid sequences SEQ IDNOs:1, 7, and 50, or variants containing up to 5 amino acid substitutions; and VL The V L Contains the amino acid sequences SEQ IDNOs:51, 52, and 57, or a variant containing up to five amino acid substitutions. In some embodiments, the anti-GM-CSFRα antibody comprises V H The V H Contains amino acid sequences SEQ ID NOs:1, 7, and 50; V L The V L It contains amino acid sequences SEQ ID NOs: 51, 52 and 57.

[0147] In some embodiments, the anti-GM-CSFRα antibody includes V H It comprises V having any of the amino acid sequences in SEQ ID NOs:80-121 and 246-287. H HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V having any of the amino acid sequences in SEQ ID NOs:122-144, 150-245, and 288-289. L LC-CDR1, LC-CDR2 and LC-CDR3.

[0148] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains one, two, or three HC-CDRs from the amino acid sequence SEQ ID NO:80. In some embodiments, the anti-GM-CSFRα antibody comprises V H The V H Contains one, two, or three HC-CDRs from the amino acid sequence SEQ ID NO:85. In some embodiments, the anti-GM-CSFRα antibody comprises V H The V H Contains one, two, or three HC-CDRs from the amino acid sequence SEQ ID NO:86. In some embodiments, the anti-GM-CSFRα antibody comprises V H The V H Contains one, two, or three HC-CDRs from the amino acid sequence SEQ ID NO:91. In some embodiments, the anti-GM-CSFRα antibody comprises V H The V H Contains one, two, or three HC-CDRs from the amino acid sequence SEQ ID NO:99. In some embodiments, the anti-GM-CSFRα antibody comprises V H The V HContains one, two, or three HC-CDRs from the amino acid sequence SEQ ID NO:101. In some embodiments, the anti-GM-CSFRα antibody comprises V H The V H Contains one, two, or three HC-CDRs from the amino acid sequence SEQ ID NO:103. In some embodiments, the anti-GM-CSFRα antibody comprises V H The V H It contains one, two, or three HC-CDRs of the amino acid sequence SEQ ID NO:121.

[0149] In some embodiments, the anti-GM-CSFRα antibody includes the V L The V L The antibody comprises one, two, or three LC-CDRs of the amino acid sequence SEQ ID NO:123. In some embodiments, the anti-GM-CSFRα antibody comprises one, two, or three LC-CDRs of the amino acid sequence SEQ ID NO:125. In some embodiments, the anti-GM-CSFRα antibody includes the V... L The V L Contains one, two, or three LC-CDRs from the amino acid sequence SEQ ID NO:126. In some embodiments, the anti-GM-CSFRα antibody comprises the V... L The V L It contains one, two, or three LC-CDRs of the amino acid sequence SEQ ID NO:122.

[0150] In some embodiments, the anti-GM-CSFRα antibody includes V H It contains V of SEQ ID NO:80 H HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V of SEQ ID NO:123 L The LC-CDR1, LC-CDR2, and LC-CDR3 are included. In some embodiments, the anti-GM-CSFRα antibody includes V. H It contains V of SEQ ID NO:85 H HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V of SEQ ID NO:125 L The LC-CDR1, LC-CDR2, and LC-CDR3 are included. In some embodiments, the anti-GM-CSFRα antibody includes V. H It contains V of SEQ ID NO:86H HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V of SEQ ID NO:126 L The LC-CDR1, LC-CDR2, and LC-CDR3 are included. In some embodiments, the anti-GM-CSFRα antibody includes V. H It contains V of SEQ ID NO:91 H HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V of SEQ ID NO:126 L The LC-CDR1, LC-CDR2, and LC-CDR3 are included. In some embodiments, the anti-GM-CSFRα antibody includes V. H It contains V of SEQ ID NO:99 H HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V of SEQ ID NO:122 L The LC-CDR1, LC-CDR2, and LC-CDR3 are included. In some embodiments, the anti-GM-CSFRα antibody includes V. H It contains V of SEQ ID NO:101 H HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V of SEQ ID NO:126 L The LC-CDR1, LC-CDR2, and LC-CDR3 are included. In some embodiments, the anti-GM-CSFRα antibody includes V. H It contains V of SEQ ID NO:103 H HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V of SEQ ID NO:123 L The LC-CDR1, LC-CDR2, and LC-CDR3 are included. In some embodiments, the anti-GM-CSFRα antibody includes V. H It contains V of SEQ ID NO:99 H HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V of SEQ ID NO:126 L The LC-CDR1, LC-CDR2, and LC-CDR3 are included. In some embodiments, the anti-GM-CSFRα antibody includes V. HIt contains V of SEQ ID NO:121 H HC-CDR1, HC-CDR2, and HC-CDR3; and V L It contains V of SEQ ID NO:126 L LC-CDR1, LC-CDR2 and LC-CDR3.

[0151] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains any of the amino acid sequences in SEQ ID NOs:80-121 and 246-287, or contains V with at least 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to any of the amino acid sequences in SEQ ID NOs:80-121 and 246-287. H Variant, and V L The V L Contains any of the amino acid sequences in SEQ ID NOs:122-144, 150-245, and 288-289, or contains V with at least 90% sequence homology to any of the amino acid sequences in SEQ ID NOs:122-144, 150-245, and 288-289. L Variant. In some embodiments, the anti-GM-CSFRα antibody comprises V containing any of the amino acid sequences in SEQ ID NOs: 80-121 and 246-287. H V, and containing any of the amino acid sequences in SEQ ID NOs:122-144, 150-245, and 288-289. L .

[0152] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains the amino acid sequence SEQ ID NO:80, or is a V with at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology. H Variant, and V L The V L Contains the amino acid sequence SEQ ID NO:123, or is a V with at least about 90% sequence homology. L Variant. In some embodiments, the anti-GM-CSFRα antibody comprises V containing the amino acid sequence SEQ ID NO:80. HAnd V containing the amino acid sequence SEQ ID NO:123 L .

[0153] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains the amino acid sequence SEQ ID NO:85, or is a V with at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology. H Variant, and V L The V L Contains the amino acid sequence SEQ ID NO:125, or is a V with at least 90% sequence homology. L Variant. In some embodiments, the anti-GM-CSFRα antibody comprises V containing the amino acid sequence SEQ ID NO:85. H And V containing the amino acid sequence SEQ ID NO:125 L .

[0154] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains the amino acid sequence SEQ ID NO:86, or is a V with at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology. H Variant, and V L The V L Contains the amino acid sequence SEQ ID NO:126, or is a V with at least 90% sequence homology. L Variant. In some embodiments, the anti-GM-CSFRα antibody comprises V containing the amino acid sequence SEQ ID NO:86. H And V containing the amino acid sequence SEQ ID NO:126 L .

[0155] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains the amino acid sequence SEQ ID NO:91, or is a V with at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology. H Variant, and V L The V LContains the amino acid sequence SEQ ID NO:126, or is a V with at least 90% sequence homology. L Variant. In some embodiments, the anti-GM-CSFRα antibody comprises V containing the amino acid sequence SEQ ID NO:91. H And V containing the amino acid sequence SEQ ID NO:126 L .

[0156] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains the amino acid sequence SEQ ID NO:99, or is a V with at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology. H Variant, and V L The V L Contains the amino acid sequence SEQ ID NO:122, or is a V with at least 90% sequence homology. L Variant. In some embodiments, the anti-GM-CSFRα antibody comprises V containing the amino acid sequence SEQ ID NO:99. H And V containing the amino acid sequence SEQ ID NO:122 L .

[0157] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains the amino acid sequence SEQ ID NO:101, or is a V that has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology. H Variant, and V L The V L Contains the amino acid sequence SEQ ID NO:126, or is a V with at least about 90% sequence homology. L Variant. In some embodiments, the anti-GM-CSFRα antibody comprises V containing the amino acid sequence SEQ ID NO:101. H And V containing the amino acid sequence SEQ ID NO:126 L .

[0158] In some embodiments, the anti-GM-CSFRα antibody includes V H The V HContains the amino acid sequence SEQ ID NO:103, or is a V with at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology. H Variant, and V L The V L Contains the amino acid sequence SEQ ID NO:123, or is a V with at least 90% sequence homology. L Variant. In some embodiments, the anti-GM-CSFRα antibody comprises V containing the amino acid sequence SEQ ID NO:103. H And V containing the amino acid sequence SEQ ID NO:123 L .

[0159] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains the amino acid sequence SEQ ID NO:99, or is a V with at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology. H Variant, and V L The V L Contains the amino acid sequence SEQ ID NO:126, or is a V with at least 90% sequence homology. L Variant. In some embodiments, the anti-GM-CSFRα antibody comprises V containing the amino acid sequence SEQ ID NO:99. H And V containing the amino acid sequence SEQ ID NO:126 L .

[0160] In some embodiments, the anti-GM-CSFRα antibody includes V H The V H Contains the amino acid sequence SEQ ID NO:121, or is a V with at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology. H Variant, and V L The V L Contains the amino acid sequence SEQ ID NO:126, or is a V with at least 90% sequence homology. L Variant. In some embodiments, the anti-GM-CSFRα antibody comprises V containing the amino acid sequence SEQ ID NO:121. H And V containing the amino acid sequence SEQ ID NO:126L .

[0161] In some embodiments, the functional epitope can be resolved using a combined alanine scanning method. In this process, the combined alanine scanning technique can be used to identify the amino acids in the GM-CSFRα protein essential for interaction with anti-GM-CSFRα antibodies. In some embodiments, the epitope is conformational, and the epitope can also be identified using the crystal structure of an anti-GM-CSFRα antibody that binds to the GM-CSFRα protein.

[0162] In some embodiments, this application relates to antibodies that competitively bind to GM-CSFRα, in conjunction with any of the anti-GM-CSFRα antibodies described herein. In some embodiments, it relates to antibodies capable of competitively binding to epitopes on GM-CSFRα, in conjunction with any of the anti-GM-CSFRα antibodies described herein. In some embodiments, it relates to anti-GM-CSFRα antibodies, which, together with V H and V L The anti-GM-CSFRα antibody molecule binds to the same epitope, wherein the V H Contains any of the amino acid sequences in SEQ ID NOs:80-121 and 246-287, and the V L Contains any of the amino acid sequences in SEQ ID NOs: 122-144, 150-245, and 288-289. In some embodiments, it relates to an anti-GM-CSFRα antibody, which contains V H and V L The anti-GM-CSFRα antibody competitively binds to GM-CSFRα, wherein the V H Contains any of the amino acid sequences in SEQ ID NOs:80-121 and 246-287, and the V L It contains any of the amino acid sequences in SEQ ID NOs:122-144, 150-245 and 288-289.

[0163] In some embodiments, a competition assay can be used to identify monoclonal antibodies that competitively bind to GM-CSFRα with the anti-GM-CSFRα antibody described herein. A competition assay can determine whether two antibodies bind to the same epitope by identifying identical or spatially overlapping epitopes or by one antibody competitively inhibiting the binding of another antibody to the antigen. In some embodiments, such a competitive antibody binds to the same epitope as the antibody described herein. Some exemplary competition assays include, but are not limited to, conventional assays as described in Harlow and Lane (1988) Antibodies: A Laboratory Manual ch. 14 (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). Detailed exemplary methods for resolving antibody-bound epitopes are described in Morris (1996) "Epitope Mapping Protocols," in Methods in Molecular Biology vol. 66 (Humana Press, Totowa, NJ). In some embodiments, if each antibody blocks 50% or more of the binding of another antibody, they are said to bind to the same epitope. In some embodiments, the antibody competing with the anti-GM-CSFRα antibody described herein is a chimeric antibody, a humanized antibody, or a humanized antibody.

[0164] Exemplary anti-GM-CSFRα antibody sequences are shown in Tables 2, 3, and 18. Figure 19A , Figure 19B As shown. Those skilled in the art will recognize that there are various known algorithms (Kabat definition) for predicting the location of CDRs and defining the variable regions of the antibody light and heavy chains. CDRs containing antibodies as described herein, V H and / or V L Sequences, but antibodies based on prediction algorithms rather than those exemplified in the table below are also within the scope of this invention.

[0165] Table 2. Exemplary anti-GM-CSFRα antibody CDR sequences

[0166]

[0167]

[0168]

[0169]

[0170]

[0171] Table 3. Exemplary Sequences

[0172]

[0173]

[0174]

[0175]

[0176]

[0177] GM-CSF: Granulocyte-Macrophage Colony-Stimulating Factor

[0178] Granulocyte-macrophage colony-stimulating factor (GM-CSF), also known as colony-stimulating factor 2 (CSF2), is produced by macrophages, T cells, mast cells, natural killer cells, endothelial cells, or fibroblasts. GM-CSF is a type I pro-inflammatory cytokine that enhances the survival, proliferation, and / or differentiation of a wide range of hematopoietic cell types, including neutrophils, eosinophils, monocytes, and macrophages. For example, it promotes bone marrow cell differentiation, recruitment and differentiation of monocyte-derived dendritic cells, and neutrophil initiation and activation. It also participates in promoting angiogenesis and tumor cell growth. Clinically, GM-CSF is commonly used to promote bone marrow recovery after radiation therapy.

[0179] GM-CSF is one of the first pro-inflammatory cytokines present at sites of inflammation and plays a crucial role in regulating inflammatory processes. For example, it can promote the differentiation of hematopoietic cell types into neutrophils, eosinophils, monocytes, and macrophages (Nature Reviews Rheumatology 2015; 7(11):415-430). By activating vascular endothelial cells, GM-CSF promotes the recruitment of monocytes and macrophages. GM-CSF also promotes the proliferation of monocytes and macrophages, such as macrophages in the synovial joints of patients with rheumatoid arthritis; it also promotes the release of cytokines from macrophages, including GM-CSF and other inflammatory cytokines such as TNF-α, IL-6, IL-1, and chemokines. GM-CSF can further participate in regulating antigen-presenting cells in inflamed tissues and promote the production of IL-23 by macrophages and dendritic cells, which, along with IL-6 and IL-1, regulates T cell differentiation; endogenous GM-CSF regulates sensory neurons by depending on pain signals and promoting sensitivity to pain. In GM-CSF knockout mice, myeloid cell development was not affected, indicating that the factor has a limited role in promoting myeloid cell development (Stanley et al. PNAS 1994; 91(12):5592-5594). However, due to the lack of an appropriate inflammatory response, GM-CSF knockout mice were more susceptible to infection (Trapnell et al. N Engl J Med 2003; 349:2527-2539; Dranoff et al. Science 1994; 264:713-716). The absence of GM-CSF reduces the incidence of rheumatoid arthritis, encephalomyelitis, and autoimmune myocarditis, indicating that GM-CSF is mainly involved in the inflammatory process (Lawlore et al. Arthritis & Rheumatism 2005; (52) 3749-3754; McQualter et al. Journal of Experimental Medicine 2001; 194(7): 873-882; Sonderegger et al. Journal of Experimental Medicine 2008; 205(10): 2281-2294).

[0180] GM-CSFR: Granulocyte-Macrophage Colony-Stimulating Factor Receptor

[0181] The GM-CSF receptor is a member of the hematopoietic receptor superfamily. It is a heterodimer composed of an α subunit and a β subunit. The α subunit is highly specific to GM-CSF, while the β subunit is shared with other cytokine receptors such as IL-3 and IL-5. The more widespread distribution of the β receptor subunit in tissues reflects this. The α subunit, GM-CSFRα, is mainly expressed on bone marrow cells and non-hematopoietic cells, such as neutrophils, macrophages, eosinophils, dendritic cells, endothelial cells, and respiratory epithelial cells. The full-length human GM-CSFRα is a 400-amino acid type I membrane glycoprotein belonging to the type I cytokine receptor family. It consists of a 22-amino acid signal peptide (positions 1-22), a 298-amino acid extracellular domain (positions 23-320), a transmembrane domain (positions 321-345), and a short 55-amino acid intracellular domain. The signal peptide is cleaved to produce the 378-amino acid mature GM-CSFRα protein. Human and mouse GM-CSFRα cDNA clones were obtained, and at the protein level, their receptor subunits shared 36% homology. GM-CSF could bind the α subunit alone with relatively low affinity (Kd 1–5 nM), but not at all with the β subunit alone. However, when the α and β subunits were present together, a high-affinity ligand-receptor complex (Kd ≈ 100 pM) was formed. GM-CSF signaling occurs through its initial binding to the GM-CSFRα chain, followed by cross-linking of the common β chain with the larger subunit, resulting in a high-affinity interaction that phosphorylates the JAK-STAT pathway. The binding of GM-CSFR to GM-CSF was reviewed in Haman et al. Journal of Biological Chemistry 1999; 274(48). This interaction can also transmit signals through tyrosine phosphorylation and activation of the MAPK pathway. Pathologically, GM-CSF has been shown to play a role in exacerbating inflammation, respiratory diseases, and autoimmune diseases. Therefore, the neutralization of the GM-CSF-CSFRα binding has become a treatment option for diseases and conditions mediated by GM-CSFR.

[0182] Full-length anti-GM-CSFRα antibody

[0183] In some embodiments, the anti-GM-CSFRα antibody is a full-length anti-GM-CSFRα antibody. In some embodiments, the full-length anti-GM-CSFRα antibody is IgA, IgD, IgE, IgG, or IgM. In some embodiments, the full-length anti-GM-CSFRα antibody includes a constant IgG region, such as a constant region of any IgG1, IgG2, IgG3, IgG4, or a variant thereof. In some embodiments, the full-length anti-GM-CSFRα antibody includes a λ light chain constant region. In some embodiments, the full-length anti-GM-CSFRα antibody includes a κ light chain constant region. In some embodiments, the full-length anti-GM-CSFRα antibody is a full-length human anti-GM-CSFRα antibody. In some embodiments, the full-length anti-GM-CSFRα antibody includes a mouse immunoglobulin Fc sequence. In some embodiments, the full-length anti-GM-CSFRα antibody includes an altered or otherwise modified Fc sequence such that it has enhanced effector functions of antibody-dependent cell-mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

[0184] Therefore, for example, in some embodiments, a full-length anti-GM-CSFRα antibody is involved, which includes an IgG1 constant region that specifically binds to GM-CSFRα. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0185] In some embodiments, a full-length anti-GM-CSFRα antibody is involved, comprising an IgG2 constant region, the anti-GM-CSFRα antibody specifically binding to GM-CSFRα. In some embodiments, the IgG2 is human IgG2. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:147.

[0186] In some embodiments, a full-length anti-GM-CSFRα antibody is involved, comprising an IgG3 constant region, the anti-GM-CSFRα antibody specifically binding to GM-CSFRα. In some embodiments, the IgG3 is human IgG3. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:147.

[0187] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed, comprising an IgG4 constant region, the anti-GM-CSFRα antibody specifically binding to GM-CSFRα. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0188] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed comprising an IgG1 constant region, wherein the anti-GM-CSFRα antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising any amino acid sequence of SEQ ID NOs: 1-4 or a variant comprising up to 3 amino acid substitutions (e.g., any one of 1, 2, or 3); an HC-CDR2 comprising any amino acid sequence of SEQ ID NOs: 5-16 or a variant comprising up to 3 amino acid substitutions (e.g., any one of 1, 2, or 3); and an HC-CDR3 comprising any amino acid sequence of SEQ ID NOs: 17-50 or a variant comprising up to 3 amino acid substitutions (e.g., any one of 1, 2, or 3); and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence SEQ ID NO: 51 or a variant comprising up to 3 amino acid substitutions (e.g., any one of about 1, 2, or 3); and an LC-CDR2 comprising the amino acid sequence SEQ ID NO: 51; and a variant ... NO:52 or a variant containing up to three amino acid substitutions (e.g., any one of 1, 2, or 3), and an LC-CDR3 containing any amino acid sequence of SEQ ID NOs:53-75 or a variant containing up to three amino acid substitutions (e.g., any one of 1, 2, or 3). In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region contains or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region contains or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region contains or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region contains or is composed of the amino acid sequence SEQ ID NO:147.

[0189] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed, comprising an IgG2 constant region, wherein the anti-GM-CSFRα antibody comprises a) a heavy chain variable domain comprising: an HC-CDR1 comprising any amino acid sequence of SEQ ID NOs: 1-4 or a variant comprising up to 3 amino acid substitutions (e.g., any one of 1, 2, or 3); an HC-CDR2 comprising any amino acid sequence of SEQ ID NOs: 5-16 or a variant comprising up to 3 amino acid substitutions (e.g., any one of 1, 2, or 3); and an HC-CDR3 comprising any amino acid sequence of SEQ ID NOs: 17-50 or a variant comprising up to 3 amino acid substitutions (e.g., any one of 1, 2, or 3); and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence SEQ ID NO: 51 or a variant comprising up to 3 amino acid substitutions (e.g., any one of 1, 2, or 3); and an LC-CDR2 comprising the amino acid sequence SEQ ID NO: 51; and a variant ... NO:52 or a variant containing up to three amino acid substitutions (e.g., any one of 1, 2, or 3), an LC-CDR3 containing any amino acid sequence of SEQ ID NOs:53-75 or a variant containing up to three amino acid substitutions (e.g., any one of 1, 2, or 3). In some embodiments, the IgG2 is human IgG2. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0190] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed, comprising an IgG3 constant region, wherein the anti-GM-CSFRα antibody comprises a) a heavy chain variable domain comprising: an HC-CDR1 comprising any amino acid sequence of SEQ ID NOs: 1-4 or a variant comprising up to 3 amino acid substitutions (e.g., any one of 1, 2, or 3); an HC-CDR2 comprising any amino acid sequence of SEQ ID NOs: 5-16 or a variant comprising up to 3 amino acid substitutions (e.g., any one of 1, 2, or 3); and an HC-CDR3 comprising any amino acid sequence of SEQ ID NOs: 17-50 or a variant comprising up to 3 amino acid substitutions (e.g., any one of 1, 2, or 3); and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence SEQ ID NO: 51 or a variant comprising up to 3 amino acid substitutions (e.g., any one of 1, 2, or 3); and an LC-CDR2 comprising the amino acid sequence SEQ ID NO: 51. NO:52 or a variant containing up to three amino acid substitutions (e.g., any one of 1, 2, or 3), and an LC-CDR3 containing any amino acid sequence of SEQ ID NOs:53-75 or a variant containing up to three amino acid substitutions (e.g., any one of 1, 2, or 3). In some embodiments, the IgG3 is human IgG3. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0191] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed comprising an IgG4 constant region, wherein the anti-GM-CSFRα antibody comprises: a) a heavy chain variable domain comprising an HC-CDR1 comprising any amino acid sequence of SEQ ID NOs: 1-4 or a variant comprising up to 3 amino acid substitutions (e.g., any one of about 1, 2, or 3), an HC-CDR2 comprising any amino acid sequence of SEQ ID NOs: 5-16 or a variant comprising up to 3 amino acid substitutions (e.g., any one of 1, 2, or 3), and an HC-CDR3 comprising any amino acid sequence of SEQ ID NOs: 17-50 or a variant comprising up to 3 amino acid substitutions (e.g., any one of 1, 2, or 3); and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence SEQ ID NO: 51 or a variant comprising up to 3 amino acid substitutions (e.g., any one of 1, 2, or 3), and an LC-CDR2 comprising the amino acid sequence SEQ ID NO: 51. NO:52 or a variant containing up to three amino acid substitutions (e.g., any one of 1, 2, or 3), and an LC-CDR3 containing any amino acid sequence of SEQ ID NOs:53-75 or a variant containing up to three amino acid substitutions (e.g., any one of 1, 2, or 3). In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region contains or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region contains or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region contains or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region contains or is composed of the amino acid sequence SEQ ID NO:147.

[0192] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed comprising an IgG1 constant region, wherein the anti-GM-CSFRα antibody comprises a) a heavy chain variable domain comprising: an HC-CDR1 comprising any of the amino acid sequences of SEQ ID NOs:1-4, an HC-CDR2 comprising any of the amino acid sequences of SEQ ID NOs:5-16, and an HC-CDR3 comprising any of the amino acid sequences of SEQ ID NOs:17-50 or a variant comprising up to three amino acid substitutions (e.g., any one of about 1, 2, or 3); and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence SEQ ID NO:51, an LC-CDR2 comprising the amino acid sequence SEQ ID NO:52, and an LC-CDR3 comprising any of the amino acid sequences of SEQ ID NOs:53-75 or a variant comprising up to three amino acid substitutions (e.g., any one of about 1, 2, or 3). In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region of the anti-GM-CSFRα antibody comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region of the anti-GM-CSFRα antibody comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0193] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed comprising an IgG4 constant region, wherein the anti-GM-CSFRα antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising any of the amino acid sequences of SEQ ID NOs:1-4, an HC-CDR2 comprising any of the amino acid sequences of SEQ ID NOs:5-16, and an HC-CDR3 comprising any of the amino acid sequences of SEQ ID NOs:17-50 or a variant comprising up to three amino acid substitutions (e.g., any one of 1, 2, or 3); and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence SEQ ID NO:51, an LC-CDR2 comprising the amino acid sequence SEQ ID NO:52, and an LC-CDR3 comprising any of the amino acid sequences of SEQ ID NOs:53-75 or a variant comprising up to three amino acid substitutions (e.g., any one of 1, 2, or 3). In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0194] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed comprising an IgG1 constant region, wherein the anti-GM-CSFRα antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising any of the amino acid sequences SEQ ID NOs:1-4, an HC-CDR2 comprising any of the amino acid sequences SEQ ID NOs:5-16, and an HC-CDR3 comprising any of the amino acid sequences SEQ ID NOs:17-50; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence SEQ ID NO:51, an LC-CDR2 comprising the amino acid sequence SEQ ID NO:52, and an LC-CDR3 comprising any of the amino acid sequences SEQ ID NOs:53-75. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0195] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed comprising an IgG4 constant region, wherein the anti-GM-CSFRα antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising any of the amino acid sequences SEQ ID NOs:1-4, an HC-CDR2 comprising any of the amino acid sequences SEQ ID NOs:5-16, and an HC-CDR3 comprising any of the amino acid sequences SEQ ID NOs:17-50; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence SEQ ID NO:51, an LC-CDR2 comprising the amino acid sequence SEQ ID NO:52, and an LC-CDR3 comprising any of the amino acid sequences SEQ ID NOs:53-75. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0196] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed comprising an IgG1 constant region, wherein the anti-GM-CSFRα antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence SEQ ID NO:1, an HC-CDR2 comprising the amino acid sequence SEQ ID NO:5, and an HC-CDR3 comprising the amino acid sequence SEQ ID NO:17; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence SEQ ID NO:51, an LC-CDR2 comprising the amino acid sequence SEQ ID NO:52, and an LC-CDR3 comprising the amino acid sequence SEQ ID NO:54. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0197] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed comprising an IgG1 constant region, wherein the anti-GM-CSFRα antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence SEQ ID NO:1, an HC-CDR2 comprising the amino acid sequence SEQ ID NO:8, and an HC-CDR3 comprising the amino acid sequence SEQ ID NO:22; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence SEQ ID NO:51, an LC-CDR2 comprising the amino acid sequence SEQ ID NO:52, and an LC-CDR3 comprising the amino acid sequence SEQ ID NO:56. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0198] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed comprising an IgG1 constant region, wherein the anti-GM-CSFRα antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence SEQ ID NO:1, an HC-CDR2 comprising the amino acid sequence SEQ ID NO:7, and an HC-CDR3 comprising the amino acid sequence SEQ ID NO:23; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence SEQ ID NO:51, an LC-CDR2 comprising the amino acid sequence SEQ ID NO:52, and an LC-CDR3 comprising the amino acid sequence SEQ ID NO:57. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0199] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed comprising an IgG1 constant region, wherein the anti-GM-CSFRα antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence SEQ ID NO:1, an HC-CDR2 comprising the amino acid sequence SEQ ID NO:6, and an HC-CDR3 comprising the amino acid sequence SEQ ID NO:27; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence SEQ ID NO:51, an LC-CDR2 comprising the amino acid sequence SEQ ID NO:52, and an LC-CDR3 comprising the amino acid sequence SEQ ID NO:57. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0200] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed, comprising an IgG1 constant region, wherein the anti-GM-CSFRα antibody comprises a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence SEQ ID NO:1, an HC-CDR2 comprising the amino acid sequence SEQ ID NO:7, and an HC-CDR3 comprising the amino acid sequence SEQ ID NO:35; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence SEQ ID NO:51, an LC-CDR2 comprising the amino acid sequence SEQ ID NO:52, and an LC-CDR3 comprising the amino acid sequence SEQ ID NO:53. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0201] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed comprising an IgG1 constant region, wherein the anti-GM-CSFRα antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence SEQ ID NO:1, an HC-CDR2 comprising the amino acid sequence SEQ ID NO:7, and an HC-CDR3 comprising the amino acid sequence SEQ ID NO:37; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence SEQ ID NO:51, an LC-CDR2 comprising the amino acid sequence SEQ ID NO:52, and an LC-CDR3 comprising the amino acid sequence SEQ ID NO:57. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0202] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed comprising an IgG1 constant region, wherein the anti-GM-CSFRα antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence SEQ ID NO:3, an HC-CDR2 comprising the amino acid sequence SEQ ID NO:6, and an HC-CDR3 comprising the amino acid sequence SEQ ID NO:39; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence SEQ ID NO:51, an LC-CDR2 comprising the amino acid sequence SEQ ID NO:52, and an LC-CDR3 comprising the amino acid sequence SEQ ID NO:54. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0203] In some embodiments, a full-length anti-GM-CSFRα antibody is involved, comprising an IgG1 constant region, wherein the anti-GM-CSFRα antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence SEQ ID NO:1, an HC-CDR2 comprising the amino acid sequence SEQ ID NO:7, and an HC-CDR3 comprising the amino acid sequence SEQ ID NO:35; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence SEQ ID NO:51, an LC-CDR2 comprising the amino acid sequence SEQ ID NO:52, and an LC-CDR3 comprising the amino acid sequence SEQ ID NO:57. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0204] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed comprising an IgG1 constant region, wherein the anti-GM-CSFRα antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence SEQ ID NO:1, an HC-CDR2 comprising the amino acid sequence SEQ ID NO:7, and an HC-CDR3 comprising the amino acid sequence SEQ ID NO:50; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence SEQ ID NO:51, an LC-CDR2 comprising the amino acid sequence SEQ ID NO:52, and an LC-CDR3 comprising the amino acid sequence SEQ ID NO:57. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0205] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed comprising an IgG4 constant region, wherein the anti-GM-CSFRα antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence SEQ ID NO:1, an HC-CDR2 comprising the amino acid sequence SEQ ID NO:5, and an HC-CDR3 comprising the amino acid sequence SEQ ID NO:17; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence SEQ ID NO:51, an LC-CDR2 comprising the amino acid sequence SEQ ID NO:52, and an LC-CDR3 comprising the amino acid sequence SEQ ID NO:54. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0206] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed comprising an IgG4 constant region, wherein the anti-GM-CSFRα antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence SEQ ID NO:1, an HC-CDR2 comprising the amino acid sequence SEQ ID NO:8, and an HC-CDR3 comprising the amino acid sequence SEQ ID NO:22; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence SEQ ID NO:51, an LC-CDR2 comprising the amino acid sequence SEQ ID NO:52, and an LC-CDR3 comprising the amino acid sequence SEQ ID NO:56. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0207] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed comprising an IgG4 constant region, wherein the anti-GM-CSFRα antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence SEQ ID NO:1, an HC-CDR2 comprising the amino acid sequence SEQ ID NO:7, and an HC-CDR3 comprising the amino acid sequence SEQ ID NO:23; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence SEQ ID NO:51, an LC-CDR2 comprising the amino acid sequence SEQ ID NO:52, and an LC-CDR3 comprising the amino acid sequence SEQ ID NO:57. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0208] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed comprising an IgG4 constant region, wherein the anti-GM-CSFRα antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence SEQ ID NO:1, an HC-CDR2 comprising the amino acid sequence SEQ ID NO:6, and an HC-CDR3 comprising the amino acid sequence SEQ ID NO:27; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence SEQ ID NO:51, an LC-CDR2 comprising the amino acid sequence SEQ ID NO:52, and an LC-CDR3 comprising the amino acid sequence SEQ ID NO:57. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0209] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed comprising an IgG4 constant region, wherein the anti-GM-CSFRα antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence SEQ ID NO:1, an HC-CDR2 comprising the amino acid sequence SEQ ID NO:7, and an HC-CDR3 comprising the amino acid sequence SEQ ID NO:35; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence SEQ ID NO:51, an LC-CDR2 comprising the amino acid sequence SEQ ID NO:52, and an LC-CDR3 comprising the amino acid sequence SEQ ID NO:53. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0210] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed comprising an IgG4 constant region, wherein the anti-GM-CSFRα antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence SEQ ID NO:1, an HC-CDR2 comprising the amino acid sequence SEQ ID NO:7, and an HC-CDR3 comprising the amino acid sequence SEQ ID NO:37; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence SEQ ID NO:51, an LC-CDR2 comprising the amino acid sequence SEQ ID NO:52, and an LC-CDR3 comprising the amino acid sequence SEQ ID NO:57. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0211] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed comprising an IgG4 constant region, wherein the anti-GM-CSFRα antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence SEQ ID NO:3, an HC-CDR2 comprising the amino acid sequence SEQ ID NO:6, and an HC-CDR3 comprising the amino acid sequence SEQ ID NO:39; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence SEQ ID NO:51, an LC-CDR2 comprising the amino acid sequence SEQ ID NO:52, and an LC-CDR3 comprising the amino acid sequence SEQ ID NO:54. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0212] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed comprising an IgG4 constant region, wherein the anti-GM-CSFRα antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence SEQ ID NO:1, an HC-CDR2 comprising the amino acid sequence SEQ ID NO:7, and an HC-CDR3 comprising the amino acid sequence SEQ ID NO:35; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence SEQ ID NO:51, an LC-CDR2 comprising the amino acid sequence SEQ ID NO:52, and an LC-CDR3 comprising the amino acid sequence SEQ ID NO:57. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0213] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed comprising an IgG4 constant region, wherein the anti-GM-CSFRα antibody comprises: a) a heavy chain variable domain comprising: an HC-CDR1 comprising the amino acid sequence SEQ ID NO:1, an HC-CDR2 comprising the amino acid sequence SEQ ID NO:7, and an HC-CDR3 comprising the amino acid sequence SEQ ID NO:50; and b) a light chain variable domain comprising: an LC-CDR1 comprising the amino acid sequence SEQ ID NO:51, an LC-CDR2 comprising the amino acid sequence SEQ ID NO:52, and an LC-CDR3 comprising the amino acid sequence SEQ ID NO:57. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0214] In some embodiments, a full-length anti-GM-CSFRα antibody is involved, comprising an IgG1 constant region, wherein the anti-GM-CSFRα antibody includes a heavy chain variable domain V. HThe heavy chain variable domain V H Contains any amino acid sequence from SEQ ID NOs:80-121 and 246-287, or contains V with at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to any amino acid sequence from SEQ ID NOs:80-121 and 246-287. H Variants, and light chain variable domain V L The light chain variable domain V L The V sequence comprises any of the amino acid sequences in SEQ ID NOs:122-144, 150-245, and 288-289, or comprises a sequence homology of at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) with any of the amino acid sequences in SEQ ID NOs:122-144, 150-245, and 288-289. L Variants. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0215] In some embodiments, a full-length anti-GM-CSFRα antibody is involved, comprising an IgG2 constant region, wherein the anti-GM-CSFRα antibody includes a heavy chain variable domain V. H The heavy chain variable domain V H Contains any amino acid sequence from SEQ ID NOs:80-121 and 246-287, or contains V with at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to any amino acid sequence from SEQ ID NOs:80-121 and 246-287. H Variants, and light chain variable domain V L The light chain variable domain V LThe V sequence comprises any of the amino acid sequences in SEQ ID NOs:122-144, 150-245, and 288-289, or comprises a sequence homology of at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) with any of the amino acid sequences in SEQ ID NOs:122-144, 150-245, and 288-289. L Variants. In some embodiments, the IgG2 is human IgG2. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:147.

[0216] In some embodiments, a full-length anti-GM-CSFRα antibody is involved, comprising an IgG3 constant region, wherein the anti-GM-CSFRα antibody includes a heavy chain variable domain V. H The heavy chain variable domain V H Contains any amino acid sequence from SEQ ID NOs:80-121 and 246-287, or contains V with at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to any amino acid sequence from SEQ ID NOs:80-121 and 246-287. H Variants, and light chain variable domain V L The light chain variable domain V L The V sequence comprises any of the amino acid sequences in SEQ ID NOs:122-144, 150-245, and 288-289, or comprises a sequence homology of at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) with any of the amino acid sequences in SEQ ID NOs:122-144, 150-245, and 288-289. L Variants. In some embodiments, the IgG3 is human IgG3. In some embodiments, the light chain constant region comprises or consists of the amino acid sequence SEQ ID NO:147.

[0217] In some embodiments, a full-length anti-GM-CSFRα antibody is involved, comprising an IgG4 constant region, wherein the anti-GM-CSFRα antibody includes a heavy chain variable domain V. H The heavy chain variable domain V HContains any of the amino acid sequences in SEQ ID NOs:80-121 and 246-287, or contains V with at least about 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to any of the amino acid sequences in SEQ ID NOs:80-121 and 246-287. H Variants, and light chain variable domain V L The light chain variable domain V L Contains any one of the amino acid sequences in SEQ ID NOs:122-144, 150-245, and 288-289, or contains V with at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to any one of the amino acid sequences in SEQ ID NOs:122-144, 150-245, and 288-289. L Variants. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0218] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed, comprising an IgG1 constant region, wherein the anti-GM-CSFRα antibody includes a heavy chain variable domain comprising any of the amino acid sequences of SEQ ID NOs: 80-121 and 246-287, and a light chain variable domain comprising any of the amino acid sequences of SEQ ID NOs: 122-144, 150-245, and 288-289. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO: 145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO: 147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO: 145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO: 147.

[0219] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed, comprising an IgG4 constant region, wherein the anti-GM-CSFRα antibody includes a heavy chain variable domain comprising any of the amino acid sequences of SEQ ID NOs: 80-121 and 246-287, and a light chain variable domain comprising any of the amino acid sequences of SEQ ID NOs: 122-144, 150-245, and 288-289. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO: 146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO: 147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO: 146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO: 147.

[0220] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed, comprising an IgG1 constant region, wherein the anti-GM-CSFRα antibody comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID NO:80, and a light chain variable domain comprising SEQ ID NO:123. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0221] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed, comprising an IgG1 constant region, wherein the anti-GM-CSFRα antibody comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID NO:85, and a light chain variable domain comprising SEQ ID NO:125. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0222] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed, comprising an IgG1 constant region, wherein the anti-GM-CSFRα antibody comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID NO:86, and a light chain variable domain comprising SEQ ID NO:126. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0223] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed, comprising an IgG1 constant region, wherein the anti-GM-CSFRα antibody comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID NO:91, and a light chain variable domain comprising SEQ ID NO:126. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0224] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed, comprising an IgG1 constant region, wherein the anti-GM-CSFRα antibody comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID NO:99, and a light chain variable domain comprising SEQ ID NO:122. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145, and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0225] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed, comprising an IgG1 constant region, wherein the anti-GM-CSFRα antibody comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID NO:101, and a light chain variable domain comprising SEQ ID NO:126. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0226] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed, comprising an IgG1 constant region, wherein the anti-GM-CSFRα antibody comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID NO:103, and a light chain variable domain comprising SEQ ID NO:123. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0227] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed, comprising an IgG1 constant region, wherein the anti-GM-CSFRα antibody comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID NO:99, and a light chain variable domain comprising SEQ ID NO:126. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0228] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed, comprising an IgG1 constant region, wherein the anti-GM-CSFRα antibody comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID NO:121, and a light chain variable domain comprising SEQ ID NO:126. In some embodiments, the IgG1 is human IgG1. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0229] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed, comprising an IgG4 constant region, wherein the anti-GM-CSFRα antibody comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID NO:80, and a light chain variable domain comprising SEQ ID NO:123. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146, and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0230] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed, comprising an IgG4 constant region, wherein the anti-GM-CSFRα antibody comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID NO:85, and a light chain variable domain comprising SEQ ID NO:125. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0231] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed, comprising an IgG4 constant region, wherein the anti-GM-CSFRα antibody comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID NO:86, and a light chain variable domain comprising SEQ ID NO:126. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146, and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0232] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed, comprising an IgG4 constant region, wherein the anti-GM-CSFRα antibody comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID NO:91, and a light chain variable domain comprising SEQ ID NO:126. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0233] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed, comprising an IgG4 constant region, wherein the anti-GM-CSFRα antibody comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID NO:99, and a light chain variable domain comprising SEQ ID NO:122. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146, and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0234] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed, comprising an IgG4 constant region, wherein the anti-GM-CSFRα antibody comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID NO:101, and a light chain variable domain comprising SEQ ID NO:126. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0235] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed, comprising an IgG4 constant region, wherein the anti-GM-CSFRα antibody comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID NO:103, and a light chain variable domain comprising SEQ ID NO:123. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0236] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed, comprising an IgG4 constant region, wherein the anti-GM-CSFRα antibody comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID NO:99, and a light chain variable domain comprising SEQ ID NO:126. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0237] In some embodiments, a full-length anti-GM-CSFRα antibody is disclosed, comprising an IgG4 constant region, wherein the anti-GM-CSFRα antibody comprises a heavy chain variable domain comprising the amino acid sequence SEQ ID NO:121, and a light chain variable domain comprising SEQ ID NO:126. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146 and the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0238] Combining affinity

[0239] K can be used to combine affinity. d K off K on or K a To indicate. As used in this article, the term "K" is used to indicate. off "K" refers to the rate constant of antibody dissociation from the antigen / antibody complex, determined by a kinetic selection device. on "K" refers to the rate constant of antibody-antigen binding to form an antigen / antibody complex. The dissociation constant "K" used in this paper... d "" refers to the dissociation constant of a specific antibody-antigen interaction. It is the antigen concentration required for the antigen to occupy half of all antibody binding sites and reach equilibrium in an antibody molecule solution, and is equal to K. off / K on K d The assay assumes all bound molecules are in solution. For antibody-cell wall binding, such as in yeast expression systems, the corresponding dissociation rate constant is expressed as EC50, which gives Kc. d A good approximation. The affinity binding constant Ka is the dissociation constant K. d The reciprocal of.

[0240] The equilibrium dissociation constant (Kd) can serve as an indicator of the affinity between the antibody moiety and the antigen. For example, a simple analysis can be performed using the Scatchard method with antibodies labeled with various markers and a Biacore instrument (manufactured by Amersham Biosciences), analyzing the interactions between biomolecules via surface plasmon resonance according to the user manual or accompanying kit. The Kd values ​​obtained using these methods are expressed in units of megohms (M). Antibodies that bind specifically to a target may have a Kd value, for example, ≤10. -7 M, ≤10 -8 M, ≤10-9 M, ≤10 -10 M, ≤10 -11 M, ≤10 -12 M or ≤10 -13 The Kd value of M.

[0241] The binding specificity of antibodies can be experimentally determined using methods known in the art. These methods include, but are not limited to, Western blots, ELISA, RIA, ECL, IRMA, EIA, BIAcore assays, and peptide scanning.

[0242] In some embodiments, the anti-GM-CSFRα antibody specifically binds to the GM-CSFα target, whose K d The value is 10 -7 M to 10 -13 M (e.g., 10) -7 M to 10 -13 M, 10 -8 M to 10 -13 M, 10 -9 M to 10 -13 M or 10 -10 M to 10 -12 M). Thus, in some embodiments, the Kd value for binding between the anti-GM-CSFRα antibody and GM-CSFRα is 10. -7 M to 10 -13 M, 1×10 -7 M to 5×10 -13 M, 10 -7 M to 10 -12 M, 10 -7 M to 10 -11 M, 10 -7 M to 10 -10 M, 10 -7 M to 10 -9 M, 10 -8 M to 10 -13 M, 1×10 -8 M to 5×10 - 13 M, 10 -8 M to 10 -12 M, 10 -8 M to 10 -11 M, 10 -8 M to 10 -10 M, 10 -8 M to 10 -9 M, 5×10 -9 M to 1×10 -13 M, 5×10 - 9M to 1×10 -12 M, 5×10 -9 M to 1×10 -11 M, 5×10 -9 M-1×10 -10 M, 10 -9 M to 10 -13 M, 10 -9 M to 10 -12 M, 10 -9 M to 10 -11 M, 10 -9 M to 10 -10 M, 5×10 -10 M to 1×10 -13 M, 5×10 -10 M to 1×10 -12 M, 5×10 -10 M to 1×10 -11 M, 10 -10 M to 10 -13 M, 1×10 -10 M to 5×10 -13 M, 1×10 -10 M to 1×10 -12 M, 1×10 -10 M to 5×10 -12 M, 1×10 - 10 M to 1×10 -11 M, 10 -11 M to 10 -13 M, 1×10 -11 M to 5×10 -13 M, 10 -11 M to 10 -12 M, 10 -12 M to 10 -13 M. In some embodiments, the anti-GM-CSFRα antibody binds to K-CSFRα. d The value is 10 -7 M to 10 -13 M.

[0243] In some embodiments, the Kd value of the anti-GM-CSFRα antibody binding to a non-target is higher than the Kd value of the anti-GM-CSFRα antibody binding to a GM-CSFRα target, and in some embodiments cited herein, the binding affinity of the anti-GM-CSFRα antibody to the target (e.g., GM-CSFRα) is higher than the binding affinity of the GM-CSFRα antibody to a non-target. In some embodiments, non-target refers to a non-GM-CSFRα antigen. In some embodiments, the Kd values ​​of the anti-GM-CSFRα antibody (against GM-CSFRα) binding to a non-GM-CSFRα target differ by at least 10-fold, for example, 10-100-fold, 100-1000-fold, or 10... 3 -10 4 times, 10 4 -10 5 times, 10 5 -10 6 times, 10 6 -10 7 times, 10 7 -10 8 times, 10 8 -10 9 times, 10 9 -10 10 times, 10 10 -10 11 times, 10 11 -10 12 times.

[0244] In some embodiments, the Kd value of the anti-GM-CSFRα antibody binding to the non-target is 10. -1 M to 10 -6 M (e.g., 10) -1 M to 10 -6 M, 10 -1 M to 10 -5 M, 10 -2 M to 10 -4 M). In some embodiments, the non-target refers to a non-GM-CSFRα antigen. Therefore, in some embodiments, the Kd value for binding between the anti-GM-CSFRα antibody and the non-GM-CSFRα target is 10. -1 M to 10 -6 M, 1×10 -1 M to 5×10 -6 M, 10 -1 M to 10 -5 M, 1×10 -1 M to 5×10 -5 M, 10 -1 M to 10 -4 M, 1×10 -1 M to 5×10-4 M, 10 -1 M to 10 -3 M, 1×10 -1 M to 5×10 -3 M, 10 -1 M to 10 -2 M, 10 -2 M to 10 -6 M, 1×10 -2 M to 5×10 -6 M, 10 -2 M to 10 -5 M, 1×10 -2 M to 5×10 -5 M, 10 -2 M to 10 -4 M, 1×10 -2 M to 5×10 -4 M, 10 -2 M to 10 -3 M, 10 -3 M to 10 -6 M, 1×10 -3 M to 5×10 -6 M, 10 -3 M to 10 -5 M, 1×10 -3 M to 5×10 -5 M, 10 -3 M to 10 -4 M, 10 -4 M to 10 -6 M, 1×10 -4 M to 5×10 -6 M, 10 -4 M to 10 -5 M, 10 -5 M to 10 -6 M.

[0245] In some embodiments, when it is mentioned that the anti-GM-CSFRα antibody specifically recognizes the GM-CSFRα target with high binding affinity and binds to non-targets with low binding affinity, the Kd value of the anti-GM-CSFRα antibody binding to the GM-CSFRα target is 10. -7 M to 10 -13 M (e.g., 10) -7 M to 10 -13 M, 10 -8 M to 10 -13 M, 10 -9 M to 10 -13 M, 10 -10 M to 10 -12 M), and the Kd value for binding to the non-target is 10.-1 M to 10 -6 M (e.g., 10) -1 M to 10 -6 M, 10 -1 M to 10 -5 M, 10 -2 M to 10 -4 M).

[0246] In some embodiments, when it is mentioned that the anti-GM-CSFRα antibody specifically recognizes GM-CSFRα, the binding affinity of the anti-GM-CSFRα antibody is compared with the binding affinity of a control anti-GM-CSFRα antibody (e.g., Mavrilimumab). In some embodiments, the Kd value of the binding between the control anti-GM-CSFRα antibody and GM-CSFRα is at least twice that of the Kd value of the binding between the anti-GM-CSFRα antibody and GM-CSFRα described in this application, for example, 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times, 10-100 times, 100-1000 times, 10 3 -10 4 times.

[0247] Nucleic acid

[0248] Nucleic acid molecules encoding anti-GM-CSFRα antibodies are also considered. In some embodiments, this relates to one (or a group of) nucleic acids encoding a full-length anti-GM-CSFRα antibody, including any of the full-length anti-GM-CSFRα antibodies described herein. In some embodiments, the nucleic acid (or group of nucleic acids) of the anti-GM-CSFRα antibody described herein may also include a nucleic acid sequence encoding a polypeptide tag (e.g., a protein purification tag, His-tag, HA tag).

[0249] This paper also considers isolated host cells containing anti-GM-CSFRα antibodies, isolated nucleic acids encoding anti-GM-CSFRα antibody polypeptide components, or vectors containing nucleic acids encoding the anti-GM-CSFRα antibody polypeptide components described herein.

[0250] This application also includes variants of these nucleic acid sequences. For example, variants include nucleotide sequences that hybridize with nucleic acid sequences encoding the anti-GM-CSFRα antibody of this application at least under moderately stringent hybridization conditions.

[0251] This application also relates to vectors into which the nucleic acid sequence of this application can be inserted.

[0252] In short, by inserting a natural or synthetic nucleic acid encoding an anti-GM-CSFRα antibody into a suitable expression vector, allowing the nucleic acid to be operatively linked to 5' and 3' regulatory elements, such as promoters (e.g., lymphocyte-specific promoters) and a 3' untranslated region (UTR), anti-GM-CSFRα antibodies (e.g., full-length anti-GM-CSFRα antibodies) can be expressed. The vector is suitable for replication and integration in eukaryotic host cells. Typical cloning and expression vectors contain transcription and translation terminators, initiation sequences, and promoters to regulate the expression of the target nucleic acid sequence.

[0253] The nucleic acids described in this application can also be used for nucleic acid immunization and gene therapy using standard gene delivery protocols. Nucleic acid delivery methods are known in the art. See, for example, US Pat. Nos. 5,399,346, 5,580,859, 5,589,466, the entire contents of which are incorporated herein by reference. In some embodiments, this application also relates to gene therapy vectors.

[0254] Nucleic acids can be cloned into many types of vectors. For example, nucleic acids can be cloned into vectors, including, but not limited to, plasmids, phage particles, phage derivatives, animal viruses, and Cosmids. Vectors of particular interest include expression vectors, replication vectors, probe generation vectors, and sequencing vectors.

[0255] In addition, expression vectors can be provided to cells in the form of viral vectors. Viral vector technology is well known in the art and described, for example, in Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York), and other virology or molecular biology manuals. Viruses that can be used as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. Typically, a suitable vector includes an origin of replication that functions in at least one organism, a promoter sequence, a convenient restriction endonuclease site, and one or more selection markers (see, for example, WO 01 / 96584; WO 01 / 29058; and US Pat. No. 6,326,193).

[0256] Numerous virus-based systems have been developed for transferring genes into mammalian cells. For example, retroviruses provide a convenient platform for gene delivery systems. Selected genes can be inserted into vectors and packaged into retroviral particles using techniques known in the art. The recombinant virus is then isolated and delivered in vivo or in vitro to the cells of a recipient. Many retroviral systems are known in the art. In some embodiments, adenoviral vectors are used. Many adenoviral vectors are known in the art. In some embodiments, lentiviral vectors are used. Vectors derived from retroviruses, such as lentiviruses, are suitable tools for achieving long-term gene transfer because they allow for long-term stable integration of transgenes and replication in daughter cells. Lentiviral vectors have additional advantages over tumor-derived retroviruses, such as mouse leukemia virus, because they can transduce non-dividing cells, such as hepatocytes. They also have the additional advantage of low immunogenicity.

[0257] Other promoter elements, such as enhancers, regulate transcription initiation frequency. They are typically located 30-110 bp upstream of the start site, although recent studies have found that many promoters also contain functional elements downstream of the start site. The spacing between promoter elements is usually flexible, so the promoter retains its function even when elements interchange or move. In the thymidine kinase (TK) promoter, activity begins to decline only when the spacing between promoter elements increases to 50 bp.

[0258] In one example, a suitable promoter is the Immediate Early Cytomegalovirus (CMV) promoter sequence. This promoter sequence is a very strong constitutive promoter sequence that can enable high-level expression of any polynucleotide sequence operatively linked to it. In another example, a suitable promoter is the Elongation Growth Factor 1α (EF-1α) promoter. However, other constitutive promoters may also be used, including but not limited to, the simian virus 40 (SV40) early promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus long terminal repeat (HIV-LTR) promoter, MoMuLV promoter, avian leukosis virus promoter, Epstein-Barr virus Immediate Early promoter, Rouss sarcoma virus promoter, and human gene promoters, such as but not limited to, actin promoter, myosin promoter, hemoglobin promoter, and creatine kinase promoter. Furthermore, this application should not be limited to the application of constitutive promoters. Inducible promoters are also a consideration in this application. The use of inducible promoters provides a molecular switch that can initiate the expression of a polynucleotide sequence, operablely linked to the polynucleotide sequence when such expression is needed, or shut down when such expression is not needed. Inducible promoters include, but are not limited to, metallothionein promoters, glucocorticoid promoters, progesterone promoters, and tetracycline promoters.

[0259] In some embodiments, expression of the anti-GM-CSFRα antibody is inducible. In some embodiments, the nucleic acid sequence encoding the anti-GM-CSFRα antibody is operatively linked to an inducible promoter, including any of the inducible promoters described herein.

[0260] Inducible promoters

[0261] The use of inducible promoters provides a molecular switch that can initiate the expression of a polynucleotide sequence, operablely linked to the polynucleotide sequence when such expression is needed, or shut off expression when such expression is not needed. Exemplary inducible promoters applicable in eukaryotic cells include, but are not limited to, hormone regulatory elements (e.g., see Mader, S. and White, JH (1993) Proc. Natl. Acad. Sci. USA 90:5603-5607), synthetic ligand regulatory elements (see Spencer, D. M et al (1993) Science 262:1019-1024), and ionizing radiation regulatory elements (see Manome, Y. et al. (1993) Biochemistry 32:10607-10613; Datta, R. et al. (1992) Proc. Natl. Acad. Sci. USA 89:1014-10153). Other exemplary inducible promoters applicable to in vivo or in vitro mammalian systems can be found in Gingrich et al. (1998) Annual Rev. Neurosci 21:377-405. In some embodiments, the inducible promoter system for expressing anti-GM-CSFRα antibodies is the Tet system. In some embodiments, the inducible promoter system for expressing anti-GM-CSFRα antibodies is the E. coli lac inhibitory system.

[0262] The exemplary inducible promoter system used in this application is the Tet system. This system is based on the Tet system described by Gossen et al. (1993). In an exemplary embodiment, the target polynucleotide is controlled by a promoter containing one or more Tet operon (TetO) sites. In the inactive state, the Tet repressor (TetR) binds to the TetO site and inhibits transcription of the promoter. In the active state, for example, in the presence of an inducer such as tetracycline (Tc), anhydrous tetracycline, doxycycline (Dox), or an active analog thereof, the inducer causes the TetR to be released from the TetO, thereby allowing transcription to occur. Doxycycline is a member of the tetracycline antibiotic family, and its chemical name is 1-dimethylamino-2,4a,5,7-pentahydroxy-11-methyl-4,6-dioxy-1,4a,11,11a,12,12a-hexahydrotetraene-3-carboxamide.

[0263] In one embodiment, TetR is codon-optimized for expression in mammalian cells, such as mouse or human cells. Due to the degeneracy of the genetic code, most amino acids are encoded by more than one codon, allowing for a large number of variations in a given nucleic acid sequence without any change in the amino acid sequence it encodes. However, many organisms exhibit differences in codon usage, also known as "codon preference" (i.e., a preference for using specific codons for a given amino acid). Codon preference is often associated with the presence of a dominant tRNA species for a particular codon, which in turn improves the efficiency of mRNA translation. Therefore, codon optimization can be used to tailor coding sequences derived from a specific species (e.g., prokaryotes) to improve their expression in different species (e.g., eukaryotes).

[0264] Other specific variants of the Tet system include the following "Tet-Off" and "Tet-On" systems. In the Tet-Off system, transcription is inactive in the presence of Tc or Dox. In this system, a tetracycline-regulated transcriptional activator protein (tTA), composed of TetR fused to the strong transcriptional activation domain of herpes simplex virus VP16, regulates the expression of target nucleic acids under the transcriptional control of tetracycline-responsive promoter elements (TREs). TRE elements consist of a TetO sequence tandemly fused to a promoter (typically a minimal promoter sequence derived from the immediate early promoter of human cytomegalovirus). In the absence of Tc or Dox, tTA binds to the TRE and activates transcription of the target gene. In the presence of Tc or Dox, tTA cannot bind to the TRE, and the target gene is not expressed.

[0265] Conversely, in the Tet-On system, transcription is activated in the presence of Tc or Dox. The Tet-On system is based on the transcriptional activator rtTA, regulated by reverse tetracycline. Like tTA, rtTA is a fusion protein composed of a TetR repressor and a VP16 transactivation domain. However, a four-amino acid change in the TetR DNA-binding region alters rtTA's binding properties, enabling it to recognize only the tetO sequence on the target transgene TRE in the presence of Dox. Therefore, in the Tet-On system, rtTA can only activate the transcription of the TRE-regulated target gene in the presence of Dox.

[0266] Another inducible promoter system is the *E. coli* lac repressor system (see Brown et al., Cell 49:603-612 (1987)). The lac repressor system functions by regulating the transcription of target polynucleotides operably linked to a promoter containing the lac operon (lacO). The lac repressor (lacR) binds to LacO, thereby preventing the transcription of the target polynucleotide. The target polynucleotide is induced to express by a suitable inducer, such as isopropyl-β-D-thiogalactopyranoside (IPTG).

[0267] To assess the expression of a peptide or its fraction, the expression vector to be introduced into cells may also contain a selectable marker gene or a reporter gene, or both, to facilitate the identification and selection of expressing cells from a population of cells transfected or infected by a viral vector. In other respects, the selectable marker may be carried on a separate DNA fragment and used in co-transfection experiments. Both the selectable marker gene and the reporter gene may be side-mounted to a suitable regulatory sequence to enable its expression in the host cell. Useful selectable markers include, for example, antibiotic resistance genes such as neo and similar genes.

[0268] Reporter genes can be used to identify potentially transfected cells and evaluate the function of regulatory sequences. Typically, a reporter gene is a gene absent from or expressed by a recipient organism or tissue, and it encodes a polypeptide whose expression exhibits easily detectable properties, such as enzymatic activity. Reporter gene expression is detected at an appropriate time after DNA is introduced into recipient cells. Suitable reporter genes may include those encoding luciferase, β-galactosidase, chloramphenicol acetyltransferase, secreted alkaline phosphatase, or green fluorescent protein (e.g., Ui-Tel et al., 2000 FEBS Letters 479:79-82). Suitable expression systems are well-known and can be prepared using known techniques or obtained commercially. Typically, a construct with the smallest 5' flanking region exhibiting the highest expression level of the reporter gene is identified as a promoter. Such promoter regions can be linked to reporter genes and used to assess the ability of certain substances to regulate promoter-driven transcription.

[0269] In some embodiments, the nucleic acid relates to a full-length anti-GM-CSFRα antibody encoding any of the full-length anti-GM-CSFRα antibodies described herein. In some embodiments, the nucleic acid comprises one or more nucleic acid sequences encoding the heavy and light chains of the full-length anti-GM-CSFRα antibody. In some embodiments, each of the one or more nucleic acid sequences is contained in a separate vector. In some embodiments, at least some nucleic acid sequences are contained in the same vector. In some embodiments, all nucleic acid sequences are contained in the same vector. The vector may be selected from, for example, mammalian expression vectors and viral vectors (such as vectors derived from retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses).

[0270] Methods for introducing and expressing genes into cells are known in the art. In the context of expression vectors, vectors can be readily introduced into host cells, such as mammalian cells, bacterial, yeast, or insect cells, by any method in the art. For example, expression vectors can be introduced into host cells by physical, chemical, or biological methods.

[0271] Physical methods for introducing polynucleotides into host cells include calcium phosphate precipitation, liposome transfection, gene gun methods, microinjection, electroporation, and the like. Methods for preparing cells containing vectors and / or exogenous nucleic acids are well known in the art. See, for example, Green and Sambrook (2013, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). In some embodiments, polynucleotides are introduced into host cells via calcium phosphate transfection.

[0272] Biological methods for introducing target polynucleotides into host cells include the use of DNA and RNA vectors. Viral vectors, particularly retroviral vectors, have become the most widely used method for inserting genes into mammalian cells, such as human cells. Other viral vectors can be derived from lentiviruses, poxviruses, herpes simplex virus type 1, adenoviruses, and adeno-associated viruses. See, for example, US Pat. Nos. 5,350,674 and 5,585,362.

[0273] Chemical methods for introducing polynucleotides into host cells include colloidal dispersion systems, such as polymeric complexes, nanocapsules, microspheres, magnetic beads, and lipid-based systems, including oil-in-water emulsions, micelles, hybrid micelles, and liposomes. An exemplary colloidal system used as a delivery carrier in vivo and in vitro is a liposome (e.g., an artificial membrane capsule).

[0274] In the use of non-viral delivery systems, an exemplary delivery vector is the liposome. Consider using lipid formulations to introduce nucleic acids into host cells (in vitro, ex vivo, or in vivo). Alternatively, the nucleic acid can bind to lipids. Lipid-bound nucleic acids can be encapsulated within the aqueous interior of a liposome, dispersed within a lipid bilayer of the liposome, linked to the liposome by linking molecules that bind to the liposome and oligonucleotides, embedded within the liposome, forming a complex with the liposome, dispersed in a solution containing lipids, mixed with lipids, bound to lipids, suspended in lipids, contained in or mixed with micelles, or otherwise bound to lipids. Lipids, lipid / DNA, or lipid / expression vector-related compositions in solution are not limited to any particular structure. For example, they may exist in a bilayer structure, as micelles, or as a “collapsed” structure. They may also be simply dispersed in solution, possibly forming aggregates of varying sizes or shapes. Lipids are fatty substances and can be naturally occurring or synthetic. For example, lipids include fat droplets that are naturally present in the cytoplasm, as well as a class of compounds containing long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes.

[0275] Regardless of the method used to introduce exogenous nucleic acids into host cells or otherwise expose cells to the inhibitors of this application, a variety of experiments can be performed to confirm the presence of the recombinant DNA sequence in the host cells. These experiments include, for example, "molecular biology" experiments well known to those skilled in the art, such as Southern and Northern blotting, RT-PCR, and PCR; and "biochemical" experiments, such as detecting the presence or absence of a specific peptide, for example, identification by immunological methods (ELISAs and Western blots) or by the experiments described herein, all of which fall within the scope of this application.

[0276] Preparation of anti-GM-CSFRα antibody

[0277] In some embodiments, the anti-GM-CSFRα antibody is a monoclonal antibody or derived from a monoclonal antibody. In some embodiments, the anti-GM-CSFRα antibody includes V derived from a monoclonal antibody. H and V L Or its variants. In some embodiments, the anti-GM-CSFRα antibody further comprises C from a monoclonal antibody. H 1 and C L Regions, or variations thereof. Monoclonal antibodies can be prepared using methods known in the art, including hybridoma cell methods, phage display methods, or methods using recombinant DNA. Furthermore, exemplary phage display methods are described herein and in the following examples.

[0278] In hybridoma cell methods, hamsters, mice, or other suitable host animals are typically immunized with an immunomodulator to induce lymphocytes that produce or are capable of producing antibodies that specifically bind to the immunomodulator. Alternatively, lymphocytes can be immunized in vitro. The immunomodulator may include a peptide or fusion protein of the target protein. Peripheral blood lymphocytes (PBLs) are typically used if human cells are desired, while spleen cells or lymph node cells are used if non-human mammalian cells are desired. Lymphocytes are fused with immortalized cell lines using a suitable fusion agent, such as polyethylene glycol, to form hybridoma cells. Immortalized cell lines are typically transformed mammalian cells, particularly rodent, bovine, and human myeloma cells. Rat or mouse myeloma cell lines are commonly used. Hybridoma cells can be cultured in a suitable culture medium, preferably containing one or more substances that inhibit the growth or survival of unfused immortalized cells. For example, if the parental cells lack hypoxanthine-guanine phosphoribosyltransferase (HGPRT or HPRT), the hybridoma cell culture medium typically includes hypoxanthine, aminopterin, and thymidine (HAT medium), which inhibits the growth of HGPRT-deficient cells.

[0279] In some embodiments, the immortalized cell lines are efficiently fused, support stable high-level antibody expression through selected antibody-producing cells, and are sensitive to certain culture media, such as HAT medium. In some embodiments, the immortalized cell lines are mouse myeloma cell lines, obtainable from, for example, the Salk Cell Collection in San Diego, California, and the American Collection of Type Cultures in Manassas, Virginia. Human myeloma and mouse-human hybrid myeloma cell lines for the preparation of human monoclonal antibodies are also described.

[0280] The presence of monoclonal antibodies against the peptide can then be determined in the culture medium for hybridoma cells. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). Such techniques or analytical methods are known in the art. The binding affinity of monoclonal antibodies can be determined, for example, by Scatchard analysis as described in Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0281] After identifying the desired hybridoma cells, the target clone can be subcloned using limiting dilution and cultured using standard methods. Suitable media for this purpose include, for example, modified Eagle medium (DMEM) and RPMI-1640 medium. Alternatively, hybridoma cells can be grown in mammals as ascites fluid.

[0282] Subclonal secreted monoclonal antibodies can be isolated or purified from culture medium or ascites using conventional immunoglobulin purification methods, such as protein A-agarose gel, hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0283] In some embodiments, according to any of the anti-GM-CSFRα antibodies described herein, the anti-GM-CSFRα antibody comprises a sequence selected from an antibody library (e.g., a phage library displaying scFv or Fab fragments). The clone can be identified by screening antibody fragment combination libraries with the desired activity. For example, various methods are known in the art for generating phage display libraries and screening these libraries to obtain antibodies with the desired binding properties. These methods have been reviewed in, for example, Hoogenboom et al., Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, 2001), and in, for example, McCafferty et al., Nature 348:552-554; Clackson et al., Nature 352:624-628 (1991); Marks et al., J. Mol. Biol. 222:581-597 (1992); Marks and Bradbury, Methods in Molecular Biology 248:161-175 (Lo, ed., Human Press, Totowa, NJ, 2003); Sidhu et al., J. Mol. Biol. 338(2):299-310 (2004); Lee et al. Further descriptions can be found in al., J.Mol.Biol.340(5):1073-1093(2004); Fellouse, Proc.Natl.Acad.Sci.USA 101(34):12467-12472(2004); and Lee et al., J.Immunol.Methods 284(1-2):119-132(2004).

[0284] In some phage display methods, V is cloned separately using polymerase chain reaction (PCR). H and V LAll components of the gene are randomly recombined in a phage library, and then phages capable of binding antigens are screened, as described in Winter et al., Ann. Rev. Immunol., 12:433-455 (1994). Phages typically display antibody fragments as scFv fragments or Fab fragments. Phages from immune-derived libraries provide high-affinity antibodies against immunogens without the need to construct hybridoma cells. Alternatively, natural libraries (e.g., from humans) can be cloned to provide a single source of antibodies against multiple non-self and self antigens without any immunization, as described in Griffiths et al., EMBO J, 12:725-734 (1993). Finally, natural libraries can also be prepared by cloning a non-rearranged V-gene fragment from stem cells, encoding the CDR3 hypervariable region using PCR primers containing random sequences, and performing rearrangement in vitro, as described in Hoogenboom and Winter, J. Mol. Biol., 227:381-388 (1992). Patent publications describing human antibody phage libraries include, for example, US Patent No. 5,750,373, and US Patent Publication Nos. 2005 / 0079574, 2005 / 0119455, 2005 / 0266000, 2007 / 0117126, 2007 / 0160598, 2007 / 0237764, 2007 / 0292936, and 2009 / 0002360.

[0285] The anti-GM-CSFRα antibody can be prepared using a phage display method to screen for anti-GM-CSFRα antibody moieties in a library that specifically bind to the target GM-CSFRα. The library can be a human scFv phage display library with at least 1 × 10⁻⁶ cells / mL. 9 (for example, at least about 1×10) 9 2.5×10 9 5×10 9 7.5×10 9 1×10 10 2.5×10 10 5×10 10 7.5×10 10 Or 1×10 11The library contains unique human antibody fragments of any kind. In some embodiments, the library is a natural human library constructed from DNA extracted from human PMBCs and spleens of healthy subjects, containing all human heavy and light chain subfamilies. In some embodiments, the library is a natural human library constructed from DNA extracted from PMBCs isolated from patients with various diseases, such as patients with autoimmune diseases, cancer patients, and patients with infectious diseases. In some embodiments, the library is a semi-synthetic human library in which the heavy chain CDR3 is completely randomized and all amino acids (except cysteine) are present at any given position with equal probability. (See, for example, Hoet, R.M. et al., Nat. Biotechnol. 23(3):344-348, 2005). In some embodiments, the heavy chain CDR3 length of the semi-synthetic human library is between about 5 and about 24 amino acids (e.g., any one of about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, or 24). In some embodiments, the library is a fully synthetic phage display library. In some embodiments, the library is a non-human phage display library.

[0286] Phage clones with high affinity for the target GM-CSFRα can be screened by iterative binding of the phage to the target GM-CSFRα, which is bound to a solid support (e.g., beads for solution panning or mammalian cells for cell panning). Unbound phages are then removed, and the specifically bound phages are eluted. The eluted phage clones are subsequently used to infect suitable host cells, such as E. coli XL1-Blue, for expression and purification. Multiple rounds of panning (e.g., any one of about 2, 3, 4, 5, 6, or more), such as solution panning, cell panning, or a combination of both, can be used to enrich phage clones that specifically bind to GM-CSFRα. The specific binding of the enriched phage clones to the target GM-CSFRα can be detected by any method known in the art, including, for example, ELISA and FACS.

[0287] Monoclonal antibodies can also be prepared using recombinant DNA methods, as described in US Patent No. 4,816,567. The DNA encoding the monoclonal antibody described in this application can be easily isolated and sequenced using conventional methods, such as using oligonucleotide probes that specifically bind to genes encoding the light and heavy chains of murine antibodies. Hybridoma cells as described above or the GM-CSFRα-specific phage clone of this application can serve as sources of this DNA. After isolation, the DNA can be placed in an expression vector, which is then transfected into host cells, such as simian COS cells, Chinese hamster ovary cancer (CHO) cells, or non-immunoglobulin-producing myeloma cells, to obtain monoclonal antibodies synthesized in recombinant host cells. The DNA can also be modified, for example, by replacing the human heavy and light chain constant regions with coding sequences and / or replacing homologous non-human sequences with frame regions (US Patent No. 4,816,567; Morrison et al., supra), or by covalently linking all or part of the coding sequences of immunoglobulins to the coding sequences of non-immunoglobulin polypeptides. This non-immunoglobulin polypeptide can replace the constant region of the antibody in this application, or can replace an antigen-binding site in the variable domain of the antibody in this application, to form a chimeric bivalent antibody.

[0288] The antibody may be a monovalent antibody. Methods for preparing monovalent antibodies are known in the art. For example, a recombinant expression method involving immunoglobulin light chains and modified heavy chains. Typically, the heavy chain is truncated at any position in the Fc region to prevent cross-linking between the heavy chains. Alternatively, relevant cysteine ​​residues are substituted with other amino acid residues or deleted to prevent cross-linking.

[0289] In vitro methods are also suitable for preparing monovalent antibodies. Digesting antibodies to produce antibody fragments, particularly Fab fragments, can be accomplished using any method known in the art.

[0290] Antibody variable domains with the desired binding specificity (antibody-antigen binding site) can be fused to immunoglobulin constant regions. The fusion is preferably fused to an immunoglobulin heavy chain constant region, which includes at least a partial hinge, CH2, and CH3 regions. In some embodiments, a first heavy chain constant region (CH1) containing the necessary light chain binding site is present in at least one fusion. DNAs encoding the immunoglobulin heavy chain fusion, and optionally DNAs encoding the immunoglobulin light chain, are inserted into separate expression vectors and co-transfected into suitable host organisms.

[0291] Humanized and whole-human antibodies

[0292] The anti-GM-CSFRα antibody (such as a full-length anti-GM-CSFRα antibody) can be a humanized antibody or a fully human antibody. The humanized form of the non-human (e.g., mouse) antibody portion is a chimeric immunoglobulin, immunoglobulin chain, or fragment thereof (e.g., Fv, Fab, Fab', F(ab')2, scFv, or other antigen-binding sequence of the antibody), which typically includes a minimal sequence derived from a non-human immunoglobulin. Humanized antibodies include human immunoglobulins, immunoglobulin chains, or fragments thereof (receptor antibodies), wherein residues from the receptor CDR are replaced by CDR residues from a non-human source (donor antibody), such as a mouse, rat, or rabbit CDR, which has the desired specificity, affinity, and performance. In some embodiments, human immunoglobulin Fv frame region residues are replaced by corresponding non-human residues. Humanized antibodies may also contain amino acid residues that are neither in the receptor antibody nor in the introduced CDR or frame region sequence. Typically, humanized antibodies contain at least one, usually two, variable domains. In these variable domains, all or substantially all of the CDR regions correspond to the CDR regions of non-human immunoglobulins, and all or substantially all of the frame regions are human immunoglobulin common sequences.

[0293] Typically, humanized antibodies contain one or more amino acid residues introduced from non-human sources. These non-human amino acid residues are generally referred to as “shift-in” residues and typically originate from the “shift-in” variable domain. According to some embodiments, humanization can be performed essentially as described by Winter and colleagues (Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)) by replacing the corresponding sequence of the human antibody with rodent CDRs or CDR sequences. Thus, this “humanized” antibody portion (US Patent No. 4, 816, 567), which is essentially less than a complete human antibody, has its variable domain replaced by the corresponding sequence from a non-human source. In practice, the humanized antibody portion is a typical human antibody portion, in which some CDR residues and possibly some framework region residues are replaced by residues from similar sites in rodent antibodies.

[0294] Fully human antibodies are an alternative to humanization. For example, transgenic animals (e.g., mice) are typically created and immunized to produce a complete fully human antibody library without generating endogenous immunoglobulins. For instance, it has been reported that homozygous deletion of the antibody heavy chain linker (JH) gene in chimeric and germline mutant mice completely suppresses the production of endogenous antibodies. Transferring human germline immunoglobulin gene arrays into germline mutant mice can produce fully human antibodies upon antigen stimulation, see, for example, akobovits et al., PNAS USA, 90:2551 (1993); Jakobovits et al., Nature, 362:255-258 (1993); Bruggemann et al., Year in Immunol., 7:33 (1993); US Patent Nos. 5,545,806, 5,569,825, 5,591,669; 5,545,807; and WO 97 / 17852. Alternatively, fully human antibodies can be prepared by introducing human immunoglobulin gene loci into transgenic animals, such as mice in which endogenous immunoglobulin genes have been partially or completely silenced. Once stimulated, the production of fully human antibodies can be found to be very similar to that in humans in all aspects, including gene rearrangement, assembly, and antibody library formation. This method has been used in, for example, US Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; and 5,661,016, and Marks et al., Bio / Technology, 10:779-783 (1992); Lonberg et al., Nature, 368:856-859 (1994); Morrison, Nature, 368:812-813 (1994); Fishwild et al., Nature Biotechnology, 14:845-851 (1996); Neuberger, Nature Biotechnology, 14:826 (1996); Lonberg and It is described in Huszar, Intern. Rev. Immunol., 13:65-93 (1995).

[0295] Fully human antibodies can also be produced by activating B cells in vitro (see US Patents 5,567,610 and 5,229,275) or by using various techniques known in the art, including phage display libraries. Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991). Techniques by Cole et al. and Boerner et al. can also be used to prepare fully human monoclonal antibodies. See Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p.77 (1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991).

[0296] Anti-GM-CSFRα antibody variant

[0297] In some embodiments, amino acid sequence variants of the anti-GM-CSFRα antibodies (e.g., full-length anti-GM-CSFRα antibodies) described herein are also considered. For example, it may be necessary to improve the binding affinity and / or other biological activities of the antibody. Amino acid sequence variants of the antibody can be prepared by introducing appropriate modifications into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues in the antibody amino acid sequence. The final construction can be accomplished by any combination of amino acid residue deletions, insertions, and substitutions to give it the desired characteristics, such as antigen binding.

[0298] In some embodiments, the invention relates to anti-GM-CSFRα antibody variants having one or more amino acid substitutions. Target sites for substitution mutations include hypervariable regions (HVRs) and frame regions (FRs). Amino acid substitutions can be introduced into the target antibody to screen for products with desired activities, such as improved biological activity, retention / improvement of antigen-binding ability, reduced immunogenicity, or improved ADCC or CDC.

[0299] Conservative substitutions are shown in Table 4 below.

[0300] Table 4 Conservative Substitution

[0301]

[0302]

[0303] Amino acids are classified into different categories based on the properties of their side chains:

[0304] Hydrophobic amino acids: Norleucine, Methionine (Met), Alanine (Ala), Valine (Val), Leucine (Leu), Isoleucine (Ile);

[0305] Neutral hydrophilic amino acids: Cysteine ​​(Cys), Serine (Ser), Threonine (Thr), Asparagine (Asn), Glutamine (Gln);

[0306] Acidic amino acids: Aspartic acid (Asp), Glutamic acid (Glu);

[0307] Basic amino acids: Histidine (His), Lysine (Lys), Arginine (Arg);

[0308] Amino acids that affect chain orientation: glycine (Gly), proline (Pro);

[0309] Aromatic amino acids: tryptophan (Trp), tyrosine (Tyr), phenylalanine (Phe).

[0310] Substitution of non-conservative amino acids involves replacing one of the above categories with another.

[0311] An exemplary substitution variant is an antibody that is readily produced through affinity maturation, for example, using phage display-based affinity maturation techniques. In short, one or more CDR residues are mutated, the variant antibody is partially displayed on a phage, and variants with specific biological activities (e.g., biological activity or binding affinity based on TF-1 cell proliferation assays) are screened. Alterations (e.g., substitutions) can be made in HVR regions to obtain improved biological activity or antibody affinity based on TF-1 cell proliferation assays. Alterations can be made in “hotspot regions” of the HVR, i.e., residues encoded by codons that are frequently mutated during somatic cell maturation (see, for example, Chowdhury, Methods Mol. Biol. 207:179-196 (2008)), and / or at specific determinant residues (SDRs), to detect the resulting variant V. H and V L Binding affinity. Methods for constructing and reselecting affinity maturities from secondary libraries have been described in some literature, for example, Hoogenboome et al. in Methods in Molecular Biology 178:1-37 (O'Brien et al., ed., Human Press, Totowa, NJ, (2001)).

[0312] In some affinity maturation embodiments, diversity is introduced into selected variable genes for affinity maturation using any of a variety of methods (e.g., error-prone PCR, strand shuffling, or oligonucleotide directed mutagenesis). A secondary library is then created. This library is screened to identify antibody variants with the desired affinity. Another approach involves HVR-mediated introduction of diversity, where several HVR residues (e.g., 4-6 residues at a time) are randomized. HVR residues involved in antigen binding are specifically identified, for example, using alanine scan mutagenesis or modeling. Typically, the CDR-H3 and CDR-L3 regions are particularly key targets.

[0313] In some embodiments, substitutions, insertions, or deletions may occur within one or more HVRs, provided that such changes do not substantially reduce the antibody’s ability to bind to the antigen. For example, conserved changes that do not substantially reduce binding affinity (e.g., conserved substitutions referred to herein) are produced in HVRs. These changes may occur outside the HVR “hotspot” or SDR regions. In some embodiments, the variant VH and VL sequences described above, each HVR, are either unchanged or contain no more than one, two, or three amino acid substitutions.

[0314] A useful method for identifying amino acid residues or regions in an antibody that can be targeted for mutation is called "alanine scanning mutation," as described by Cunningham and Wells (1989) Science, 244:1081-1085. In this method, the substitution of one or a group of residues in the target residue (e.g., charged residues such as arginine, aspartic acid, histidine, lysine, and glutamic acid) with a neutral or negatively charged amino acid (e.g., alanine or aspartic acid, glutamic acid) determines whether the antibody-antigen interaction is affected. Further substitutions can be introduced at the amino acid position to demonstrate that the position is functionally sensitive to the initial substitution. Alternatively / additionally, contact sites between the antibody and antigen can be identified by the crystal structure of the antigen-antibody complex. These contact site residues and adjacent residues can be targeted or eliminated as substitution candidates. Variants are screened to determine if they possess the desired properties.

[0315] Amino acid sequence insertions include peptides with fusion lengths ranging from one residue to 100 or more residues at the amino and / or carboxyl ends, and also include the insertion of one or more amino acid residues within the sequence. Examples of terminal insertions include an antibody having a methionyl residue at its N-terminus. Other insertion variants of antibody molecules include N-terminal or C-terminal fusion enzymes (e.g., ADEPT) of antibody molecules or peptides that increase the serum half-life of the antibody.

[0316] In some embodiments, an anti-GM-CSFRα antibody is involved, which comprises VH The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:6, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:27, or in V H Variants containing up to 5 amino acid substitutions; and V L The V L Contains: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:57, or in V L It contains variants with up to 5 amino acid substitutions.

[0317] In some embodiments, an anti-GM-CSFRα antibody is involved, which comprises V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:6, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:27, wherein V H It contains an amino acid residue selected from E, H, N, G, D, M, S, P, F, Y, A, V, K, W, R, or C at position 31.

[0318] In some embodiments, an anti-GM-CSFRα antibody is involved, which comprises V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:6, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:27, wherein V H It contains an amino acid residue selected from T, H, V, E, P, L, M, S, W, C, A, G, N, or K at position 28; and / or contains an amino acid residue selected from T, P, D, E, Y, W, V, M, N, L, Q, G, S, A, K, or R at position 30.

[0319] In some embodiments, an anti-GM-CSFRα antibody is involved, which comprises V L The V LContains: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:57, wherein V L The amino acid residue selected from S, L, N, A, K, R, I, Q, G, T, H, M, or C is contained at position 26; and / or at position 27, an amino acid residue selected from Q, Y, P, A, I, F, T, R, V, L, E, S, or C is contained; and / or at position 28, an amino acid residue selected from S, H, W, L, R, K, T, P, I, F, V, E, A, or Q is contained; and / or at position 30, an amino acid residue selected from S, L, W, M, A, Y, K, R, G, T, E, V, N, F, or C is contained; and / or at position 31, an amino acid residue selected from S, T, R, A, H, Q, P, The amino acid residues selected from M, L, or G; and / or the amino acid residue selected from Y, L, or F at position 32; and / or the amino acid residue selected from G or T at position 50; and / or the amino acid residue selected from A, G, R, H, K, S, T, M, F, N, or V at position 51; and / or the amino acid residue selected from S, A, W, R, L, T, Q, F, Y, H, or N at position 52; and / or the amino acid residue selected from D, A, Q, or W at position 92; and / or the amino acid residue selected from N, D, E, T, Y, G, A, M, F, S, I, or L at position 93.

[0320] In some embodiments, any amino acid substitution or combination thereof shown in Table 15 is within the scope of consideration.

[0321] In some embodiments, the anti-GM-CSFRα antibody comprises V H The V H Contains the amino acid sequence SEQ ID NO:250, or contains V with at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to SEQ ID NO:250. H Variant, and V L The V L Contains the amino acid sequence SEQ ID NO:241, or contains V that has at least 90% sequence homology with SEQ ID NO:241. L Variant. In some embodiments, the anti-GM-CSFRα antibody comprises: V containing the amino acid sequence SEQ ID NO:250. H And V containing the amino acid sequence SEQ ID NO:241 L .

[0322] In some embodiments, the anti-GM-CSFRα antibody comprises V H The V H Contains the amino acid sequence SEQ ID NO:250, or contains V with at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to SEQ ID NO:250. H Variant, and V L The V L Contains the amino acid sequence SEQ ID NO:193, or contains V that has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with SEQ ID NO:193. L Variant. In some embodiments, the anti-GM-CSFRα antibody comprises: V containing the amino acid sequence SEQ ID NO:250. H And V containing the amino acid sequence SEQ ID NO:193 L .

[0323] In some embodiments, the anti-GM-CSFRα antibody comprises V H The V H Contains the amino acid sequence SEQ ID NO:248, or contains V with at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to SEQ ID NO:248. H Variant, and V L The V L Contains the amino acid sequence SEQ ID NO:188, or contains V that has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with SEQ ID NO:188. L Variant. In some embodiments, the anti-GM-CSFRα antibody comprises: V containing the amino acid sequence SEQ ID NO:248. H And V containing the amino acid sequence SEQ ID NO:188 L .

[0324] In some embodiments, the anti-GM-CSFRα antibody comprises V H The V HContains the amino acid sequence SEQ ID NO:248, or contains V that has at least 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with SEQ ID NO:248. H Variant, and V L The V L Contains the amino acid sequence SEQ ID NO:193, or contains V that has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with SEQ ID NO:193. L Variant. In some embodiments, the anti-GM-CSFRα antibody comprises: V containing the amino acid sequence SEQ ID NO:248. H And V containing the amino acid sequence SEQ ID NO:193 L .

[0325] In some embodiments, the anti-GM-CSFRα antibody comprises V H The V H Contains the amino acid sequence SEQ ID NO:250, or contains V with at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology to SEQ ID NO:250. H Variant, and V L The V L Contains the amino acid sequence SEQ ID NO:288, or contains V that has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with SEQ ID NO:288. L Variant. In some embodiments, the anti-GM-CSFRα antibody comprises: V containing the amino acid sequence SEQ ID NO:250. H And V containing the amino acid sequence SEQ ID NO:288 L .

[0326] In some embodiments, the anti-GM-CSFRα antibody comprises V H The V H Contains the amino acid sequence SEQ ID NO:250, or contains V that has at least 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with SEQ ID NO:250. H Variant, and VL The V L Contains the amino acid sequence SEQ ID NO:188, or contains V that has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with SEQ ID NO:188. L Variant. In some embodiments, the anti-GM-CSFRα antibody comprises: V containing the amino acid sequence SEQ ID NO:250. H And V containing the amino acid sequence SEQ ID NO:188 L .

[0327] In some embodiments, the anti-GM-CSFRα antibody comprises V H The V H Contains the amino acid sequence SEQ ID NO:250, or contains V that has at least 90% (e.g., at least about 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with SEQ ID NO:250. H Variant, and V L The V L Contains the amino acid sequence SEQ ID NO:236, or contains V that has at least about 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with SEQ ID NO:236. L Variant. In some embodiments, the anti-GM-CSFRα antibody comprises: V containing the amino acid sequence SEQ ID NO:250. H And V containing the amino acid sequence SEQ ID NO:236 L .

[0328] In some embodiments, the anti-GM-CSFRα antibody comprises V H The V H Contains the amino acid sequence SEQ ID NO:91, or contains V that has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with SEQ ID NO:91. H Variant, and V L The V LContains the amino acid sequence SEQ ID NO:288, or contains V that has at least 90% (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) sequence homology with SEQ ID NO:288. L Variant. In some embodiments, the anti-GM-CSFRα antibody comprises: V containing the amino acid sequence SEQ ID NO:91. H And V containing the amino acid sequence SEQ ID NO:288 L .

[0329] Fc region variants

[0330] In some embodiments, one or more amino acid modifications are introduced into the Fc region of an antibody described herein (e.g., a full-length anti-GM-CSFRα antibody or an anti-GM-CSFRα antibody fusion protein), thereby producing an Fc region variant. In some embodiments, the Fc region variant has enhanced ADCC efficacy, generally associated with receptors (FcRs) that bind to the Fc. In some embodiments, the Fc region variant has reduced ADCC efficacy. There are numerous examples of how alterations or mutations in the Fc sequence affect its efficacy; for example, WO 00 / 42072 and Shields et al. J Biol. Chem. 9(2):6591-6604 (2001) describe antibody variants that enhance or weaken binding to FcRs. The contents of these publications are incorporated herein by reference.

[0331] Antibody-dependent cell-mediated cytotoxicity (ADCC) is the mechanism of action of therapeutic antibodies against tumor cells. ADCC is a cell-mediated immune defense mechanism in which effector cells of the immune system actively lyse target cells (e.g., cancer cells) when antigens on the surface of target cells are bound by specific antibodies (e.g., anti-GM-CSFRα antibodies). Typically, ADCC effects involve antibody-activated NK cells. NK cells express the Fc receptor CD16. This receptor recognizes and binds to the Fc portion of antibody molecules bound to the surface of target cells. The most common Fc receptors on the surface of NK cells are CD16 or FcγRIII. Binding of the Fc receptor to the Fc region of the antibody leads to NK cell activation, release of cytolytic granules, and subsequent apoptosis of the target cells. The cytotoxic effect of ADCC on tumor cells can be determined by specificity assays using NK-92 cells transfected with high-affinity FcRs. The results were compared with those of wild-type NK-92 cells that do not express FcRs.

[0332] In some embodiments, this application also relates to anti-GM-CSFRα antibody variants (e.g., full-length anti-GM-CSFRα antibody variants) containing an Fc region that has some, but not all, effector functions, resulting in an extended half-life in vivo, where the specific effector function (e.g., CDC or ADCC) is non-essential or detrimental. Such anti-GM-CSFRα antibodies are ideal candidates for this application. The reduction / depletion of CDC and / or ADCC activity is confirmed by in vitro and / or in vivo cytotoxicity assays. For example, an Fc receptor (FcR) binding assay confirms that the antibody lacks FcγR binding capacity (and therefore may lack ADCC activity) but still retains FcRn binding capacity. In the main cells mediating ADCC, NK cells express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. The expression of FcR on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and KinetAnnu.Rev.Immunol.9:457-492 (1991). Non-restrictive examples of assessing ADCC activity of target molecules in vitro are described in US Pat. No. 5, 500, 362 (see, for example, Hellstrom, I. et al.Proc.Nat'l Acad.Sci.USA 83:7059-7063 (1986)) and Hellstrom, I et al.,Proc.Nat'l Acad.Sci.USA 82:1499-1502 (1985); US Pat. No. 5, 821, 337 (see Bruggemann, M. et al.,J.Exp.Med.166:1351-1361 (1987)). Alternatively, non-radioactive detection methods can be used (see, for example, the ACTITM flow cytometry non-radioactive cytotoxicity assay (Cell Technology, Inc. Mountain View, Calif.) and the CYTOTOX 96TM non-radioactive cytotoxicity assay (Promega, Madison, Wis.)). Effector cells used in such assays include peripheral blood mononuclear cells (PBMCs) and natural killer cells (NK cells). Alternatively, the ADCC activity of the target molecule can be detected in vivo, for example in animal models, as described in Clynes et al. Proc. Nat'l Acad. Sci. USA 95:652-656 (1998). A C1q binding assay can also be performed to confirm that the antibody does not bind to C1q, thus lacking CDC activity. See, for example, C1q and C3c binding ELISAs in WO2006 / 029879 and WO 2005 / 100402.To assess complement activation, CDC detection was performed (see, for example, Gazzano-Santoro et al., J. Immunol. Methods 202:163 (1996); Cragg, MS et al., Blood 101:1045-1052 (2003); and Cragg, MS and MJ Glennie, Blood 103:2738-2743 (2004)). FcRn binding and in vivo clearance / half-life were determined using methods known in the art (see, for example, Petkova, S B et al., Int'l. Immunol. 18(12):1759-1769 (2006)).

[0333] Antibodies with reduced effector function include those with one or more residue substitutions at positions 238, 265, 269, 270, 297, 327, and 329 in the Fc region (US Pat. No. 6,737,056). These Fc variants include those with two or more residue substitutions at positions 265, 269, 270, 297, and 327, including the Fc variant known as “DANA,” which has alanine substitutions at positions 265 and 297 (US Pat. No. 7,332,581).

[0334] Antibody variants with increased or decreased binding affinity to FcRs have been described (see, for example, US Pat. No. 6, 737, 056; WO 2004 / 056312, and Shields et al., J. Biol. Chem. 9(2): 6591-6604 (2001)).

[0335] In some embodiments, a variant of an anti-GM-CSFRα antibody (e.g., a full-length anti-GM-CSFRα antibody) is involved, comprising an Fc region variant having one or more amino acid substitutions capable of enhancing ADCC effects. In some embodiments, the Fc region variant comprises one or more amino acid substitutions capable of enhancing ADCC effects, these substitutions being located at positions 298, 333, and / or 334 (EU residue number) of the Fc region. In some embodiments, the anti-GM-CSFRα antibody variant (e.g., a full-length anti-GM-CSFRα antibody) includes amino acid substitutions at positions S298A, E333A, and K334A of the Fc region.

[0336] In some embodiments, alterations in the Fc region result in alterations (i.e., enhancement or attenuation) of C1q binding and / or complement-dependent cytotoxicity (CDC), as described in US Pat. No. 6, 194, 551, WO 99 / 51642, and Idusogie et al., J. Immunol. 164: 4178-4184 (2000).

[0337] In some embodiments, a variant of an anti-GM-CSFRα antibody (e.g., a full-length anti-GM-CSFRα antibody) is involved, comprising an Fc region variant having one or more amino acid substitutions that can prolong the half-life or enhance binding to the Fc receptor (FcRn). Antibodies with prolonged half-life and improved FcRn binding are described in US2005 / 0014934A1 (Hinton et al.). These antibodies contain one or more amino acid substitutions in the Fc region that enhance the binding of the Fc region to the FcRn. These Fc variants contain one or more substitutions for residues at positions 238, 256, 265, 272, 286, 303, 305, 307, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 382, ​​413, 424, or 434 in the Fc region, such as the substitution of residue at position 434 in the Fc region (US Pat. No. 7, 371, 826).

[0338] See also examples of other Fc region variants in Duncan & Winter, Nature 322:738-40 (1988); US Pat. No. 5, 648, 260; US Pat. No. 5, 624, 821 and WO 94 / 29351.

[0339] This application considers anti-GM-CSFRα antibodies (e.g., full-length anti-GM-CSFRα antibodies) including any of the Fc variants or combinations thereof described herein.

[0340] Glycosylation variants

[0341] In some embodiments, the anti-GM-CSFRα antibody (e.g., full-length anti-GM-CSFRα antibody) described herein is modified to increase or decrease the degree of glycosylation of the anti-GM-CSFRα antibody. Adding or deleting glycosylation sites on the anti-GM-CSFRα antibody can be conveniently achieved by altering the amino acid sequence of the anti-GM-CSFRα antibody or its polypeptide moiety to increase or remove one or more glycosylation sites.

[0342] The anti-GM-CSFRα antibody contains an Fc region, to which the attached sugar can be modified. Natural antibodies produced by mammalian cells typically contain branched biantennary oligosaccharides, which are usually linked to the CH2 domain Asn297 of the Fc region via N-linkage, see, for example, Wright et al., TIBTECH 15:26-32 (1997). The oligosaccharide can contain various sugars, such as mannose, N-acetylglucosinolate (GlcNAc), galactose, and sialic acid, as well as trehalose linked to the GlcNAc in the "stem" of the biantennary oligosaccharide structure. In some embodiments, the anti-GM-CSFRα antibody of this application can be modified with oligosaccharides to produce anti-GM-CSFRα antibody variants with certain improved properties.

[0343] The N-glycans linked to the CH2 domain of the Fc region are heterogeneous. Antibodies or Fc fusion proteins produced in CHO cells are fucosylated via fucosyltransferase activity (see Shoji-Hosaka et al., J. Biochem. 2006, 140:777-83). Typically, a small subset of naturally occurring non-fucosylated IgGs can be detected in human serum. N-glycosylation of the Fc region is important for its binding to FcRIIIa; and non-fucosylated N-glycans enhance the binding affinity of Fc to FcRIIIa. This enhanced binding affinity to FcRIIIa enhances ADCC effects, which is advantageous in certain antibody therapeutic applications requiring cytotoxicity.

[0344] In some embodiments, enhanced effector function may be detrimental when Fc-mediated cytotoxicity is not required. In some embodiments, the Fc fragment or CH2 domain is non-glycosylated. In some embodiments, glycosylation is prevented by mutating the N-glycosylation site in the CH2 domain.

[0345] In some embodiments, variants of anti-GM-CSFRα antibodies (e.g., full-length anti-GM-CSFRα antibodies) are involved, comprising an Fc region wherein the glycosylation structure linked to the Fc region has reduced or absent fucose, which may enhance ADCC function. Specifically, this document relates to anti-GM-CSFRα antibodies that have reduced fucose relative to the same anti-GM-CSFRα antibody produced by wild-type CHO cells. That is, they are characterized by having less fucose than antibodies produced by native CHO cells (e.g., CHO cells producing the native glycosylated form, CHO cells containing the native FUT8 gene). In some embodiments, the N-linked glycan of the anti-GM-CSFRα antibody has less than 50%, 40%, 30%, 20%, 10%, or 5% fucose. For example, the fucose content of the anti-GM-CSFRα antibody may be 1%-80%, 1%-65%, 5%-65%, or 20%-40%. In some embodiments, the N-linked glycan of the anti-GM-CSFRα antibody does not contain fucose, i.e., the anti-GM-CSFRα antibody is completely fucose-free, or contains no fucose, or is defucosylated. The fucose content is determined by calculating the average fucose content within the glycan chain linked to Asn297 relative to the total amount of all sugar structures (such as complex, hybrid, or mannose structures) linked to Asn297 as measured by MALDI-TOF mass spectrometry, as described in WO 2008 / 077546. Asn297 refers to the asparagine residue at position 297 of the Fc region (EU Fc region residue numbering system). However, due to minor sequence variations in the antibody, Asn297 may also be located approximately ±3 amino acids upstream or downstream of position 297, i.e., between positions 294 and 300. These fucosylated variants may possess enhanced ADCC function. See, for example, US Patent Publication Nos. US 2003 / 0157108 (Presta, L.) and US 2004 / 0093621 (Kyowa Hakko Kogyo Co., Ltd.).Examples of publications related to antibody variants that are “defucosylated” or “fucosylated” include US2003 / 0157108; WO 2000 / 61739; WO 2001 / 29246; US 2003 / 0115614; US 2002 / 0164328; US2004 / 0093621; US ​​2004 / 0132140; US 2004 / 0110704; US 2004 / 0110282; US 2004 / 0109865; WO2003 / 085119; WO 2003 / 084570; WO 2005 / 035586; WO 2005 / 035778; WO2005 / 053742; WO2002 / 031140; Okazaki et al. Cell lines capable of producing defucosylation antibodies include Lec13CHO cells lacking protein fucosylation function (Ripka et al. Arch. Biochem. Biophys. 249:533-545 (1986); US Pat Appl No. US 2003 / 0157108A1, Presta, L; and WO 2004 / 056312 A1, Adams et al., especially Example 11), and gene knockout cell lines, such as CHO cells with α-1,6-fucosylation gene and FUT8 gene knockout (see Yamane-Ohnuki et al. Biotech. Bioeng. 87:614 (2004); Kanda, Y. et al., Biotechnol. Bioeng. 94(4):680-688 (2006); and WO2003 / 085107).

[0346] Anti-GM-CSFRα antibody variants (e.g., full-length anti-GM-CSFRα antibodies) further involve bimeric oligosaccharides, for example, wherein the biantennary oligosaccharide linked to the Fc region of the anti-GM-CSFRα antibody is bimeric by GlcNAc. Such anti-GM-CSFRα antibody variants (e.g., full-length anti-GM-CSFRα antibodies) may have reduced fucosylation and / or enhanced ADCC function. Examples of such antibody variants are described in WO 2003 / 011878 (Jean-Mairet et al.); US Pat. No. 6,602,684 (Umana et al.); US 2005 / 0123546 (Umana et al.); and Ferrara et al., Biotechnology and Bioengineering, 93(5):851-861 (2006). Also involved are variants of anti-GM-CSFRα antibodies (e.g., full-length anti-GM-CSFRα antibodies) that have at least one galactose residue in the oligosaccharide linked to the Fc region. These anti-GM-CSFRα antibody variants may have enhanced CDC function. Such variants are described, for example, in WO 1997 / 30087 (Patel et al.); WO 1998 / 58964 (Raju, S.); and WO 1999 / 22764 (Raju, S.).

[0347] In some embodiments, the anti-GM-CSFRα antibody variant containing the Fc region (e.g., a full-length anti-GM-CSFRα antibody) can bind to FcγRIII. In some embodiments, the anti-GM-CSFRα antibody variant containing the Fc region (e.g., a full-length anti-GM-CSFRα antibody) has ADCC activity in the presence of human effector cells (e.g., T cells), or has enhanced ADCC activity in the presence of human effector cells compared to other identical anti-GM-CSFRα antibodies (e.g., full-length anti-GM-CSFRα antibodies) having a human wild-type IgG1 Fc region.

[0348] Cysteine ​​engineered variants

[0349] In some embodiments, it is necessary to prepare a cysteine-engineered anti-GM-CSFRα antibody (e.g., a full-length anti-GM-CSFRα antibody) in which one or more amino acid residues are replaced by cysteine ​​residues. In some embodiments, the substituted residues are located at accessible sites on the anti-GM-CSFRα antibody. By replacing those residues with cysteine, an active thiol group is located at an accessible site on the anti-GM-CSFRα antibody, which can be used to conjugate the anti-GM-CSFRα antibody to other parts, such as a pharmaceutical part or a linker-pharmaceutical part, to prepare an anti-GM-CSFRα immunoconjugate as further described herein. Cysteine-engineered anti-GM-CSFRα antibodies (e.g., full-length anti-GM-CSFRα antibodies) can be prepared according to, for example, US Pat. No. 7,521,541.

[0350] derivative

[0351] In some embodiments, the anti-GM-CSFRα antibody (e.g., full-length anti-GM-CSFRα antibody) described herein may be further modified to include other non-protein moieties known in the art and readily available. Moieties suitable for derivatizing anti-GM-CSFRα antibodies include, but are not limited to, water-soluble polymers. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), ethylene glycol / propylene glycol copolymers, carboxymethyl cellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1,3-dioxolane, poly-1,3,6-trioxane, ethylene / maleic anhydride copolymers, polyamino acids (homogeneous or random copolymers), dextran or poly(n-vinylpyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide / ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde is advantageous in manufacturing due to its stability in water. The polymer can be of any molecular weight and can be branched or unbranched. The number of polymers linked to an anti-GM-CSFRα antibody can vary, and if more than one polymer is linked, they can be the same or different molecules. Typically, the number and / or type of polymers used for derivatization can be determined based on considerations including, but not limited to, the need to improve the properties or function of the anti-GM-CSFRα antibody, and whether the anti-GM-CSFRα antibody derivative is intended for treatment under specific conditions.

[0352] Pharmaceutical Composition

[0353] This document also relates to compositions comprising any anti-GM-CSFRα antibody (e.g., a full-length anti-GM-CSFRα antibody), a nucleic acid encoding the antibody, a vector comprising the nucleic acid encoding the antibody, or a host cell comprising the nucleic acid or vector described herein (e.g., pharmaceutical compositions, also referred to herein as formulations). In some embodiments, a pharmaceutical composition is described comprising any anti-GM-CSFRα antibody described herein and a pharmaceutically acceptable vector.

[0354] Suitable anti-GM-CSFRα antibody formulations can be obtained by mixing anti-GM-CSFRα antibodies of the desired purity with optional pharmaceutically acceptable carriers, excipients, or stabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), prepared as lyophilized or liquid formulations. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the doses and concentrations used, including buffers such as phosphates, citric acid, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (e.g., octadecyl dimethyl benzyl ammonium chloride; hexamethyl ammonium chloride; benzalkonium chloride; benzyl chloride; phenol; butanol or benzyl alcohol; alkyl esters of parabens, such as methylparaben or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol and m-cresol); low molecular weight (less than about 10 residues) peptides; proteins, such as serum albumin, gelatin, or immunoglobulins. Hydrophilic polymers, such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextrin; chelating agents such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (such as zinc-protein complexes); and / or nonionic surfactants such as TWEENTM, PLURONICS™, or polyethylene glycol (PEG); exemplary formulations as described in WO98 / 56418, which are expressly incorporated herein by reference. Lyophilized formulations suitable for subcutaneous administration are described in WO97 / 04801. Such lyophilized formulations can be reconstituted into high-protein-concentration formulations with suitable diluents, and the reconstituted formulations can be administered subcutaneously to the individuals to be treated herein. Cationic liposomes or liposomes can be used to deliver the anti-GM-CSFRα antibody of this application to cells.

[0355] In addition to an anti-GM-CSFRα antibody (e.g., a full-length anti-GM-CSFRα antibody), the formulations described herein may contain one or more other active substances necessary for treating a specific condition, preferably substances with complementary activities and no adverse interactions with each other. For example, in addition to the anti-GM-CSFRα antibody, it may be necessary to further include antitumor agents, growth inhibitors, cytotoxic agents, or chemotherapeutic agents. These molecules are present in a combination of amounts effective for the intended purpose. The effective amount of other substances depends on the content of the anti-GM-CSFRα antibody in the formulation, the type of disease or condition or treatment, and other factors as described above. These drugs are typically used at the same dosage and route of administration as described herein, or at approximately 1% to 99% of the currently used dosage.

[0356] The anti-GM-CSFRα antibody (e.g., full-length anti-GM-CSFRα antibody) can also be encapsulated in microcapsules, for example, prepared by coagulation techniques and interfacial polymerization, such as hydroxymethyl cellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules) or crude emulsions. Sustained-release formulations can be prepared.

[0357] Sustained-release formulations of anti-GM-CSFRα antibodies (e.g., full-length anti-GM-CSFRα antibodies) can be prepared. Suitable examples of sustained-release formulations include solid hydrophobic polymer semi-permeable matrices containing antibodies (or fragments thereof), which are in the form of molded articles, such as films or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate) or poly(vinyl alcohol)), polylactic acid (US Pat. No. 3,773,919), L-glutamic acid and L-glutamic acid ethyl ester copolymers, non-degradable ethylene-vinyl acetate copolymers, and degradable lactic acid-glycolic acid copolymers such as LUPRON DEPOT. TM (Injectable microspheres composed of lactic-glycolic acid copolymer and leuprolide acetate) and poly-D(-)-3-hydroxybutyric acid. While polymer molecules such as ethylene-vinyl acetate and lactic-glycolic acid can release proteins over 100 days, some hydrogels can release proteins in a shorter time. When encapsulated antibodies remain in vivo for extended periods, they may denature or aggregate due to exposure to a humid environment at 37°C, leading to loss of biological activity or potential alteration of immunogenicity. Appropriate strategies can be designed to stabilize anti-GM-CSFRα antibodies based on the corresponding mechanisms. For example, if the aggregation mechanism is found to be the formation of intermolecular SS bonds through thiodisulfide exchange, stabilization can be achieved by modifying thiol residues, lyophilizing in acidic solutions, controlling water content, using appropriate additives, and developing specific polymer matrix compositions.

[0358] In some embodiments, the anti-GM-CSFRα antibody (e.g., full-length anti-GM-CSFRα antibody) is formulated in a buffer containing citrate, sodium chloride, acetate, succinate, glycine, polysorbate 80 (Tween 80), or any combination thereof.

[0359] Formulations intended for internal administration must be sterile. This can be easily achieved, for example, by applying a sterile filter membrane.

[0360] Treatment using anti-GM-CSFRα antibodies

[0361] Anti-GM-CSFRα antibodies (e.g., full-length anti-GM-CSFRα antibodies) and / or the compositions described herein can be administered to individuals (e.g., mammals, such as humans) to treat diseases and / or conditions associated with high expression of GM-CSF and / or GM-CSFRα, as well as diseases and / or conditions resulting from dysregulation of GM-CSF and / or GM-CSFRα, such as autoimmune diseases and / or inflammatory diseases or cancers characterized by high expression of GM-CSF and / or GM-CSFRα and / or abnormal GM-CSF / GM-CSFRα function, for example, rheumatoid arthritis, asthma, and myeloid leukemia-related lung diseases. Therefore, in some embodiments of this application, a method for treating autoimmune diseases and / or inflammatory conditions or cancers (e.g., rheumatoid arthritis, asthma, myeloid leukemia) characterized by high expression of GM-CSF and / or GM-CSFRα and / or abnormal GM-CSF / GM-CSFRα function is provided, comprising administering to an individual an effective amount of a composition (e.g., a pharmaceutical composition) containing an anti-GM-CSFRα antibody (e.g., a full-length anti-GM-CSFRα antibody), such as any of the anti-GM-CSFRα antibodies described herein (e.g., a full-length anti-GM-CSFRα antibody).

[0362] In some embodiments, the disease or condition is selected from, for example, rheumatoid arthritis, asthma, chronic obstructive pulmonary disease, allergic reactions, multiple sclerosis, myeloid leukemia, or atherosclerosis. In some embodiments, the individual is a human.

[0363] For example, in some embodiments, a method relates to treating an individual suffering from an autoimmune disease and / or inflammatory condition or cancer (e.g., rheumatoid arthritis, asthma, or myeloid leukemia) characterized by high expression of GM-CSF and / or GM-CSFRα and / or abnormal GM-CSF / GM-CSFRα function, comprising administering to the individual an effective amount of a pharmaceutical composition comprising a GM-CSFRα antibody (e.g., a full-length anti-GM-CSFRα antibody) that specifically binds to an epitope of human GM-CSFRα, wherein the epitope comprises amino acid residues at Val50, Glu59, Lys194, Lys195, Arg283, and Ile284 of human GM-CSFRα. In some embodiments, the anti-GM-CSFRα antibody described herein specifically binds to an epitope on human GM-CSFRα, the epitope comprising amino acid residues at Val50, Glu59, Lys194, Lys195, Arg283, Ile284, Val51, Thr63, and Ile196 of human GM-CSFRα. In some embodiments, the anti-GM-CSFRα antibody described herein specifically binds to an epitope on human GM-CSFRα, the epitope comprising amino acid residues at Val50, Glu59, Lys194, Lys195, Arg283, Ile284, Leu191, and Ile196 of human GM-CSFRα. In some embodiments, the anti-GM-CSFRα antibody described herein specifically binds to an epitope on human GM-CSFRα, the epitope comprising amino acid residues at Val50, Glu59, Lys194, Lys195, Arg283, Ile284, Arg49, Val51, Asn57, and Ser61 of human GM-CSFRα. In some embodiments, the anti-GM-CSFRα antibody is a full-length antibody. In some embodiments, the full-length anti-GM-CSFRα antibody is an IgG1 or IgG4 antibody. In some embodiments, the disease or condition is selected from rheumatoid arthritis, asthma, chronic obstructive pulmonary disease, allergic reactions, multiple sclerosis, myeloid leukemia, or atherosclerosis. In some embodiments, the individual is a human.

[0364] In some embodiments, a method of treating an individual suffering from an autoimmune disease and / or inflammatory disease or cancer (e.g., rheumatoid arthritis, asthma, or myeloid leukemia) characterized by high expression of GM-CSF and / or GM-CSFRα and / or abnormal GM-CSF / GM-CSFRα function is involved, comprising administering to the individual an effective amount of a pharmaceutical composition comprising an anti-GM-CSFRα antibody (e.g., a full-length anti-GM-CSFRα antibody), wherein the anti-GM-CSFRα antibody comprises V H The V HContains: an HC-CDR1 containing the sequence X1LX2X3H (SEQ ID NO:76), wherein X1 is E, N, G, D, M, S, P, F, Y, A, V, K, W, R, or C, X2 is S, C, or P, and X3 is I or M; an HC-CDR2 containing the sequence GFDX1X2X3X4EX5X6YAQKX7QG (SEQ ID NO:77), wherein X1 is P, G, T, S, or V, X2 is E, D, G, or A, X3 is D, G, I, W, S, or V, X4 is G, E, D, or H, X5 is T or A, X6 is N or I, and X7 is S or F; and a HC-CDR2 containing the sequence GRYX1X2X3X4X5X6YGFDY (SEQ ID NO:77). NO:78) of HC-CDR3, wherein X1 is C, T, S, I, A or V; X2 is S, G, E, F, W, H, I, V, N, Y, T or R; X3 is T, H, L, F, P, I, S, Y, K, A, D, V, N or G; X4 is D, A, M, Y, F, S, T, G or W; X5 is T, S, F, Q, A, N, L, E, I, G or M; X6 is C, T, N, S or A; and V L The V L Contains: an LC-CDR1 containing the sequence RAX1X2X3VX4X5X6LA (SEQ ID NO:293), wherein X1 is S, L, N, A, K, R, I, Q, G, T, H, M, or C; X2 is Q, Y, P, A, I, F, T, R, V, L, E, S, or C; X3 is S, H, W, L, R, K, T, P, I, F, V, E, A, or Q; X4 is S, L, W, M, A, Y, K, R, G, T, E, V, N, F, or C; X5 is S, T, R, A, H, Q, P, M, L, or G; and X6 is Y, L, or F; and a LC-CDR1 containing the sequence X1X2X3LA (SEQ ID NO:293), wherein X1 is S, L, N, A, Q, P, M, L, or G; and X6 is Y, L, or F. LC-CDR2 (NO:294), wherein X1 is G or T, X2 is A, G, R, H, K, S, T, M or F, and X3 is S, A, W, R, L, T, Q, F, Y, H or N; and LC-CDR3 containing the sequence QQYX1X2X3PX4T (SEQ ID NO:79), wherein X1 is N, D, S, R, A, T, L, Y, Q, W or G, X2 is N, D, E, T, Y, G, A, M, F, S, I or L, X3 is W, S, P, V, G or R, and X4 is P, Y, H, S, F, N, D, V or G.

[0365] In some embodiments, a method of treating an individual suffering from an autoimmune disease and / or inflammatory condition or cancer (e.g., rheumatoid arthritis, asthma, or myeloid leukemia) characterized by high expression of GM-CSF and / or GM-CSFRα and / or abnormal GM-CSF / GM-CSFRα function, comprising administering to the individual an effective amount of a composition comprising an anti-GM-CSFRα antibody, wherein the antibody comprises V H The V H Contains: an HC-CDR1 comprising any of the amino acid sequences of SEQ ID NOs:1-4, an HC-CDR2 comprising any of the amino acid sequences of SEQ ID NOs:5-16, and an HC-CDR3 comprising any of the amino acid sequences of SEQ ID NOs:17-50, or a variant comprising up to 5 amino acid substitutions; and V L The V L It comprises: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing any of the amino acid sequences SEQ ID NOs:53-75, or a variant containing up to 5 amino acid substitutions.

[0366] In some embodiments, a method of treating an individual suffering from an autoimmune disease and / or inflammatory condition or cancer (e.g., rheumatoid arthritis, asthma, or myeloid leukemia) characterized by high expression of GM-CSF and / or GM-CSFRα and / or abnormal GM-CSF / GM-CSFRα function, comprising administering to the individual an effective amount of a composition comprising an anti-GM-CSFRα antibody, wherein the antibody comprises V H The V H Contains any of the amino acid sequences in SEQ ID NOs:80-121 and 246-287, or contains V with at least 90% sequence homology to any of the amino acid sequences in SEQ ID NOs:80-121 and 246-287. H Variant; and V L The V L The amino acid sequence comprising any one of SEQ ID NOs:122-144, 150-245, and 288-289, or comprising V having at least 90% sequence homology with any one of the amino acid sequences in SEQ ID NOs:122-144, 150-245, and 288-289. L Variants.

[0367] In some embodiments, the anti-GM-CSFRα antibody described herein is a full-length anti-GM-CSFRα antibody comprising a constant region of IgG1 or IgG4. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0368] In some embodiments, a method of treating an individual suffering from an autoimmune disease and / or inflammatory condition or cancer (e.g., rheumatoid arthritis, asthma, or myeloid leukemia) characterized by high expression of GM-CSF and / or GM-CSFRα and / or abnormal GM-CSF / GM-CSFRα function is included, comprising administering to the individual an effective amount of a composition comprising an anti-GM-CSFRα antibody, wherein the antibody comprises V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:5, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:17, or a variant containing up to 5 amino acid substitutions; and V L The V L It comprises: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:54, or a variant containing up to 5 amino acid substitutions.

[0369] In some embodiments, the anti-GM-CSFRα antibody described herein comprises: V containing the amino acid sequence SEQ ID NO:80 H And V containing the amino acid sequence SEQ ID NO:123 L In some embodiments, the anti-GM-CSFRα antibody described herein is a full-length anti-GM-CSFRα antibody containing a constant region of IgG1 or IgG4. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0370] In some embodiments, a method of treating an individual suffering from an autoimmune disease and / or inflammatory condition or cancer (e.g., rheumatoid arthritis, asthma, or myeloid leukemia) characterized by high expression of GM-CSF and / or GM-CSFRα and / or abnormal GM-CSF / GM-CSFRα function, comprising administering to the individual an effective amount of a composition comprising an anti-GM-CSFRα antibody, wherein the antibody comprises V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:8, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:22, or a variant containing up to 5 amino acid substitutions; and V L The V L It comprises an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:56, or a variant containing up to 5 amino acid substitutions.

[0371] In some embodiments, the anti-GM-CSFRα antibody described herein comprises: V containing the amino acid sequence SEQ ID NO:85 H And V containing the amino acid sequence SEQ ID NO:125 L In some embodiments, the anti-GM-CSFRα antibody described herein is a full-length anti-GM-CSFRα antibody containing a constant region of IgG1 or IgG4. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0372] In some embodiments, a method of treating an individual suffering from an autoimmune disease and / or inflammatory condition or cancer (e.g., rheumatoid arthritis, asthma, or myeloid leukemia) characterized by high expression of GM-CSF and / or GM-CSFRα and / or abnormal GM-CSF / GM-CSFRα function, comprising administering to the individual an effective amount of a composition comprising an anti-GM-CSFRα antibody, wherein the antibody comprises V H The V HContains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:7, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:23, or a variant containing up to 5 amino acid substitutions; and V L V L It includes: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:57, or a variant containing up to 5 amino acid substitutions.

[0373] In some embodiments, the anti-GM-CSFRα antibody described herein comprises: V containing the amino acid sequence SEQ ID NO:86 H And V containing the amino acid sequence SEQ ID NO:126 L In some embodiments, the anti-GM-CSFRα antibody described herein is a full-length anti-GM-CSFRα antibody containing a constant region of IgG1 or IgG4. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0374] In some embodiments, a method of treating an individual suffering from an autoimmune disease and / or inflammatory condition or cancer (e.g., rheumatoid arthritis, asthma, or myeloid leukemia) characterized by high expression of GM-CSF and / or GM-CSFRα and / or abnormal GM-CSF / GM-CSFRα function, comprising administering to the individual an effective amount of a composition comprising an anti-GM-CSFRα antibody, wherein the antibody comprises V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:6, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:27, or a variant containing up to 5 amino acid substitutions; and V L The V LIt includes: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:57, or a variant containing up to 5 amino acid substitutions.

[0375] In some embodiments, the anti-GM-CSFRα antibody described herein comprises: V containing the amino acid sequence SEQ ID NO:91 H And V containing the amino acid sequence SEQ ID NO:126 L In some embodiments, the anti-GM-CSFRα antibody described herein is a full-length anti-GM-CSFRα antibody containing a constant region of IgG1 or IgG4. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0376] In some embodiments, a method of treating an individual suffering from an autoimmune disease and / or inflammatory condition or cancer (e.g., rheumatoid arthritis, asthma, or myeloid leukemia) characterized by high expression of GM-CSF and / or GM-CSFRα and / or abnormal GM-CSF / GM-CSFRα function, comprising administering to the individual an effective amount of a composition comprising an anti-GM-CSFRα antibody, wherein the antibody comprises V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:7, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:35, or a variant containing up to 5 amino acid substitutions; and V L The V L It includes: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:53, or a variant containing up to 5 amino acid substitutions.

[0377] In some embodiments, the anti-GM-CSFRα antibody described herein comprises: V containing the amino acid sequence SEQ ID NO:99 HAnd V containing the amino acid sequence SEQ ID NO:122 L In some embodiments, the anti-GM-CSFRα antibody described herein is a full-length anti-GM-CSFRα antibody containing a constant region of IgG1 or IgG4. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0378] In some embodiments, a method of treating an individual suffering from an autoimmune disease and / or inflammatory condition or cancer (e.g., rheumatoid arthritis, asthma, or myeloid leukemia) characterized by high expression of GM-CSF and / or GM-CSFRα and / or abnormal GM-CSF / GM-CSFRα function, comprising administering to the individual an effective amount of a composition comprising an anti-GM-CSFRα antibody, wherein the antibody comprises V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:7, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:37, or a variant containing up to 5 amino acid substitutions; and V L The V L It includes: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:57, or a variant containing up to 5 amino acid substitutions.

[0379] In some embodiments, the anti-GM-CSFRα antibody described herein comprises: V containing the amino acid sequence SEQ ID NO:101. H And V containing the amino acid sequence SEQ ID NO:126 LIn some embodiments, the anti-GM-CSFRα antibody described herein is a full-length anti-GM-CSFRα antibody containing a constant region of IgG1 or IgG4. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0380] In some embodiments, a method of treating an individual suffering from an autoimmune disease and / or inflammatory condition or cancer (e.g., rheumatoid arthritis, asthma, or myeloid leukemia) characterized by high expression of GM-CSF and / or GM-CSFRα and / or abnormal GM-CSF / GM-CSFRα function, comprising administering to the individual an effective amount of a composition comprising an anti-GM-CSFRα antibody, wherein the antibody comprises V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:3, an HC-CDR2 containing the amino acid sequence SEQ ID NO:6, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:39, or a variant containing up to 5 amino acid substitutions; and V L The V L It comprises: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:54, or a variant containing up to 5 amino acid substitutions.

[0381] In some embodiments, the anti-GM-CSFRα antibody described herein comprises: V containing the amino acid sequence SEQ ID NO:103. H And V containing the amino acid sequence SEQ ID NO:123 L In some embodiments, the anti-GM-CSFRα antibody described herein is a full-length anti-GM-CSFRα antibody containing a constant region of IgG1 or IgG4. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0382] In some embodiments, a method of treating an individual suffering from an autoimmune disease and / or inflammatory disease or cancer (e.g., rheumatoid arthritis, asthma, or myeloid leukemia) characterized by high expression of GM-CSF and / or GM-CSFRα and / or abnormal GM-CSF / GM-CSFRα function, comprising administering to the individual an effective amount of a composition comprising an anti-GM-CSFRα antibody, wherein the antibody comprises V H The V H Contains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:7, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:35, or a variant containing up to 5 amino acid substitutions; and V L The V L It includes: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:57, or a variant containing up to 5 amino acid substitutions.

[0383] In some embodiments, the anti-GM-CSFRα antibody described herein comprises: V containing the amino acid sequence SEQ ID NO:99 H And V containing the amino acid sequence SEQ ID NO:126 L In some embodiments, the anti-GM-CSFRα antibody described herein is a full-length anti-GM-CSFRα antibody containing a constant region of IgG1 or IgG4. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0384] In some embodiments, a method of treating an individual suffering from an autoimmune disease and / or inflammatory condition or cancer (e.g., rheumatoid arthritis, asthma, or myeloid leukemia) characterized by high expression of GM-CSF and / or GM-CSFRα and / or abnormal GM-CSF / GM-CSFRα function, comprising administering to the individual an effective amount of a composition comprising an anti-GM-CSFRα antibody, wherein the antibody comprises V H The V HContains: an HC-CDR1 containing the amino acid sequence SEQ ID NO:1, an HC-CDR2 containing the amino acid sequence SEQ ID NO:7, and an HC-CDR3 containing the amino acid sequence SEQ ID NO:50, or a variant containing up to 5 amino acid substitutions; and V L The V L It includes: an LC-CDR1 containing the amino acid sequence SEQ ID NO:51, an LC-CDR2 containing the amino acid sequence SEQ ID NO:52, and an LC-CDR3 containing the amino acid sequence SEQ ID NO:57, or a variant containing up to 5 amino acid substitutions.

[0385] In some embodiments, the anti-GM-CSFRα antibody described herein comprises: V containing the amino acid sequence SEQ ID NO:121 H And V containing the amino acid sequence SEQ ID NO:126 L In some embodiments, the anti-GM-CSFRα antibody described herein is a full-length anti-GM-CSFRα antibody containing a constant region of IgG1 or IgG4. In some embodiments, the IgG1 is human IgG1. In some embodiments, the IgG4 is human IgG4. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:145. In some embodiments, the heavy chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:146. In some embodiments, the light chain constant region comprises or is composed of the amino acid sequence SEQ ID NO:147.

[0386] In some embodiments, the individual is a mammal (e.g., human, non-human primate, rat, mouse, cow, horse, pig, sheep, goat, dog, cat, etc.). In some embodiments, the individual is a human. In some embodiments, the individual is a clinical patient, clinical trial volunteer, laboratory animal, etc. In some embodiments, the individual is less than about 60 years of age (including, for example, less than about 50, 40, 30, 25, 20, 15, or 10 years of age). In some embodiments, the individual is more than about 60 years of age (including, for example, more than about 70, 80, 90, or 100 years of age). In some embodiments, the individual is diagnosed with or is genetically predisposed to one or more diseases or conditions described herein (e.g., rheumatoid arthritis, asthma, chronic obstructive pulmonary disease, allergic reactions, multiple sclerosis, myeloid leukemia, or atherosclerosis). In some embodiments, the individual has one or more risk factors associated with one or more diseases or conditions described herein.

[0387] In some embodiments, this application relates to a method of delivering an anti-GM-CSFR antibody (such as any of the anti-GM-CSFRα antibodies described herein, such as isolated anti-GM-CSFRα antibodies) to cells expressing GM-CSFRα on the surface of an individual, the method comprising administering to the individual a composition comprising the anti-GM-CSFRα antibody.

[0388] Many diagnostic methods for other diseases or cancers exhibiting abnormal expression of GM-CSF and / or GM-CSFRα, and their clinical descriptions, are known in the art. Such methods include, but are not limited to, immunohistochemistry, PCR, and fluorescence in situ hybridization (FISH).

[0389] In some embodiments, the anti-GM-CSFRα antibody (e.g., full-length anti-GM-CSFRα antibody) and / or composition described herein are used in combination with a second, third, or fourth agent (including, for example, an antitumor agent, a growth inhibitor, a cytotoxic agent, or a chemotherapeutic agent) to treat diseases or conditions associated with abnormal expression of GM-CSF and / or GM-CSFRα.

[0390] Cancer treatment can be evaluated using metrics such as tumor regression, reduction in tumor weight or size, time to progression, overall survival, progression-free survival, overall response rate, duration of response, quality of life, protein expression levels, and / or activity. Methods for determining treatment efficacy can be employed, including, for example, detecting a response via radiographic imaging.

[0391] In some embodiments, the efficacy of treatment is assessed as the percentage of tumor growth inhibition (%TGI), calculated using the equation 100 - (T / C × 100), where T is the relative mean tumor volume of treated tumors and C is the relative mean tumor volume of untreated tumors. In some embodiments, %TGI is approximately 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, or greater than 95%. In some embodiments, the efficacy of treatment is assessed by changes in granulocyte morphology and / or an increase in the number of surviving granulocytes. In some embodiments, the efficacy of treatment is assessed by an increase in cytokine secretion by monocytes.

[0392] Dosage and administration method of anti-GM-CSFRα antibody.

[0393] The dosage of an anti-GM-CSFRα antibody (e.g., isolated anti-GM-CSFRα antibody) composition administered to an individual (e.g., a human) may vary depending on the specific composition, route of administration, and type of disease being treated. In some embodiments, the amount of the composition (e.g., a composition containing an anti-GM-CSFRα antibody) may be effective in producing an objective response (e.g., a partial or complete response) in cancer treatment. In some embodiments, the amount of the anti-GM-CSFRα antibody composition is sufficient to produce a complete response in an individual. In some embodiments, the amount of the anti-GM-CSFRα antibody composition is sufficient to produce a partial response in an individual. In some embodiments, the administered dose of the anti-GM-CSFRα antibody composition (e.g., when administered alone) is sufficient to produce an overall response rate higher than any one of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 64%, 65%, 70%, 75%, 80%, 85%, or 90% in a population of individuals treated with the anti-GM-CSFRα antibody composition. An individual's response to the treatment methods described in this article can be determined, for example, by the level of RECIST.

[0394] In some embodiments, the amount of the composition (e.g., a composition comprising isolated anti-GM-CSFRα antibody) is sufficient to prolong an individual's progression-free survival. In some embodiments, the amount of the composition is sufficient to prolong an individual's overall survival. In some embodiments, in a population of individuals treated with the anti-GM-CSFRα antibody composition, the amount of the composition (e.g., when administered alone) is sufficient to produce a clinical benefit greater than about 50%, 60%, 70%, or 77%.

[0395] In some embodiments, the amount of the composition (e.g., a composition comprising an isolated anti-GM-CSFRα antibody), used alone or in combination with a second, third, and / or fourth agent, is sufficient to reduce tumor size, cancer cell number, or tumor growth rate by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or 100% compared to the corresponding tumor size, cancer cell number, or tumor growth rate before treatment in the same subject, or compared to the corresponding activity in other untreated subjects. The magnitude of this efficacy can be measured using standard methods, such as in vitro detection of purified enzymes, cell-based assays, animal models, or human trials.

[0396] In some embodiments, the amount of anti-GM-CSFRα antibody (e.g., full-length anti-GM-CSFRα antibody) in the composition is below the level that would cause a toxic effect (i.e., an effect above the clinically acceptable level of toxicity), or is at a level where potential side effects can be controlled or tolerated when the composition is administered to an individual.

[0397] In some embodiments, following the same dosing regimen, the amount of composition is close to the maximum tolerated dose (MTD) of the composition. In some embodiments, the amount of composition is higher than 80%, 90%, 95%, or 98% of the MTD.

[0398] In some embodiments, the content of anti-GM-CSFRα antibody (e.g., full-length anti-GM-CSFRα antibody) in the composition is in the range of 0.001 μg to 1000 μg.

[0399] In any of the embodiments described above, the effective amount of GM-CSFRα antibody (e.g., full-length anti-GM-CSFRα antibody) in the composition, calculated based on body weight, is in the range of 0.1 μg / kg to 100 mg / kg.

[0400] The anti-GM-CSFRα antibody composition can be administered to an individual (e.g., a human) via various routes, including, for example, intravenous injection, intra-arterial administration, intraperitoneal injection, intrapulmonary administration, oral administration, inhalation administration, intravascular administration, intramuscular administration, intratracheal administration, subcutaneous injection, intraocular administration, intrathecal administration, mucosal administration, or transdermal administration. In some embodiments, a sustained-release formulation of the composition is used. In some embodiments, the composition is administered intravenously. In some embodiments, the composition is administered via an artery. In some embodiments, the composition is administered intraperitoneally. In some embodiments, the composition is administered intrahepatically. In some embodiments, the composition is administered via hepatic artery infusion. In some embodiments, the composition is administered at a site remote from the first lesion.

[0401] Products and reagent kits

[0402] In some embodiments of this application, an article of manufacture is disclosed comprising a substance capable of treating autoimmune diseases and / or inflammatory conditions or cancers (e.g., rheumatoid arthritis, asthma, or myeloid leukemia) characterized by high expression of GM-CSF and / or GM-CSFRα and / or abnormal GM-CSF / GM-CSFRα function, or for delivering an anti-GM-CSFRα antibody (e.g., a full-length anti-GM-CSFRα antibody) to cells expressing GM-CSFRα on their surface. The article of manufacture may include a container and a label or packaging instructions on or accompanying the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container may be made of a variety of materials, such as glass or plastic. Typically, the container contains a composition capable of effectively treating the diseases or conditions described herein and has a sterile port (e.g., the container may be an intravenous infusion bag or a vial with a puncture cap via a hypodermic needle). The composition contains at least one active substance, namely the anti-GM-CSFRα antibody described in this application. The label or package insert indicates the specific condition for which the composition can be used to treat. The label or package insert further includes instructions for administering the anti-GM-CSFRα antibody composition to a patient. Products and kits, including those for combination therapy, are within the scope of this discussion.

[0403] Instructions for use are instructions typically included in the commercial packaging of a therapeutic product, containing information about the indications, usage, dosage, administration, contraindications, and / or warnings for using the product. In some embodiments, the instructions for use indicate that the composition can be used to treat autoimmune diseases and / or inflammatory conditions (e.g., rheumatoid arthritis, asthma, chronic obstructive pulmonary disease, allergic reactions, multiple sclerosis, myeloid leukemia, or atherosclerosis). In some embodiments, the instructions for use indicate that the composition can be used to treat cancer (e.g., myeloid leukemia).

[0404] In addition, the article may include a second container containing a pharmaceutically acceptable buffer solution, such as bacteriostatic water for injection (BWFI), phosphate buffer, Green's solution, or glucose solution. It may also include other materials required from a commercial and user perspective, including additional buffer solutions, diluents, filters, needles, and syringes.

[0405] The invention also relates to kits that can be used for various purposes, such as for treating autoimmune diseases and / or inflammatory conditions or cancers (e.g., rheumatoid arthritis, asthma, or myeloid leukemia) characterized by high expression of GM-CSF and / or GM-CSFRα and / or abnormal GM-CSF / GM-CSFRα function, or for delivering anti-GM-CSFRα antibodies (e.g., full-length anti-GM-CSFRα antibodies) to cells expressing GM-CSFRα on the surface, optionally in combination with other products. Kits of this application include one or more containers containing an anti-GM-CSFRα antibody composition (or a single-dose form and / or product), and in some embodiments, further include another agent (e.g., the agent described herein) and / or instructions for use consistent with any of the methods described herein. The kit may further include a description suitable for the individual being treated. Instructions for use accompanying the kits of this application are typically written instructions on a label or packaging (e.g., paper pages included within the kit), and machine-readable instructions (e.g., instructions on a magnetic or optical storage disc) are also acceptable.

[0406] For example, in some embodiments, the kit includes a composition comprising an anti-GM-CSFRα antibody (e.g., a full-length anti-GM-CSFRα antibody). In some embodiments, the kit includes: a) a composition comprising any of the anti-GM-CSFRα antibodies described herein, and b) at least one effective amount of another agent capable of enhancing the effect of the anti-GM-CSFRα antibody (e.g., therapeutic effect, detection effect). In some embodiments, the kit includes: a) a composition comprising any of the anti-GM-CSFRα antibodies described herein, and b) instructions for use in administering the anti-GM-CSFRα antibody composition to an individual for the treatment of autoimmune diseases and / or inflammatory conditions or cancers (e.g., rheumatoid arthritis, asthma, or myeloid leukemia) characterized by high expression of GM-CSF and / or GM-CSFRα and / or abnormal GM-CSF / GM-CSFRα function. In some embodiments, the kit includes: a) a composition comprising any of the anti-GM-CSFRα antibodies described herein; b) at least one effective amount of another agent capable of enhancing the effect of the anti-GM-CSFRα antibody (e.g., therapeutic effect, detection effect); and c) instructions for use to administer the anti-GM-CSFRα antibody composition and other substances to an individual for the treatment of autoimmune diseases and / or inflammatory diseases or cancers (e.g., rheumatoid arthritis, asthma, or myeloid leukemia) characterized by high expression of GM-CSF and / or GM-CSFRα and / or abnormal GM-CSF / GM-CSFRα function. The anti-GM-CSFRα antibody and other substances may be present in separate containers or in the same container. For example, the kit may include one specific composition or two or more compositions, wherein one composition comprises an anti-GM-CSFRα antibody and another composition comprises another agent.

[0407] In some embodiments, the kit comprises a nucleic acid (or set of nucleic acids) encoding an anti-GM-CSFRα antibody (e.g., a full-length anti-GM-CSFRα antibody). In some embodiments, the kit comprises: a) a nucleic acid (or set of nucleic acids) encoding an anti-GM-CSFRα antibody (e.g., a full-length anti-GM-CSFRα antibody), and b) a host cell expressing the nucleic acid (or set of nucleic acids). In some embodiments, the kit comprises: a) a nucleic acid (or set of nucleic acids) encoding an anti-GM-CSFRα antibody (e.g., a full-length anti-GM-CSFRα antibody), and b) instructions for use suitable for: i) expressing anti-GM-CSFRα antibodies in host cells, ii) preparing a composition comprising anti-GM-CSFRα antibodies, and iii) administering the composition comprising anti-GM-CSFRα antibodies to an individual for the treatment of autoimmune diseases and / or inflammatory conditions or cancers (e.g., rheumatoid arthritis, asthma, or myeloid leukemia) characterized by high expression of GM-CSF and / or GM-CSFRα and / or abnormal GM-CSF / GM-CSFRα function. In some embodiments, the kit includes: a) a nucleic acid (or a group of nucleic acids) encoding an anti-GM-CSFRα antibody (e.g., a full-length anti-GM-CSFRα antibody); b) a host cell expressing the nucleic acid (or nucleic acid group); and c) instructions for use suitable for: i) expressing an anti-GM-CSFRα antibody in a host cell; ii) preparing a composition containing an anti-GM-CSFRα antibody; and iii) administering the composition containing an anti-GM-CSFRα antibody to an individual for the treatment of autoimmune diseases and / or inflammatory conditions or cancers (e.g., rheumatoid arthritis, asthma, or myeloid leukemia) characterized by high expression of GM-CSF and / or GM-CSFRα and / or abnormal GM-CSF / GM-CSFRα function.

[0408] The kit described in this application is packaged in a suitable form. Suitable packaging includes, but is not limited to, vials, bottles, wide-mouth flasks, flexible packaging (e.g., sealed polyester film or plastic bags), etc. The kit may optionally include other components, such as buffer solutions and instruction information. Therefore, this application also provides articles of manufacture including vials, bottles, wide-mouth flasks, flexible packaging (e.g., sealed polyester film or plastic bags), etc.

[0409] Instructions for use of anti-GM-CSFRα antibody compositions typically include information such as dosage, dosing cycle, and route of administration. Containers may be single-dose, bulk (e.g., multi-dose packs), or subunit doses. For example, a kit may be provided containing a sufficient dose of the anti-GM-CSFRα antibody (e.g., full-length anti-GM-CSFRα antibody) as described herein for long-term effective treatment of an individual, such as for one week, 8 days, 9 days, 10 days, 11 days, 12 days, 13 days, 2 weeks, 3 weeks, 4 weeks, 6 weeks, 8 weeks, 3 months, 4 months, 5 months, 7 months, 8 months, 9 months, or longer. Kits may also contain multiple unit doses of anti-GM-CSFRα antibody, the pharmaceutical composition, and instructions for use, and be packaged in sufficient quantities for storage and use in a pharmacy, such as a hospital pharmacy or a multi-dose pharmacy.

[0410] Those skilled in the art will recognize several possible embodiments within the scope and spirit of this application. The application will now be described in more detail with reference to the following non-limiting embodiments. These embodiments further illustrate the application but should not be construed as limiting its scope in any way. Detailed Implementation

[0411] In the embodiments disclosed below, the following abbreviations are used: GMF (human GM-CSF); GMRα (human GM-CSFRα); GMRb (human GM-CSFRβ); Mab (Mavrilimumab); GMRαh (human GM-CSFRα-6His); BGMRα (Biotin-Avi-GM-CSFRα); mGMRα (cynomolgus monkey GM-CSFRα); mGMRαh (cynomolgus monkey GM-CSFRα-6His).

[0412] Example 1: Preparation of recombinant human GM-CSFRα and screening for anti-GM-CSFRα single-chain antibodies (scFv)

[0413] Preparation of recombinant GM-CSFRα antigen

[0414] The full-length gene encoding human GM-CSFRα (hereinafter referred to as GMRα) was subcloned from the pSC-GM-CSFRα (Shanghai Jierui) vector into the eukaryotic expression vector pTT5 using the restriction endonuclease restriction sites HindIII and XhoI. A His tag or other tags commonly used by those skilled in the art were added to GMRα. The following expression vectors were constructed: pTT5-GMRα-6his(ECD), pTT5-Avi-10His-GMRα(ECD), and pTT5-GMRα(400aa). Here, "ECD" represents the extracellular region, "his" or "His" represents the His tag, and "Avi" represents the avidin tag.

[0415] In addition, the recombinant cynomolgus monkey GM-CSFRα construct was also cloned. Primers were designed based on the cynomolgus monkey GM-CSFRα sequence (XM_024791666.1) from the NCBI database, and RNA was extracted from cynomolgus monkey peripheral blood mononuclear cells (PBMCs). cDNA was obtained through reverse transcription, and the extracellular coding sequence of the cynomolgus monkey GM-CSFRα extracellular segment was amplified using primers and cloned into the eukaryotic vector pTT5, completing the construction of the pTT5-mGMRα-6his(ECD) vector.

[0416] Following the instrument manufacturer's and kit instructions, recombinant human GM-CSFRα, including GMRα-6his (ECD), Avi-10His-GMRα (ECD), and mGMRα-6his (ECD), was expressed and purified. In short, the expression vector was transfected into 293F cells, and the cells were cultured at 37°C, 8% CO2, and 120 rpm for 5 days. The cell culture medium was collected, and the His-tagged proteins were purified using a (Ni) nickel column according to the instructions. The specific procedure was as follows: Immobilized metal affinity chromatography (IMAC) was performed using a QIAGEN Ni-NTA. First, the nickel column was equilibrated with buffer A1 (50 mM Na3PO4, 0.15 M NaCl, pH 7.2) at a flow rate of 150 cm / h. The pH of the culture supernatant was adjusted to 7.2, and the sample was loaded at room temperature at a flow rate of 150 cm / h. Subsequently, the column was reequilibrated with 6 column volumes of A1 buffer at a flow rate of 150 cm / h. Finally, elution was performed using 10 column volumes of 50 mM PB solution (containing 0.15 M NaCl and 0.2 M imidazole, pH 7.2), and the eluent was collected.

[0417] Preparation of biotinylated GM-CSFRα antigen

[0418] Following the instructions, Avi-10His-GMRα was biotinylated using biotinylated ligase B0101A (GeneCopoeia). In short, the Avi-10His-GMRα antigen was incubated at 30°C for 2 hours after adding the corresponding Buffer A / B and BirA ligases. The biotinylated GMRα was named Bavih-GMRα. Biotinylation efficiency was assessed using an ELISA method. In short, the initial concentration of Bavih-GMRα was set at 500 ng / ml, and serially diluted 1:2 before coating ELISA plates. SA-HRP was used for detection, with biotinylated standards as controls. The biotinylation labeling efficiency of Bavih-GMRα was 70%. The biological activity of Bavih-GMRα was confirmed using TF-1 cell proliferation assays.

[0419] Screening for anti-GM-CSFRα single-chain antibodies (scFv)

[0420] Construction of scFv antibody yeast display library: RNA was extracted from 2000 human blood samples, and cDNA was obtained by reverse transcription. V H and V K Specific primer amplification V H and V K The fragment, after gel recovery and purification, was ligated with V... H and V K We constructed scFv and cloned it into the yeast display plasmid PYD1. Subsequently, we electroporated the plasmid into yeast cells to obtain the scFv antibody yeast display library.

[0421] Screening for anti-GM-CSFRα single-chain antibodies (scFv): scFvs recognizing GM-CSFRα were isolated from a yeast display library. In short, yeast cells expressing GM-CSFRαscFV were enriched using MACS magnetic bead sorting. Yeast cells at 1000 OD were centrifuged at 2500 g for 5 minutes. The resulting cell pellet was resuspended in 1 L of SGCAA medium at an initial concentration of OD600 = 1 and induced to express for 40-48 hours at 20°C and 250 rpm. After centrifugation and washing with PBSM solution, the cell pellet was resuspended in 5-10 volumes of PBSM solution containing 1 μM Bavih-GMRα and incubated at 4°C for 1 hour. After centrifugation and PBSM washing, unbound antigen was washed away with PBSM solution. Magnetic beads were added, and the mixture was thoroughly mixed and then incubated on a 4°C vortex mixer for 30 minutes. Centrifuge at 2500g for 5 minutes, discard the supernatant, and resuspend the pellet in 5-10 times its volume of PBSM solution. Add 7ml of cell suspension to the column each time until all the cell suspension flows through the column. Collect the cells bound to the column and culture them further to extract plasmids.

[0422] Preparation of phage display libraries and screening of scFv antibodies: PCR amplification of plasmids obtained from a yeast library was performed using scFv-F and scFv-R primers. The obtained scFv antibody fragments were cloned into the phage display vector pDAN5 via SfiI, ligated, and then transformed into TG1 phage display electroporation competent cells to obtain an scFv antibody phage display library. After a series of repeated screening steps, scFv antibodies specifically binding to GM-CSFRα were isolated from the phage display library. In short, 2 x 10⁻⁶ sq m phage display libraries were prepared. 11 PFU-bound phage scFv libraries were added to biotinylated GM-CSFRα and incubated at 37°C for 2 hours. Phages binding to GM-CSFRα were captured by streptavidin-coated magnetic beads, while unbound phages were washed away. After washing 8-15 times with TBST solution (the number of washes increased with each screening round), phages specifically binding to GM-CSFRα were eluted with Glycine-HCl solution (pH 2.2). These phages were used to infect exponentially growing TG1 cells, and after 1 hour of incubation with ampicillin, helper phages were added and the cells were incubated overnight at 28°C and 200 rpm on a shaker. The culture medium was collected the next day, centrifuged, and the supernatant was used for the next round of screening until a positive scFv antibody library was obtained.

[0423] Ligand binding assays were performed to screen scFv monoclonal antibodies. The first experiment was designed to verify that scFv antibodies could bind to human GM-CSFRα and / or cynomolgus monkey GM-CSFRα. In short, GMRαh (human GM-CSFRα) or mGMRαh (cynomolgus monkey GM-CSFRα) antigen was dissolved in PBS and coated into 96-well plates at 0.2 μg / well, incubated overnight at 4°C. Before adding the scFv antibody, the plates were washed with TBST solution, blocked with 5% milk at 37°C for 1-2 hours, and then washed with TBST solution. Each scFv sample was first diluted to 40 μg / mL, and 150 μL was added to the wells in the first row. Subsequently, the 40 μg / mL scFv sample was serially diluted 1:3 and added to the remaining wells. After incubation at 37°C for 1 hour, the plates were washed 6 times with TBST solution. Add 100 μl of a mixture of primary and secondary antibodies (mouse anti-flag (1:2500) and anti-mouse FC-AP (1:2000)) to each well, incubate at 37°C for 1 hour, and wash three times with TBST solution. Add 50 μL of PNPP to each well and incubate at 37°C for 10–20 minutes. Terminate the reaction with 3M NaOH. Analyze the ELISA results (OD410) and generate binding curves using PRISM.

[0424] The second experiment was designed to verify that the scFv antibody could block the binding of GM-CSF to GM-CSFRα using an ELISA competition assay. In short, 0.5 μg / well of GM-CSF and 5% milk were used to coat a 96-well plate, incubated at 37°C for 1-2 hours, and washed with TBST solution. First, each scFv antibody sample was diluted to 40 μg / mL, with 100 μL added to the first well. Then, the 40 μg / mL scFv antibody sample was serially diluted 1:2 and added to the remaining wells. 50 μL of PBS solution containing 2.5 μg / mL Bavih-GMRa was added to each well. After incubation at 37°C for 1 hour, the plate was washed 6 times with TBST solution. 100 μL of LSA-HRP (1:20000) was added to each well, and the plate was incubated at 37°C for 1 hour. After washing 6 times with TBST solution, 50 μL of TMB was added to each well, and the plate was incubated at 37°C for 5-10 minutes. The reaction was terminated by adding 2M H2SO4. The ELISA results (OD450) were analyzed, and a competition inhibition curve was generated using PRISM.

[0425] TF-1 Cell Proliferation Assay: In the TF-1 cell proliferation assay, the inhibitory effect of scFv antibody on GM-CSF-stimulated TF-1 cell proliferation was assessed to evaluate its biological activity in inhibiting the binding of GM-CSF to GM-CSFRα. TF-1 cells are a human premyeloid cell line established from patients with erythroleukemia. The survival and proliferation of this cell line are factor-dependent and typically require human GM-CSF to maintain its proliferation. Briefly, TF-1 cells were cultured in RPMI 1640, 10% FBS, 10 ng / mL GM-CSF medium and passaged twice weekly. Cells were washed three times with GM-CSF-free medium (RPMI 1640, 10% FBS) and resuspended in the same medium. Approximately 10,000 cells per well were added to 96-well plates and cultured overnight. The next day, the scFv antibody was serially diluted 1:10 (from 10 μg / mL to 0.0001 μg / mL) and added to 96-well plates. After incubation at 37°C for 1 hour, GM-CSF (Peprotech) was added to a final concentration of 200 pg / mL. Cell viability was assessed the following day using a Celltiter-glo assay kit (Promega). IC50 was calculated using PRISM. 50 .

[0426] Example 2: Preparation and characterization of full-length human GM-CSFRα antibody

[0427] Preparation of full-length anti-GM-CSFRα antibody

[0428] The most promising scFv antibodies were constructed into human IgG1 or IgG4 antibody molecules containing the heavy chain constant region of human IgG1 or IgG4 and the light chain constant region of human kappa. V was amplified from a prokaryotic expression vector. L and V HThe eukaryotic expression vectors pTT5-L (containing the kappa constant region) and pTT5-H1 (containing the IgG1 heavy chain constant region) or pTT5-H4 (containing the IgG4 heavy chain constant region) were constructed, respectively. Plasmids expressing both the light and heavy chains were extracted and transfected into 293F cells. Cells were cultured at 37°C, 8% CO2, and 120 rpm for 5 days. The culture medium was then purified using a Protein A affinity chromatography column. In short, the Protein A column was first equilibrated with 6 column volumes of PBS buffer (pH 7.2) containing 0.15 M NaCl and 50 mM PBS at a flow rate of 150 cm / h. The pH of the culture supernatant was adjusted to 7.2, and the sample was loaded at a flow rate of 150 cm / h. After loading, the column was further equilibrated, and finally eluted with 50 mM citrate-sodium citrate buffer (pH 3.5), and the eluent was collected. Among the constructed full-length antibodies, T119 was selected as the lead parent antibody. A phage display library containing CDR region mutations was prepared using T119 scFv. The biological activity of antibody variants that bind to human GM-CSFRα with high affinity and low dissociation rate was evaluated in TF-1 cell proliferation assays. Compared to T119 scFv, scFv antibodies with enhanced biological activity were selected to construct full-length antibodies. A new round of screening for full-length antibodies was conducted using TF-1 cell proliferation assays, and the selected lead optimized antibodies underwent further biochemical and biological analyses.

[0429] Affinity of anti-GM-CSFRα antibody

[0430] The affinity of the lead parent antibody T119 and the optimized lead antibody (reconstructed into the adult IgG1 form) for human GM-CSFRα was evaluated using ELISA. Figure 1A-1C As shown, compared with T119, the lead optimized antibodies all exhibited improved binding affinity. Next, ELISA was used to evaluate the affinity of the lead parent antibody T119 and the lead optimized antibodies E35, E200a, E87, and E108 (reconstructed into IgG4 form) for cynomolgus monkey GM-CSFRα (mGMRαh). Figure 2 As shown, the anti-GM-CSFRα antibody cross-reacts with cynomolgus monkey GM-CSFRα.

[0431] Specificity of anti-GM-CSFRα antibody

[0432] Cross-reactivity with homologous proteins: ELISA was used to detect the cross-reactivity of E35, E87, and E108 (reconstructed into IgG4 form) with GM-CSFRα homologs IL3RA, IL5RA, and G-CSFR. Figure 3As shown, compared with other homologous proteins detected, the antibody specifically binds to GMRαh, indicating that the prepared anti-GM-CSFRα antibody is specific for GM-CSFRα.

[0433] Specificity of binding to WIL2S cells expressing GM-CSFRα: Further evaluation of the specificity of the anti-GM-CSFRα antibody E35-IgG4 binding to WIL2S cells expressing GM-CSFRα was conducted. Following the manufacturer's instructions, the anti-GM-CSFRα antibody E35-IgG4 was fluorescently labeled using GLY-650 (Dylight Amine-Reactive Dyes, Thermo Fisher). WIL2S cells were electrotransfected with an expression vector containing full-length GM-CSFRα, with untransfected WIL2S cells serving as a control. Forty-eight hours after electrotransfection, transfected and untransfected cells were collected into 15 mL centrifuge tubes, centrifuged at 1000 rpm for 5 minutes, and resuspended in DPBS. 1 x 102 6 One cell was added to each Eppendorf tube and centrifuged at 1000g for 5 minutes. WIL2S cells expressing GM-CSFRα (GMRα-E35) were treated with 100 μL of 1% BSA solution containing 15 μg / mL E35-IgG4, while control WIL2S cells were treated with 100 μL of 1% BSA solution (CK) and 100 μL of 1% BSA solution containing 5 μg / mL E35-IgG4 (NC-E35). All three cell groups were incubated at 37°C for 40 minutes, washing the cells twice with 1 mL PBS each time, then resuspending them in 0.2 mL PBS before FACS analysis. Figure 4 As shown, E35-IgG4 does not bind to control WIL2S cells, but binds strongly to WIL2S cells expressing GM-CSFRα.

[0434] Characterization of binding affinity and dissociation constant (Kd) of anti-GM-CSFRα antibody

[0435] The binding affinity of anti-GM-CSFRα antibodies E35 and E87b (reconstructed into human IgG4 form) was detected using a Biacore T200 (GE) sensor. E35 and E87b were immobilized on a CM5 sensor chip. The affinity of the antibodies for GMRαh was measured at different concentrations, including 10, 5, 2.5, 1.25, 0.625, 0.3125, 0.15625, 0.078, 0.039, 0.0195, and 0 nM, with 0.625 and 0 nM values ​​repeated once each. The binding and dissociation rates of the antibodies were measured using SPR technology to determine the binding affinity. Table 5 lists the kon, koff, and Kd values ​​for the E35 and E87b antibodies.

[0436] Table 5

[0437] E35 5.37E+06 4.78E-05 8.90E-12 E87b 3.58E+06 2.42E-05 6.75E-12

[0438] Anti-GM-CSFRα antibody competitively binds to GM-CSF with GM-CSFRα

[0439] A competitive ELISA assay (as described in Example 1) was used to evaluate the ability of the GM-CSFRα antibody to recognize the GM-CSFRα ligand binding site and compete with GM-CSF for binding to GM-CSFRα. Figures 5A-5D As shown, the lead parent antibody T119 and the lead optimized antibody (reconstructed into human IgG4 form) can block the binding of GM-CSF to human GM-CSFRα, indicating that the antibody competitively binds to human GM-CSFRα with GM-CSF.

[0440] Anti-GM-CSFRα antibody stability experiment

[0441] Thermal stability analysis: The thermal stability of T119-IgG1, E35-IgG1, E35b-IgG1, and Mab-IgG1 was analyzed using the UNcle platform. The melting temperature (T) of each antibody was measured. m ) and accumulation temperature (T agg ). T m The value represents the temperature at which the antibody unfolds during the heating process, T. agg The value represents the temperature at which antibody aggregation occurs during the heating process. See Table 6 and... Figures 6A-6B As shown, compared with the control antibody Mab-IgG1, T119-IgG1, E35-IgG1, and E35b-IgG1 have higher T values. m Value and T agg The values ​​were also higher for E35-IgG1 and E35b-IgG1 than for the lead antibody T119-IgG1. m Value and T agg These results indicate that E35-IgG1 and E35b-IgG1 exhibit better thermal stability compared to the lead antibody T119-IgG1 and the control antibody Mab-IgG1.

[0442] Table 6

[0443] H1 2mg / ml Mab-IgG1 67.8 64.56 65.6 I1 2mg / ml T119-IgG1 69.53 56.65 70.01 J1 2mg / ml E35-IgG1 73.52 67.15 77.37 K1 2mg / ml E35b-IgG1 70.42 69.01 77.64

[0444] TF-1 cell proliferation experiment

[0445] The ability of the lead antibody T119 and the lead optimized antibody (reconstructed into human IgG4 form) to inhibit TF-1 cell proliferation was detected using a TF-1 cell proliferation assay (as described in Example 1). Results are as follows: Figure 7 As shown, compared with the lead antibody T119, the lead-optimized antibody has a comparable or superior ability to inhibit TF-1 cell proliferation.

[0446] Granulocyte deformability experiment

[0447] Human granulocyte deformability assay was used to further evaluate the anti-GM-CSFRα antibody. In short, 10 mL of human peripheral blood was collected, and mononuclear cells were removed using Ficoll density gradient centrifugation. After removing the mononuclear cell layer and Ficoll solution, erythrocytes were lysed using cell lysis buffer. The remaining cells were washed with PBS and cell culture medium, and 100,000 cells were added to each well of a 96-well plate and incubated at 37°C for 30 minutes. Cells were then treated with 100 pg / mL GM-CSF, and GM-CSFRα antibody diluted 1:10 (10 μg / mL to 0.0001 μg / mL) was added. The cells were incubated at 37°C for 3 hours, and FACS analysis was performed. Granulocyte deformability was assessed based on the Forward Scatter GEO mean. Figure 8 As shown, E35, E108, and E87b (reconstructed into the human IgG4 form) can all prevent granulocyte deformability, and the antibody IC50 values ​​are shown in Table 7 below.

[0448] Table 7

[0449] IC50 (μg / mL) 0.002050 0.002066 0.001505

[0450] The anti-GM-CSFRα antibody E35 (reconstructed into human IgG4 form) was further evaluated using a cynomolgus monkey granulocyte deformability assay. Granulocytes were purified from whole blood of cynomolgus monkeys, treated with 100 pg / mL GM-CSF, and then serially diluted with E35 antibody at 1:10 ratios (10 μg / mL to 0.0001 μg / mL). Cells were analyzed using FACS, and granulocyte deformability was assessed based on the forward Scatter GEO mean. Results showed that the E35-IgG4 antibody effectively prevented cynomolgus monkey granulocyte deformability. Figure 9 Its IC50 value is 0.002527 μg / mL.

[0451] Human granulocyte survival experiment

[0452] Granulocytes survive for a longer period in the presence of GM-CSF. In a human granulocyte survival assay, the inhibitory effect of the anti-GM-CSFRα antibody on this effect was evaluated. Briefly, granulocytes were isolated from human peripheral blood and treated with 100 pg / mL GM-CSF. The antibody was serially diluted 1:10 (10 μg / mL to 0.0001 μg / mL) and added to the cells. After incubation for 48 hours, cell viability was analyzed using a Celltiter-glo assay kit (Promega). Figure 10 As shown, E35, E108, and E87b effectively inhibited granulocyte survival. Table 8 shows the IC50 values ​​of different antibodies inhibiting granulocyte survival.

[0453] Table 8

[0454] IC50 (μg / mL) 0.004200 0.005521 0.002528

[0455] Inhibitory activity of cytokine release

[0456] Inhibition of CD11b expression: The inhibitory effects of anti-GM-CSFRα antibodies E35 and E87b, and the control antibody Mab on CD11b expression in human peripheral blood cells were evaluated. In short, 50 μL of human peripheral blood was added to each well of a 96-well plate, along with serially diluted antibodies (10 μg / mL to 0.0001 μg / mL) at a 1:10 ratio. Incubation was carried out at 37°C for 1 hour, followed by incubation for another hour with 10 ng / mL GM-CSF. Subsequently, CD11b was labeled using FITC-conjugated anti-CD11b antibody (BD53310) at 4°C for 30 minutes. Red blood cells were lysed using 1 mL of cell lysis buffer (BD349202), washed twice with PBS, and CD11b expression was analyzed using FACS. Figure 11 As shown in Table 9, E35 and E87b exhibit stronger inhibitory activity against CD11b expression compared to the control antibody Mab-IgG4. The IC50 values ​​of the antibodies are shown in Table 9.

[0457] Table 9

[0458] IC50 (μg / mL) 0.1813 0.1111 0.1439

[0459] Inhibition of Cytokine Production: To evaluate the inhibitory effect of anti-GM-CSFRα antibody on cytokine production, 10 mL of human peripheral blood was collected, and mononuclear cells were isolated using Ficoll density gradient centrifugation. Cells were washed twice with PBS, resuspended in cell culture medium, and 1,000,000 cells (100 μL) and 50 μL of serially diluted GM-CSFRα antibody (100 μg / mL to 0.001 μg / mL) were added to each well of a 96-well plate. The plates were incubated at 37°C for 1 hour. LPS and GM-CSF were then added to final concentrations of 100 ng / mL and 50 ng / mL, respectively. After incubation at 37°C for 48 hours, the supernatant was collected, and the levels of TNFα and IL-1β were analyzed using a Human Macrophage / Microglia Panel (Biolegend, 740503). Figure 12A As shown in Table 10, E35-IgG4 and E87b-IgG4 exhibited stronger TNFα inhibitory activity compared to the control antibody Mab-IgG4. Figure 13 As shown in Table 11, E35-IgG4 and E87b-IgG4 exhibited stronger IL-1β inhibitory activity compared to the control antibody Mab-IgG4.

[0460] Table 10

[0461] IC50 (μg / mL) 3.094 0.2777 0.06664

[0462] Table 11

[0463] IC50 (μg / mL) 0.01263 0.003535 0.01333

[0464] Simultaneously, the level of TNFα in the supernatant was further analyzed using ELISA. The ELISA results confirmed that, compared with the control antibody Mab-IgG4, E35-IgG4 and E87b-IgG4 had stron...

Claims

1. An isolated anti-GM-CSFRα antibody, comprising: Heavy chain variable structural domain (V H ), the V H Contains: HC-CDR1, whose amino acid sequence is shown in SEQ ID NO: 1; HC-CDR2, whose amino acid sequence is shown in SEQ ID NO: 6; and HC-CDR3, whose amino acid sequence is shown in SEQ ID NO: 27; and Light chain variable structural domain (V L ), the V L It includes: LC-CDR1, whose amino acid sequence is shown in SEQ ID NO: 51; LC-CDR2, whose amino acid sequence is shown in SEQ ID NO: 52; and LC-CDR3, whose amino acid sequence is shown in SEQ ID NO: 57, wherein the numbers are defined according to EU Kabat.

2. The isolated anti-GM-CSFRα antibody according to claim 1, comprising: V H It comprises the amino acid sequence SEQ ID NO: 91 or a variant thereof, said variant having at least 90% sequence identity with the amino acid sequence SEQ ID NO: 91; and V L It contains the amino acid sequence SEQ ID NO: 126 or a variant thereof, the variant having at least 90% sequence identity with the amino acid sequence SEQ ID NO:

126.

3. The isolated anti-GM-CSFRα antibody according to claim 1, wherein: (i) V H The 31st amino acid residue is E, N, G, D, M, S, P, F, Y, A, V, K, W, R, or C; or (ii) V L The 26th amino acid residue is S, L, N, A, K, R, I, Q, G, T, H, M, or C; or (iii) V L The 27th amino acid residue is Q, Y, P, A, I, F, T, R, V, L, E, S, or C; or (iv) V L The 28th amino acid residue is S, H, W, L, R, K, T, P, I, F, V, E, A, or Q; or (v) V L The 30th amino acid residue is S, L, W, M, A, Y, K, R, G, T, E, V, N, F, or C; or (vi) V L The 31st amino acid residue is S, T, R, A, H, Q, P, M, L, or G; or (vii) V L The 32nd amino acid residue is Y, L, or F; or (viii) V L The 50th amino acid residue is G or T; or (ix) V L The 51st amino acid residue is A, G, R, H, K, S, T, M, F, N, or V; or (x) V L The 52nd amino acid residue is S, A, W, R, L, T, Q, F, Y, H, or N; or (xi) V L The 92nd amino acid residue is D, A, Q, or W; or (xii) V L The 93rd amino acid residue is N, D, E, T, Y, G, A, M, F, S, I, or L; or (xiii) V H The 28th amino acid residue is T, H, V, E, P, L, M, S, W, C, A, G, N, or K; or (xiv) V H The 30th amino acid residue is T, P, D, E, Y, W, V, M, N, L, Q, G, S, A, K, or R; The numbering is defined according to EU Kabat.

4. An isolated anti-GM-CSFRα antibody, comprising: (i) V H Its amino acid sequence is SEQ ID NO: 250; and V L Its amino acid sequence is SEQ ID NO: 241; (ii) V H Its amino acid sequence is SEQ ID NO: 250; and V L Its amino acid sequence is SEQ ID NO: 193; (iii) V H Its amino acid sequence is SEQ ID NO: 248; and V L Its amino acid sequence is SEQ ID NO: 188; (iv) V H Its amino acid sequence is SEQ ID NO:248; and V L Its amino acid sequence is SEQ ID NO: 193; (v) V H Its amino acid sequence is SEQ ID NO: 250; and V L Its amino acid sequence is SEQ ID NO: 288; (vi) V H Its amino acid sequence is SEQ ID NO: 250; and V L Its amino acid sequence is SEQ ID NO: 188; or (vii) V H Its amino acid sequence is SEQ ID NO: 250; and V L Its amino acid sequence is SEQ ID NO:

236.

5. The isolated anti-GM-CSFRα antibody according to any one of claims 1-4, wherein the anti-GM-CSFRα antibody comprises an Fc fragment.

6. The isolated anti-GM-CSFRα antibody according to claim 5, wherein the anti-GM-CSFRα antibody is a full-length IgG antibody.

7. The isolated anti-GM-CSFRα antibody according to claim 6, wherein the anti-GM-CSFRα antibody is a full-length IgG1 or IgG4 antibody.

8. The isolated anti-GM-CSFRα antibody according to any one of claims 1-4, wherein the anti-GM-CSFRα antibody is chimeric, fully human, or humanized.

9. The isolated anti-GM-CSFRα antibody according to any one of claims 1-4, wherein the anti-GM-CSFRα antibody is an antigen-binding fragment selected from the group consisting of Fab, Fab', F(ab)'2, Fab'-SH, single-chain antibody (scFv), Fv fragment, and double-chain antibody.

10. A nucleic acid molecule encoding the anti-GM-CSFRα antibody according to any one of claims 1-9.

11. A carrier comprising the nucleic acid molecule of claim 10.

12. An isolated host cell comprising the anti-GM-CSFRα antibody of any one of claims 1-9, the nucleic acid molecule of claim 10, or the vector of claim 11.

13. A method for preparing an anti-GM-CSFRα antibody, comprising: a) Culture the host cells as described in claim 12 under conditions that enable effective expression of anti-GM-CSFRα antibody; and b) Obtain the expressed anti-GM-CSFRα antibody from the host cell.

14. A pharmaceutical composition comprising the anti-GM-CSFRα antibody of any one of claims 1-9, the nucleic acid molecule of claim 10, the vector of claim 11, the isolated host cell of claim 12, or the antibody prepared by the method of claim 13, and a pharmaceutically acceptable vector.

15. The use of the antibody of any one of claims 1-9, the nucleic acid molecule of claim 10, the vector of claim 11, the host cell of claim 12, the antibody prepared by the method of claim 13, or the pharmaceutical composition of claim 14 in the preparation of a medicament for treating a desired individual disease or condition, wherein the disease or condition is rheumatoid arthritis or asthma.

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