Antibodies to granulocyte-macrophage colony-stimulating factor and uses thereof

Abstract

Disclosed herein are anti-GM-CSF antibodies capable of binding to and blocking the biological activity of human GM-CSF. Also provided herein are pharmaceutical compositions comprising the anti-GM-CSF antibodies and therapeutic and diagnostic uses of such antibodies.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Application No. 62 / 745,602, filed October 15, 2018, and U.S. Provisional Application No. 62 / 830,754, filed April 8, 2019. [Background technology]

[0002] Granulocyte-macrophage colony-stimulating factor (GM-CSF), also known as colony-stimulating factor 2 (CSF2), is a monomeric glycoprotein secreted by macrophages, T cells, mast cells, natural killer cells, endothelial cells, and fibroblasts, and functions as a cytokine. GM-CSF stimulates hematopoietic stem cells to differentiate into myeloid lineage cells and produce granulocytes and monocytes.

[0003] In addition to its hematopoietic activity, GM-CSF induces the differentiation, proliferation, and activation of macrophages and dendritic cells, which are required for the subsequent activation of type 1 helper T cells and cytotoxic T lymphocytes. The hematopoietic and non-hematopoietic functions of GM-CSF have proinflammatory and immunoregulatory potential.

[0004] GM-CSF signals through the signal transducer and activator of transcription 5 (STAT5) and stimulates the expression of STAT5 target genes. GM-CSF / STAT5 signaling plays a central role not only in maintaining immune homeostasis but also in exacerbating inflammatory responses.

[0005] There is therefore great interest in the development of new GM-CSF antagonists for use in treating diseases associated with GM-CSF signaling. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] WO 99 / 58572 [Patent Document 2] U.S. Patent No. 5,565,332 [Patent Document 3] U.S. Patent No. 5,580,717 [Patent Document 4] U.S. Patent No. 5,733,743 [Patent Document 5] U.S. Patent No. 6,265,150 [Patent Document 6] WO 87 / 04462 [Patent Document 7] U.S. Patent No. 4,946,778 [Patent Document 8] U.S. Patent No. 4,704,692 [Patent Document 9] U.S. Patent No. 4,485,045 [Patent Document 10] U.S. Patent No. 4,544,545 [Patent Document 11] U.S. Patent No. 5,013,556 [Patent Document 12] U.S. Patent No. 3,773,919 [Patent Document 13] WO 00 / 53211 [Patent Document 14] U.S. Patent No. 5,981,568 [Patent Document 15] WO 90 / 07936 [Patent Document 16] WO 94 / 03622 [Patent Document 17] WO 93 / 25698 [Patent Document 18] WO 93 / 25234 [Patent Document 19] WO 93 / 11230 [Patent Document 20] WO 93 / 10218 [Patent Document 21] WO 91 / 02805 [Patent Document 22] U.S. Patent No. 5,219,740 [Patent Document 23] U.S. Patent No. 4,777,127 [Patent Document 24] British Patent No. 2,200,651 [Patent Document 25] European Patent No. 0 345 242 [Patent Document 26] WO 94 / 12649 [Patent Document 27] WO 93 / 03769 [Patent Document 28] WO 93 / 19191 [Patent Document 29] WO 94 / 28938 [Patent Document 30] WO 95 / 11984 [Patent Document 31] WO 95 / 00655 [Patent Document 32] U.S. Patent No. 5,814,482 [Patent Document 33] WO 95 / 07994 [Patent Document 34] WO 96 / 17072 [Patent Document 35] WO 95 / 30763 [Patent Document 36] WO 97 / 42338 [Patent Document 37] WO 90 / 11092 [Patent Document 38] U.S. Patent No. 5,580,859 [Patent Document 39] U.S. Patent No. 5,422,120 [Patent Document 40] WO 95 / 13796 [Patent Document 41] WO 94 / 23697 [Patent Document 42] WO 91 / 14445 [Patent Document 43] European Patent No. 0524968 [Patent Document 44] U.S. Patent No. 8,017,748

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[0008] The present disclosure is based, at least in part, on the development of anti-GM-CSF antibodies, such as 1D2 and its variants, which have demonstrated high binding affinity and specificity for human GM-CSF and potent activity in inhibiting GM-CSF-induced cell proliferation and cytokine production (e.g., IL8 and CCL17).

[0009] Accordingly, one aspect of the present disclosure relates to an isolated anti-GM-CSF antibody (anti-GM-CSF antibody) that binds to human GM-CSF. The anti-GM-CSF antibody disclosed herein is (i) a heavy chain complementarity determining region 1 (HC CDR1), described as GYTFTX1X2Y, where X1 can be D, E, S, P, G, A, V, N, Q, C, W, R, T, or L; and X2 can be K, V, L, E, P, S, T, C, D, R, H, A, F, Q, or N; (ii) a heavy chain complementarity determining region 2 (HC CDR2) as described as INPX3SGGT, where X3 can be K, L, M, E, H, S, V, N, C, F, P, W, G, A, I, R, T, or Q; and (iii) ARGX4DX5X6DX7GAADL, where X4 can be R, V, S, A, C, L, P, I, T, G, M, E, or Q; X5 can be Y, I, L, W, F, T, V, A, G, N, Y, or S; X6 can be Y, N, P, S, K, R, G, W, D, Q, L, H, F, A, T, V, or M; and X7 can be Q, S, T, M, N, G, V, L, E, or W. A heavy chain variable domain (V H ).

[0010] The anti-GM-CSF antibodies disclosed herein are (i) a light chain complementarity determining region 1 (LC CDR1) described as QGINSX8, where X8 can be V, L, G, E, R, F, P, H, or Y; (ii) the light chain complementarity determining region 2 (LC CDR2), represented as AAS; and (iii) QQYYSX9X 10 RT, where X can be P, R, N, or C; 10 Light chain complementarity determining region 3 (LC CDR3), which may be V, C, D, L, E, P, or R. A light chain variable domain (V L ).

[0011] In some embodiments, the isolated antibody comprises the same HC and LC CDRs as a reference anti-GM-CSF antibody, such as 1D2. In some embodiments, the isolated antibody disclosed herein comprises a V CDR comprising the amino acid sequence of SEQ ID NO: 1, 3 or 4. H and / or V comprising the amino acid sequence of SEQ ID NO:2 L may include.

[0012] Any of the antibodies disclosed herein may specifically bind to human GM-CSF. Alternatively, the antibodies may cross-react with non-human GM-CSF, such as GM-CSF of a non-human primate (e.g., Rhesus macaque).

[0013] Additionally, the disclosure provides isolated antibodies that bind to the same epitope as antibody 1D2 or compete with antibody 1D2 for binding to human GM-CSF. In some examples, the isolated antibodies disclosed herein may comprise HC CDR1, HC CDR2, and HC CDR3 that contain a combined total of no more than 10 amino acid changes, preferably no more than 8 amino acid changes, and more preferably no more than 5, 4, 3, 2, or 1 amino acid changes, compared to the HC CDR1, HC CDR2, and HC CDR3 of antibody 1D2. The GM-CSF antibodies disclosed herein may further comprise LC CDR1, LC CDR2, and LC CDR3 that contain a combined total of no more than 10 amino acid changes, preferably no more than 8 amino acid changes, and more preferably no more than 5, 4, 3, 2, or 1 amino acid changes, compared to the LC CDR1, LC CDR2, and LC CDR3 of antibody 1D2.

[0014] Alternatively, or in addition, the isolated antibodies disclosed herein comprise a heavy chain variable domain (V) that is at least 85% identical to the heavy chain variable domain of antibody 1D2. H ), and a light chain variable domain (V L ).

[0015] Any isolated antibody disclosed herein may be a human or humanized antibody. In some examples, any anti-GM-CSF antibody described herein may be a full-length antibody (e.g., an IgG molecule). Alternatively, the anti-GM-CSF antibody may be an antigen-binding fragment thereof.

[0016] Any of the anti-GM-CSF antibodies disclosed herein can be conjugated to a detectable label.

[0017] In another aspect, provided herein is a nucleic acid or a set of nucleic acids that together encode an antibody that binds any of the GM-CSF antibodies described herein. A set of nucleic acids refers to two nucleic acid molecules, one encoding the heavy chain and the other encoding the light chain of a multi-chain GM-CSF antibody disclosed herein. In some examples, the nucleic acid or set of nucleic acids may be a vector or a vector set, for example, an expression vector or an expression vector set. Also provided herein is a host cell comprising a vector or a vector set disclosed herein. Such a host cell may be a bacterial cell, a yeast cell, an insect cell, a plant cell, or a mammalian cell.

[0018] Additionally, the present disclosure is directed to a pharmaceutical composition comprising (a) a monoclonal antibody or antigen-binding fragment that binds GM-CSF as disclosed herein, or an encoding nucleic acid, and (b) a pharma- ceutically acceptable carrier. In some embodiments, the pharma- ceutically acceptable carrier may include a buffer, a surfactant, a salt, an amino acid, an antioxidant, a sugar derivative (e.g., a non-reducing sugar, a sugar alcohol, a polyol, a disaccharide, or a polysaccharide). Such a pharmaceutical composition may be used to treat any of the target diseases also disclosed herein. Additionally, the present disclosure provides for the use of an antibody, an encoding nucleic acid, or other aspects of an antibody disclosed herein for the manufacture of a medicament for use in treating a target disease.

[0019] The disclosure further features a method for modulating a GM-CSF-mediated immune response in a subject, the method comprising administering an effective amount of an anti-GM-CSF antibody or a pharmaceutical composition comprising same to a subject in need thereof, in some examples, the subject is a human patient having, suspected of having, or at risk for having a disease that is an inflammatory disease, an autoimmune disease, or a cancer.

[0020] Exemplary autoimmune diseases include, but are not limited to, rheumatoid arthritis, juvenile rheumatoid arthritis, spondyloarthropathy, ankylosing spondylitis, multiple sclerosis, psoriasis, plaque psoriasis, acute gouty arthritis, or osteoarthritis.

[0021] Exemplary inflammatory diseases include, but are not limited to, Kawasaki disease, chimeric antigen receptor T cell (CAR-T)-induced cytokine release syndrome, CAR-T-induced associated encephalopathy, diffuse interstitial lung disease (DPLD), chronic obstructive pulmonary disease (COPD), aortic aneurysm, neuropathic pain, or graft-versus-host disease (GVHD).

[0022] Representative cancers include, but are not limited to, leukemia, gastric cancer, adenocarcinoma, mesothelioma, breast cancer, pancreatic ductal adenocarcinoma, colorectal cancer (CAC, e.g., colitis-associated), or hypereosinophilic syndrome (HES). In some examples, the leukemia can be juvenile myelomonocytic leukemia (JMML), chronic myelomonocytic leukemia (CMML), or chronic eosinophilic leukemia.

[0023] In any of the methods disclosed herein, the subject has undergone or is undergoing additional treatment for the disease.

[0024] The disclosure further provides a method of producing an antibody that binds human GM-CSF, the method comprising the steps of (i) culturing a host cell expressing an anti-GM-CSF antibody disclosed herein under conditions that allow expression of the antibody that binds human GM-CSF; and (ii) recovering the cultured host cell or the culture medium for harvesting the antibody that binds human GM-CSF. The method may further comprise the step of (iii) purifying the antibody that binds human GM-CSF.

[0025] The present disclosure further provides a method of detecting the presence of GM-CSF, the method comprising the steps of (i) contacting a biological sample suspected of containing GM-CSF with an antibody disclosed herein, and (ii) measuring binding of the antibody to GM-CSF in the sample. The biological sample can be obtained from a human subject suspected of having or at risk of having a disease associated with GM-CSF. The contacting step can be carried out by administering an effective amount of an anti-GM-CSF antibody to the subject.

[0026] The details of one or more embodiments of the invention are set forth in the detailed description below. Other features and advantages of the invention will be apparent from the following drawings and detailed description of certain embodiments, and from the appended claims. [Brief description of the drawings]

[0027] [Figure 1A] Figure 1A is a chart showing the binding curve of antibody 1D2 to human granulocyte-macrophage colony-stimulating factor (GM-CSF) by surface plasmon resonance (SPR) using a Biacore 3000 instrument (GE Healthcare). Figure 1A is a chart showing the binding curve of 1D2 to GM-CSF. [Figure 1B] Figure 1B is a chart showing the binding curve of antibody 1D2 to human granulocyte-macrophage colony-stimulating factor (GM-CSF) by surface plasmon resonance (SPR) using a Biacore 3000 instrument (GE Healthcare). Figure 1B is a chart showing the binding curve of antibody AMG203 (namilumab) to GM-CSF. [Figure 1C] Figure 1C is a chart showing the binding curve of antibody 1D2 to human granulocyte-macrophage colony-stimulating factor (GM-CSF) by surface plasmon resonance (SPR) using a Biacore 3000 instrument (GE Healthcare). Figure 1C is a chart showing the binding curve of MOR103 (otilimab) to GM-CSF. [Figure 2A]Figure 2A includes diagrams showing that antibody 1D2 inhibits GM-CSF-induced phosphorylation of STAT5 in human peripheral blood mononuclear cells (PBMCs), but not IL-3-induced STAT5 phosphorylation.Figure 2A includes flow cytometry charts showing that antibody 1D2 inhibits GM-CSF-induced phosphorylation of STAT5 in human peripheral blood mononuclear cells (PBMCs). [Figure 2B] Figure 2B includes diagrams showing that antibody 1D2 inhibits GM-CSF-induced, but not IL-3-induced, phosphorylation of STAT5 in human peripheral blood mononuclear cells (PBMCs). Figure 2B is a chart showing the mean fluorescence intensity of the flow cytometry charts of Figure 2A. [Diagram 3] FIG. 3 is a chart showing that representative antibody 1D2 inhibits GM-CSF-induced TF-1 cell proliferation in a dose-dependent manner. [Figure 4] FIG. 4 is a chart showing that representative antibody 1D2 inhibits GM-CSF-induced CCL17 secretion from PBMCs in a dose-dependent manner. [Figure 5A] Figure 5A is a chart showing that 1D2 SDR mutant antibodies can inhibit GM-CSF TF-1 cell proliferation, but the effect is weaker than that of the wild-type 1D2 antibody. Figure 5A is a chart showing the inhibition of GM-CSF-induced TF-1 cell proliferation by 1D2-WT and SDR mutants (103N, 103N / 32R, and 103R / 32R). [Figure 5B] Figure 5B is a chart showing that 1D2 SDR mutant antibodies can inhibit GM-CSF TF-1 cell proliferation, but the effect is weaker than that of the wild-type 1D2 antibody. Figure 5B is a chart showing the inhibition of GM-CSF-induced TF-1 cell proliferation by 1D2-WT and SDR mutants (32R, 54G, 94N, 100G, 103N, 103R, 105N, 54G / 32R, 103N / 32R, 103R / 32R). [Figure 6A]Figure 6A is a chart showing the characteristics of the D89E and D90E mutants compared to 1D2. Figure 6A is a schematic diagram showing the D89E (ED) and D90E (DE) mutant antibodies of 1D2. [Figure 6B] Figure 6B is a chart showing the characteristics of the D89E and D90E mutants compared to 1D2. Figure 6B is the binding curve of wild type antibody 1D2 to GM-CSF by surface plasmon resonance (SPR) using a Biacore 3000 instrument. [Figure 6C] Figure 6C is a chart showing the characteristics of the D89E and D90E mutants compared to 1D2. Figure 6C is a binding curve of the ED mutant antibody to GM-CSF by surface plasmon resonance (SPR) using a Biacore 3000 instrument. [Figure 6D] Figure 6D is a chart showing the characteristics of the D89E and D90E mutants compared to 1D2. Figure 6D is a binding curve of the DE mutant antibody to GM-CSF by surface plasmon resonance (SPR) using a Biacore 3000 instrument. [Figure 6E] Figure 6E is a chart showing the characteristics of the D89E and D90E mutants compared to 1D2. Figure 6E is a chart showing that the ED and DE mutant antibodies inhibit GM-CSF-induced TF-1 cell proliferation at levels comparable to the wild type 1D2 antibody. [Figure 6F] Figure 6F is a chart showing the characteristics of the D89E and D90E mutants compared to 1D2. Figure 6F is a chart showing that the ED and DE mutant antibodies inhibit GM-CSF-induced IL-8 secretion by U937 cells at levels comparable to the wild-type 1D2 antibody. [Figure 6G] Figure 6G is a chart showing the characteristics of the D89E and D90E mutants compared to 1D2, and shows that the ED and DE mutant antibodies inhibit GM-CSF-induced CCL17 secretion by PBMCs at levels comparable to the wild-type 1D2 antibody. [Figure 6H]Figure 6H is a chart showing the characteristics of the D89E and D90E mutants compared to 1D2. Figure 6H is a diagram showing the hydrophobicity profiles of 1D2, ED and DE mutant antibodies by hydrophobic interaction chromatography (HIC). [Figure 6I] Figure 6I is a chart showing the characteristics of the D89E and D90E mutants compared to 1D2. Figure 6I is a diagram showing the aggregation state of 1D2, ED and DE mutant antibodies by dynamic light scattering (DLS) analysis. [Figure 7A] Figure 7A is a chart showing the cross-reactivity of anti-GM-CSF antibodies to GM-CSF antigens from different species.Figure 7A is a chart showing the binding of AMG203 and 1D2 to human GM-CSF, mouse GM-CSF, and rhesus GM-CSF. [Figure 7B] Figure 7B is a chart showing the cross-reactivity of anti-GM-CSF antibodies to GM-CSF antigens from different species.Figure 7B is a chart showing the binding of 1D2, ED and DE mutants to human GM-CSF. [Figure 7C] Figure 7C is a chart showing the cross-reactivity of anti-GM-CSF antibodies to GM-CSF antigens from different species.Figure 7C is a chart showing the binding of 1D2, ED and DE mutants to rhesus monkey GM-CSF. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0028] The present disclosure is based, at least in part, on the development of anti-GM-CSF antibodies, e.g., 1D2 and variants thereof, which have unexpected and superior characteristics compared to known therapeutic anti-GM-CSF antibodies, e.g., AMG203 and MOR103. For example, antibody 1D2 exhibited higher binding affinity to human GM-CSF compared to AMG203 and MOR103 as determined by surface plasmon resonance (SPR) (e.g., about 5-9 fold increase); potent blocking activity against GM-CSF signaling (e.g., inhibition of STAT5 phosphorylation); and potent inhibitory effect of GM-CSF-induced cytokine production (e.g., IL-8 and CCL17) from immune cells (e.g., monocytes). Because of the superior properties of antibody 1D2, it was believed that this antibody and its functional variants would have favorable properties for blocking GM-CSF signaling and thus be beneficial in treating diseases associated with GM-CSF, such as those described herein.

[0029] Thus, provided herein are antibodies capable of binding to human GM-CSF, as well as nucleic acids encoding such antibodies, and their uses for therapeutic and diagnostic purposes. Also provided herein are kits for the therapeutic and / or diagnostic use of the antibodies, and methods for producing anti-GM-CSF antibodies.

[0030] I. Anti-GM-CSF antibody The present disclosure provides isolated antibodies that bind to human granulocyte-macrophage colony-stimulating factor (GM-CSF), for example, secreted GM-CSF.

[0031] Antibodies (used interchangeably in the plural) are immunoglobulin molecules capable of specifically binding to a target antigen (e.g., GM-CSF of the present disclosure) through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" encompasses intact (i.e., full-length) polyclonal or monoclonal antibodies, as well as antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chain (scFV), variants thereof, fusion proteins comprising antibody portions, humanized antibodies, chimeric antibodies, bispecific antibodies, nanobodies, linear antibodies, single chain antibodies, multispecific antibodies (e.g., bispecific antibodies), and any other modified conjugates of immunoglobulin molecules that contain an antigen recognition site of the required specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies include antibodies of any class, e.g., IgD, IgE, IgG, IgA, or IgM (or subclasses thereof), and antibodies that need not be of any particular class. Depending on the antibody amino acid sequence of the constant domain of its heavy chain, immunoglobulins can be assigned to different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, and several of these can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains that correspond to the different classes of immunoglobulins are called alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and three-dimensional shapes of the different classes of immunoglobulins are well known. As used herein, the term "isolated antibody" refers to an antibody that is substantially free of naturally associated molecules, i.e., naturally associated molecules that constitute up to 20% by dry weight of a preparation containing the antibody. Purity can be measured by any appropriate method, including column chromatography, polyacrylamide gel electrophoresis, and HPLC.

[0032] A typical antibody molecule contains a heavy chain variable region (V H ) and the light chain variable region (V L ) is included. V Hand V L The regions can be further subdivided into regions of hypervariability, also known as "complementarity determining regions" ("CDRs"), interspersed with regions that are more conserved, known as "framework regions" ("FRs"). H and V Lis typically composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The extent of framework regions and CDRs can be precisely identified using methodologies known in the art, for example, by the Kabat definition, the IMGT definition, the Chothia definition, the AbM definition, and / or the contact definition, all of which are well known in the art.See, e.g., Kabat, EA et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242; IMGT®, the international ImMunoGeneTics information system®, http: / / www.imgt.org; Lefranc, M.-P. et al., Nucleic Acids Res., 27:209-212 (1999); Ruiz, M. et al., Nucleic Acids Res., 28:219-221 (2000); Lefranc, M.-P., Nucleic Acids Res., 29:207-209 (2001); Lefranc, M.-P., Nucleic Acids Res., 31:307-310 (2003); Lefranc, M.-P. et al., In Silico Biol., 5, 0006 (2004) [Epub], pp. 5:45-60 (2005); Lefranc, M.-P. et al., Nucleic Acids Res., 33:D593-597 (2005); Lefranc, M.-P. et al., Nucleic Acids Res., 37:D1006-1012 (2009); Lefranc, M.-P. et al., Nucleic Acids Res., 43:D413-422 (2015); Chothia et al., (1989) Nature 342:877; Chothia, C. et al. (1987) J. Mol. Biol. 196:901-917, Al-lazikani et al. (1997) J. Molec. Biol. 273:927-948; and Almagro, J. Mol. Recognit. 17:132-143 (2004). See also hgmp.mrc.ac.uk and bioinf.org.uk / abs. As used herein, CDRs may refer to CDRs defined by any method known in the art.Two antibodies having the same CDR means that the two antibodies have the same amino acid sequence of that CDR as determined by the same method, for example, the IMGT definition.

[0033] In some embodiments, the anti-GM-CSF antibodies isolated as described herein are capable of binding to GM-CSF and inhibiting its activity by at least 50% (e.g., 60%, 70%, 80%, 90%, 95% or more). The apparent inhibition constant (Kiapp or Ki,app), which provides a measure of inhibitory potency, is related to the concentration of inhibitor required to reduce enzyme activity and is independent of enzyme concentration. The inhibitory activity of the anti-GM-CSF antibodies described herein can be determined by routine methods known in the art.

[0034] Any antibody described herein may be either monoclonal or polyclonal. A "monoclonal antibody" refers to a homogeneous antibody population, whereas a "polyclonal antibody" refers to a heterogeneous antibody population. These two terms do not limit the origin of the antibody or the manner in which it is made.

[0035] In some embodiments, the anti-GM-CSF antibodies described herein bind to the same epitope of the GM-CSF antigen as a reference antibody disclosed herein (e.g., 1D2) or compete with the reference antibody for binding to the GM-CSF antigen. "Epitope" refers to the site on a target compound to which an antibody, e.g., a Fab or full length antibody, binds. An epitope may be linear and typically 6-15 amino acids in length. Alternatively, an epitope may be conformational. An antibody that binds to the same epitope as a reference antibody described herein may bind to exactly the same epitope as the reference antibody or to a substantially overlapping epitope (e.g., fewer than three non-overlapping amino acid residues, fewer than two non-overlapping amino acid residues, or only one non-overlapping amino acid residue). Whether two antibodies compete with each other for binding to the same antigen can be determined by competition assays well known in the art. Such antibodies can be identified as known to those of skill in the art, e.g., those that have substantially similar structural characteristics (e.g., complementarity determining regions) and / or those identified by assays known in the art. For example, a competition assay can be performed using one of the reference antibodies to determine whether a candidate antibody binds to the same epitope as the reference antibody or competes for its binding to the GM-CSF antigen.

[0036] In one example, the antibody used in the methods described herein may be a humanized antibody. Humanized antibodies refer to forms of non-human (e.g., murine) antibodies that are specific chimeric immunoglobulins, immunoglobulin chains, or antigen-binding fragments thereof that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a complementarity determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody), such as mouse, rat, or rabbit, having the desired specificity, affinity, and capacity. In some examples, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may contain residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences, but are included to further refine and optimize antibody performance. Generally, a humanized antibody comprises substantially all of at least one, typically two, variable domains, with all or substantially all of the CDR regions corresponding to those of a non-human immunoglobulin, and all or substantially all of the FR regions being of a human immunoglobulin consensus sequence. A humanized antibody will optimally also comprise at least a portion of an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin. The antibody may have an altered Fc region as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (one, two, three, four, five, or six) that have been altered with respect to the original antibody, also referred to as one or more CDRs "derived from" one or more CDRs from the original antibody. A humanized antibody may also include affinity maturation.

[0037] In some embodiments, the anti-GM-CSF antibodies described herein specifically bind to the corresponding target antigen or epitope thereof. An antibody that "specifically binds" to an antigen or epitope is a term well understood in the art. A molecule is said to exhibit "specific binding" if it reacts with a particular target antigen more frequently, faster, with better persistence and / or with better affinity than it reacts with alternative targets. An antibody "specifically binds" to a target antigen or epitope if it binds with better affinity, avidity, more readily, and / or with better persistence than it binds to other substances. For example, an antibody that specifically (or preferentially) binds to an antigen (e.g., human GM-CSF) or an antigenic epitope therein is an antibody that binds to this target antigen with better affinity, avidity, more readily, and / or with better persistence than it binds to other antigens or other epitopes within the same antigen. By this definition, it is also understood that, for example, an antibody that specifically binds to a first target antigen may or may not specifically or preferentially bind to a second target antigen. Thus, "specifically binds" or "preferentially binds" does not necessarily require (although it can include) exclusive binding. In some instances, an antibody that "specifically binds" to a target antigen or epitope thereof may not bind to other antigens or other epitopes within the same antigen (e.g., the binding is not detectable by conventional assays).

[0038] In some embodiments, the antibodies described herein specifically bind to GM-CSF of a particular species (e.g., human GM-CSF) compared to GM-CSF from other species. For example, the antibodies described herein may specifically bind to human GM-CSF compared to mouse GM-CSF. In other embodiments, the antibodies described herein may cross-react with human GM-CSF and one or more GM-CSFs from a non-human species (e.g., non-human primates such as rhesus monkeys). In some embodiments, the antibodies cross-react with similar binding affinity to humans and rhesus monkeys, but have significantly lower binding affinity to mouse GM-CSF. In some embodiments, the anti-GM-CSF antibodies described herein have suitable binding affinity for a target antigen (e.g., human GM-CSF) or antigenic epitope thereof.

[0039] As used herein, "binding affinity" refers to the apparent association constant or KA, which is the ratio of the association constant and the dissociation constant, K-on and K-off, respectively. A is the dissociation constant (K D The anti-GM-CSF antibodies described herein have a binding affinity of at least 10 to a target antigen or antigenic epitope. -8 , 10 -9 , 10 -10 M, 10 -11 Binding affinity (K D For example, the anti-GM-CSF antibody may have a 10 -9 M, 10 -10 The increased binding affinity can be expressed as K D A higher affinity binding of an antibody to a first antigen compared to a second antigen corresponds to a decrease in the value of K (or K D The K value for binding the first antigen is higher (or lower) than the K value for binding the first antigen. DIn such cases, the antibody has specificity for a first antigen (e.g., a first protein or a mimetic thereof in a first structure) compared to a second antigen (e.g., the same first protein or a mimetic thereof in a second structure; or a second protein). In some embodiments, the anti-GM-CSF antibodies described herein have a higher binding affinity (higher K A or low K D In some embodiments, an anti-GM-CSF antibody may have a higher binding affinity for GM-CSF of a particular species (e.g., human GM-CSF) than for GM-CSF from a different species (e.g., mouse). The difference in binding affinity (e.g., specificity or other comparison) may be at least 1.5, 2, 2.5, 3, 4, 5, 10, 15, 20, 37.5, 50, 70, 80, 91, 100, 500, 1,000, 5,000, 10,000, or 10 5 In some embodiments, any of the anti-GM-CSF antibodies can be further affinity matured to increase the binding affinity of the antibody to the target antigen or antigenic epitope thereof.

[0040] Binding affinity (or binding specificity) can be determined by a variety of methods, including equilibrium dialysis, equilibrium binding, gel filtration, ELISA, surface plasmon resonance (SPR), fluorescence activated cell sorting (FACS) or spectroscopy (e.g., using a fluorescence assay). Exemplary conditions for assessing binding affinity are HBS-P buffer (10 mM HEPES pH 7.4, 150 mM NaCl, 0.005% (v / v) surfactant P20) and PBS buffer (10 mM PO4 -3 , 137 mM NaCl, and 2.7 mM KCl). These techniques can be used to measure the concentration of bound protein as a function of target protein concentration. The concentration of bound protein ([Bound]) is generally related to the concentration of free target protein ([Free]) by the following formula: [Bound]=[Free] / (Kd+[Free])

[0041] It is not necessary to make an exact determination of the K, but it is sufficient to obtain a qualitative measure of affinity, e.g., determined using methods such as ELISA or FACS analysis, so that it is proportional to the K and can then be used to obtain a quantitative measure of affinity by comparison, e.g., to determine whether a high affinity is, e.g., two-fold higher, or to obtain an estimate of affinity, e.g., by activity in a functional assay, e.g., an in vitro or in vivo assay.

[0042] Provided below is a representative anti-GM-CSF antibody 1D2, including its heavy and light chain CDR sequences (as per IMGT definition) and heavy and light chain variable domain sequences.

[0043] [Table 1]

[0044] Heavy chain variable domain sequence of 1D2 (CDRs in bold): QVQLVQSGAEVKKPGASVKVSCEASGYTFTDKYLHWVRQAPGQGLEWMAWINPKSGGTFYAQNFKGRVTVTRDTSINTAYLELTSLRLDDTSTYYCARGRDYYDQGAADLWGQGTMVTVSS (SEQ ID NO: 1)

[0045] Light chain variable domain of 1D2: DIQLTQSPSSLSASVGDRVTITCRASQGINSVLAWYQQKPGKAPKLLLYAASKLESGVPSRFSGSGSGTDYTLTISSLQPEDFGTYYCQQYYSPVRTFGQGTKVEIKR (SEQ ID NO: 2)

[0046] In some embodiments, an isolated anti-GM-CSF antibody disclosed herein may contain the same regions / residues responsible for antigen binding as a reference antibody (e.g., 1D2), e.g., the same specificity determining residues (SDRs) of a CDR or the entire CDR. Regions / residues responsible for antigen binding can be identified from the amino acid sequence of the heavy / light chain sequences of the reference antibody by methods known in the art. See, e.g., www.bioinf.org.uk / abs; Almagro, J. Mol. Recognit. 17:132-143 (2004); Chothia et al., J. Mol. Biol. 227:799-817 (1987), as well as others known in the art or disclosed herein. In some embodiments, an isolated anti-GM-CSF antibody disclosed herein may contain the same V and VD residues as a reference antibody, e.g., 1D2. H and / or V L In some embodiments, the anti-GM-CSF antibodies disclosed herein have the same heavy chain and / or light chain CDRs as a reference antibody, such as 1D2.

[0047] Additionally, the antibody may contain specificity determining residues that are not found in the CDR sequences of the reference antibody (e.g., 1D2) but are included to develop an antibody with equivalent functionality to the reference antibody or to further refine and optimize antibody performance. As used herein, such antibodies are referred to as SDR-mutated antibodies. Generally, the antibody contains substantially all of at least one, typically two, variable domains, including all or substantially all of the CDR regions and all or substantially all of the FR region consensus sequences. The antibody may have one or more CDRs (one, two, three, four, five, six) that are altered from the original antibody. Such residues can be identified by in vitro affinity maturation of the reference antibody (e.g., 1D2). Methods for performing in vitro affinity maturation of a reference antibody are known in the art, see, for example, Li et al., Mabs, 2014 Mar-Apr;6(2):437-45.

[0048] In some embodiments, the SDR mutant antibody has at least 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 100%, 105% or more binding affinity for GM-CSF compared to a reference antibody, e.g., 1D2.

[0049] In some embodiments, the isolated anti-GM-CSF antibody comprises a heavy chain variable region comprising a heavy chain CDR1 (HC CDR1), a heavy chain CDR2 (HC CDR2), and a heavy chain CDR3 (HC CDR3).

[0050] In some embodiments, according to the IMGT definition, HC CDR1 may comprise the amino acid sequence of GYTFTX1X2Y. X1 may be D, E, S, P, G, A, V, N, Q, C, W, R, T, or L. For example, X1 may be D, V, N, Q, C, W, or R. In one example, X1 may be D. Alternatively or in addition, X2 may be K, V, L, E, P, S, T, C, D, R, H, A, F, Q, or N. In some embodiments, X2 may be K, V, E, P, S, T, C, D, R, H, Q, or N. In one example, X2 may be K. Alternatively or in addition, HC CDR2 may comprise the amino acid sequence of INPX3SGGT, where X3 may be K, L, M, E, H, S, V, N, C, F, P, W, G, A, I, R, T, or Q. In some examples, X3 may be K, L, M, E, H, S, V, N, C, P, W, G, A, I, R, T, or Q. In one example, X3 may be K. Alternatively or in addition, HC CDR3 may comprise the amino acid sequence of ARGX4DX5X6DX7GAADL, where X4 may be R, V, S, A, C, L, P, I, T, G, M, E, or Q. In some examples, X4 may be R, V, S, A, C, P, I, T, G, M, or E. In one example, X4 may be R. Alternatively or in addition, X5 may be Y, I, L, W, F, T, V, A, G, N, Y, or S. In some examples, X5 may be Y, L, W, F, V, G, N, Y, or S. In one example, X5 may be Y. Alternatively or in addition, X6 may be Y, N, P, S, K, R, G, W, D, Q, L, H, F, A, T, V, or M. In some examples, X6 may be Y, N, P, S, K, R, G, W, D, Q, H, F, A, T, V, or M. In one example, X6 may be Y. Alternatively or in addition, X7 may be Q, S, T, M, N, G, V, L, E, or W. In some examples, X7 may be Q, S, T, M, N, G, V, or E. In one example, X7 can be Q.

[0051] The anti-GM-CSF antibody may comprise a light chain variable region comprising a light chain CDR1 (LC CDR1), a light chain CDR2 (LC CDR2), and a light chain CDR3 (LC CDR3). In some embodiments, according to the IMGT definition, LC CDR1 may comprise an amino acid sequence of QGINSX8, where X8 may be V, L, G, E, R, F, P, H, or Y. In one example, X8 may be V. Alternatively or additionally, LC CDR2 may comprise an amino acid sequence of AAS. Alternatively or additionally, LC CDR3 may comprise an amino acid sequence of QQYYSX9X. 10 RT, where X9 can be P, R, N, or C. In some examples, X9 can be P, R, or C. In one example, X9 can be P. Alternatively or in addition, X 10 may be V, C, D, L, E, P, or R. In one example, X 10 may be V.

[0052] Also within the scope of the present disclosure are functional variants of any of the representative anti-GM-CSF antibodies disclosed herein. H and / or V L A reference antibody may contain one or more amino acid residue changes in one or more HC CDRs and / or one or more LC CDRs compared to the reference antibody, but maintain substantially similar binding and biological activity (e.g., substantially similar binding affinity, binding specificity, inhibitory activity, anti-inflammatory activity, or a combination thereof) to the reference antibody.

[0053] Representative WT (e.g., 1D2 in the second column) and residues X1 to X2 above 10 SDR mutants containing amino acids corresponding to are listed in Table 2. The binding affinity of the SDR mutant antibody containing each residue is listed below the residue. For example, an SDR mutant contains a single mutation at the X1 residue of HC CDR1, which is E, which has a binding affinity for GM-CSF of 63% compared to a reference antibody (e.g., 1D2).

[0054] [Table 2]

[0055] In some examples, an isolated anti-GM-CSF antibody disclosed herein comprises HC CDR1, HC CDR2, and HC CDR3, and may contain 10 or fewer amino acid changes (e.g., 9, 8, 7, 6, 5, 4, 3, 2, or 1 or fewer amino acid changes) in total compared to HC CDR1, HC CDR2, and HC CDR3 of a reference antibody, e.g., 1D2. "In total" means that the total number of amino acid changes in all three HC CDRs is within the defined range. In some examples, an anti-GM-CSF antibody disclosed herein may comprise HC CDR1, HC CDR2, and HC CDR3, at least one of which contains 5 or fewer amino acid changes (e.g., 4, 3, 2, or 1 or fewer amino acid changes) with the corresponding HC CDR of a reference antibody, e.g., 1D2. In a particular example, the antibody comprises HC CDR3, and may contain 5 or fewer amino acid changes (e.g., 4, 3, 2, or 1 or fewer amino acid changes) with the HC CDR3 of a reference antibody, e.g., 1D2.

[0056] Alternatively or additionally, the isolated anti-GM-CSF antibody may comprise an LC CDR1, an LC CDR2, and an LC CDR3, which together contain no more than 10 amino acid changes (e.g., no more than 9, 8, 7, 6, 5, 4, 3, 2, or 1 amino acid changes) compared to the LC CDR1, LC CDR2, and LC CDR3 of the reference antibody. In some examples, the anti-GM-CSF antibody may comprise an LC CDR1, an LC CDR2, and an LC CDR3, at least one of which contains no more than 5 amino acid changes (e.g., no more than 4, 3, 2, or 1 amino acid changes) from the corresponding LC CDR of the reference antibody. In particular examples, the antibody comprises an LC CDR3, which contains no more than 5 amino acid changes (e.g., no more than 4, 3, 2, or 1 amino acid changes) from the LC CDR3 of the reference antibody.

[0057] In some embodiments, an isolated anti-GM-CSF antibody disclosed herein may comprise heavy chain CDRs that, taken together, are at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to the heavy chain CDRs of a reference antibody, e.g., 1D2. Alternatively, or in addition, the antibody may comprise light chain CDRs that, taken together, are at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to the light chain CDRs of the reference antibody. In some embodiments, an anti-GM-CSF antibody may comprise a heavy chain variable region that is at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to the heavy chain variable region of a reference antibody, e.g., 1D2, and / or a light chain variable region that is at least 80% (e.g., 85%, 90%, 95%, or 98%) identical to the light chain variable region of the reference antibody.

[0058] The "percent identity" of two amino acid sequences is determined using the algorithm of Karlin and Altschul Proc. Natl. Acad. Sci. USA 87:2264-68, 1990, modified as in Karlin and Altschul Proc. Natl. Acad. Sci. USA 90:5873-77, 1993. Such an algorithm is incorporated into the NBLAST and XBLAST programs (version 2.0) of Altschul et al., J. Mol. Biol. 215:403-10, 1990. BLAST protein searches are performed with the XBLAST program, score=50, wordlength=3, to obtain amino acid sequences homologous to a protein molecule of interest. When gaps exist between the two sequences, Gapped BLAST can be utilized as described in Altschul et al., Nucleic Acids Res. 25(17):3389-3402, 1997. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used.

[0059] In some embodiments, the anti-GM-CSF antibodies may contain modifications that improve antibody properties, such as stability, oxidation, isomerization, and deamidation. In some examples, the antibodies may contain residues that are not found in the framework (FR region) sequences of the reference antibody (e.g., 1D2).

[0060] In some embodiments, the FR region of the heavy chain variable region of the reference antibody (e.g., 1D2) comprises an aspartic acid-aspartic acid (DD) motif. As used herein, a DD motif refers to two consecutive aspartic acid residues in the amino acid sequence of an antibody. During production, storage, and in vivo, therapeutic antibodies are at risk of degradation by many routes (e.g., chemical degradation). For example, isomerization of D residues and deamination of asparagine (N) residues are common routes of antibody degradation. Non-limiting examples of D and N degradation sequence motifs are DD, NG, NN, NS, NT, DG, DS, DT, DH. In some embodiments, the antibodies described herein comprise a DD motif at positions 89 and 90 of the heavy chain variable domain.

[0061] In some instances, the amino acid residue change may be a conservative amino acid residue substitution. As used herein, "conservative amino acid substitution" refers to an amino acid substitution that does not change the relative charge or size characteristics of the protein in which the amino acid substitution is made. Variants may be prepared according to methods known to those skilled in the art for modifying polypeptide sequences, for example, those methods found in compiled references, such as Molecular Cloning: A Laboratory Manual, J. Sambrook et al. eds., Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1989, or Current Protocols in Molecular Biology, FM Ausubel et al. eds., John Wiley & Sons, Inc., New York. In some instances, conservative amino acid substitutions may include substitutions made between amino acids within the following groups: (a) E, D; (b) M, I, L, V; (c) F, Y, W; (d) K, R, H; (e) A, G; (f) S, T; and (g) Q, N.

[0062] In some embodiments, the antibodies described herein comprise mutations in the DD motifs at positions 89 and 90 of the heavy chain variable domain. In some embodiments, the antibodies described herein comprise a D89E (ED) or D90E (DE) mutation in the heavy chain variable domain.

[0063] An exemplary heavy chain variable domain sequence containing a D89E (ED) or D90E (DE) mutation is: Heavy chain variable domain sequence with D89E(ED) mutation (mutation in bold): QVQLVQSGAEVKKPGASVKVSCEASGYTFTDKYLHWVRQAPGQGLEWMAWINPKSGGTFYAQNFKGRVTVTRDTSINTAYLELTSLRL ED TSTYYCARGRDYYDQGAADLWGQGTMVTVSS (SEQ ID NO:3) Heavy chain variable domain sequence with D90E(DE) mutation: QVQLVQSGAEVKKPGASVKVSCEASGYTFTDKYLHWVRQAPGQGLEWMAWINPKSGGTFYAQNFKGRVTVTRDTSINTAYLELTSLRL DE TSTYYCARGRDYYDQGAADLWGQGTMVTVSS (SEQ ID NO: 4)

[0064] In some embodiments, the antibodies described herein comprise a VH comprising the amino acid sequence of SEQ ID NO:1, 3 or 4, and / or a VL comprising the amino acid sequence of SEQ ID NO:2.

[0065] In some embodiments, the heavy chain of any of the anti-GM-CSF antibodies described herein may further comprise a heavy chain constant region (5 CH) or a portion thereof (e.g., CH1, CH2, CH3, or a combination thereof). The heavy chain constant region may be of any suitable origin, such as human, mouse, rat, or rabbit. In one particular example, the heavy chain constant region is from human IgG (gamma heavy chain), such as IgG1, IgG2, or IgG4. In one example, the heavy chain constant region is of subclass IgG1.

[0066] The light chain of any of the anti-GM-CSF antibodies described herein may further comprise a light chain constant region (CL), which may be any CL known in the art. In some examples, the CL is a kappa light chain. In other examples, the CL is a lambda light chain. Antibody heavy and light chain constant regions are known in the art, such as those provided in the IMGT database (www.imgt.org) or www.vbase2.org / vbstat.php., both of which are incorporated herein by reference.

[0067] In one particular example, the anti-GM-CSF antibodies disclosed herein are full-length IgG1 / kappa antibodies.

[0068] As described herein, anti-GM-CSF antibodies may be any antibody format, including, but not limited to, intact (i.e., full-length) antibodies, antigen-binding fragments thereof (e.g., Fab, Fab', F(ab')2, Fv), single chain antibodies, bispecific antibodies, or nanobodies.

[0069] II. Preparation of anti-GM-CSF antibodies As described herein, antibodies capable of binding to GM-CSF can be generated by any method known in the art, see, e.g., Harlow and Lane, (1998) Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York.

[0070] In some embodiments, antibodies specific for a target antigen (e.g., GM-CSF) can be generated by conventional hybridoma technology. Full-length target antigens or fragments thereof can be optionally conjugated to a carrier protein, e.g., KLH, and used to immunize a host animal to generate antibodies that bind to the antigen. The route and schedule of immunization of the host animal generally follows established and conventional techniques of antibody stimulation and production, as further described herein. General techniques for the production of mouse, humanized, and human antibodies are known in the art and described herein. Any mammalian subject, including humans, or antibody-producing cells derived therefrom, are contemplated to be engineered and serve as the basis for the production of mammalian hybridoma cell lines, including humans. Typically, the host animal is inoculated intraperitoneally, intramuscularly, orally, subcutaneously, intraplantarly, and / or intradermally with an immunogen comprising an amount described herein.

[0071] If desired, the antibody of interest (monoclonal or polyclonal) (e.g., produced by a hybridoma) can be sequenced and the polynucleotide sequence can then be cloned into a vector for expression or propagation. The sequence encoding the antibody of interest can be maintained in a vector within a host cell, and the host cell can then be propagated and frozen for future use. Alternatively, the polynucleotide sequence can be used for genetic engineering to "humanize" the antibody or improve the affinity (affinity maturation) or other characteristics of the antibody. For example, the constant region can be engineered to be more similar to human constant regions to avoid immune responses when the antibody is used in clinical trials and human treatment. It is desirable to genetically engineer the antibody sequence to obtain better affinity for the target antigen and better efficacy in inhibiting the activity of GM-CSF. It is clear to one of skill in the art that one or more polynucleotide changes can be made to an antibody and still maintain its binding specificity to the target antigen.

[0072] In other embodiments, fully human antibodies can be obtained using commercially available mice engineered to express specific human immunoglobulin proteins. Transgenic animals designed to produce a more desirable (e.g., fully human antibodies) or stronger immune response can also be used to generate humanized or human antibodies. Examples of such technology are XenomouseR™ from Amgen, Inc. (Fremont, CA) and HuMAb-MouseR™ and TC Mouse™ from Medarex, Inc. (Princeton, NJ), or H2L2 mouse from Harbour Antibodies BV (Holland). In another alternative, antibodies can be made recombinantly by phage display or yeast technology. See, e.g., U.S. Patent Nos. 5,565,332; 5,580,717; 5,733,743; and 6,265,150; and Winter et al., (1994) Annu. Rev. Immunol. 12:433-455. Alternatively, human antibodies and antibody fragments can be produced in vitro, from immunoglobulin variable (V) domain gene repertoires from unimmunized donors, using phage display technology (McCafferty et al., (1990) Nature 348:552-553).

[0073] Antigen-binding fragments of intact antibodies (full-length antibodies) can be prepared by routine methods. For example, F(ab')2 fragments can be produced by pepsin digestion of antibody molecules, and Fab fragments can be generated by reducing disulfide bonds of F(ab')2 fragments. Engineered antibodies, such as humanized antibodies, chimeric antibodies, single-chain antibodies, and bispecific antibodies, can be produced, for example, by conventional recombinant techniques. In one example, DNA encoding a monoclonal antibody specific for a target antigen can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the monoclonal antibody). Hybridoma cells serve as a preferred source of such DNA. Once isolated, the DNA is placed into one or more expression vectors and then transfected into host cells that do not otherwise produce immunoglobulin proteins, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, human HEK293 cells, or myeloma cells, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. See, for example, PCT application WO 87 / 04462. The DNA can then be modified, for example, by substituting the coding sequences for human heavy and light chain constant domains in place of the homologous murine sequences, Morrison et al. (1984) Proc. Nat. Acad. Sci. 81:6851, or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. In this manner, engineered antibodies, such as "chimeric" or "hybrid" antibodies, can be prepared, which have the binding specificity of the target antigen.

[0074] Single chain antibodies can be prepared recombinantly by linking a nucleotide sequence encoding a heavy chain variable region with a nucleotide sequence encoding a light chain variable region, preferably incorporating a flexible linker between the two variable regions.

[0075] Alternatively, techniques described for the production of single chain antibodies (U.S. Pat. Nos. 4,946,778 and 4,704,692) can be adapted to produce phage or yeast scFv libraries, and GM-CSF specific scFv clones can be identified from the libraries following routine procedures. Positive clones can be subjected to further screening to identify those that inhibit GM-CSF activity.

[0076] Antibodies obtained according to methods known in the art and described herein can be characterized using methods known in the art. For example, one method is the identification of the epitope to which an antigen binds, or "epitope mapping". There are many methods known in the art for mapping and characterizing the location of epitopes in proteins, including analysis of the crystal structure of an antibody-antigen complex, competitive assays, gene fragment expression assays, and synthetic peptide-based assays, as described, for example, in Chapter 11 of Harlow and Lane, Using Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1999. In one example, epitope mapping can be achieved using H / D-Ex (hydrogen deuterium exchange) in combination with proteolysis and mass spectrometry. In a further example, epitope mapping can be used to determine the sequence to which an antibody binds. Epitopes may be linear epitopes contained in a single chain of amino acids, or structural epitopes formed by three-dimensional interactions of amino acids that may not necessarily be contained in a single chain (linear sequence of primary structure). Peptides of various lengths (e.g., at least 4-6 amino acids long) can be isolated or synthesized (e.g., recombinantly) and used in binding assays with antibodies. In another example, the epitope to which an antibody binds can be determined in a systematic screen by using overlapping peptides derived from the target antigen sequence and determining binding by the antibody. With gene fragment expression assays, an open reading frame encoding a target antigen is fragmented, either randomly or by a specific gene construct, and the reactivity of the expressed fragments of the antigen with the antibody being tested is determined. For example, gene fragments can be produced by PCR and then transcribed and translated into protein in vitro in the presence of radioactive amino acids. Binding of the antibody to the radiolabeled antigen fragments is then determined by immunoprecipitation and gel electrophoresis.Specific epitopes can also be identified by using large libraries of random peptide sequences displayed on the surface of phage particles (phage libraries). Alternatively, defined libraries of overlapping peptide fragments can be tested for binding to the test antibody in simple binding assays. In further examples, mutagenesis of antigen-binding domains, domain swapping experiments, and alanine scanning mutagenesis can be performed to identify residues required, sufficient, and / or necessary for epitope binding. For example, domain swapping experiments can be performed using mutants of the target antigen in which various fragments of the GM-CSF polypeptide are replaced (swapped) with sequences from closely related but antigenically distinct proteins (e.g., CD28 protein). The importance of specific antigen fragments to antibody binding can be assessed by assessing the binding of the antibody to mutant GM-CSF. Alternatively, competitive assays can be performed using other antibodies known to bind to the same antigen to determine whether the antibody binds to the same epitope as the other antibodies. Competitive assays are well known to those skilled in the art.

[0077] In some examples, the anti-GM-CSF antibody is prepared by recombinant techniques, as exemplified below. The nucleic acids encoding the heavy and light chains of the anti-GM-CSF antibody described herein can be cloned into an expression vector, with each nucleotide sequence being in operable linkage with a suitable promoter. In one example, the respective nucleotide sequences encoding the heavy and light chains are in operable linkage with different promoters. Alternatively, the nucleotide sequences encoding the heavy and light chains can be in operable linkage with a single promoter, with both the heavy and light chains being expressed from the same promoter. If necessary, an internal ribosome entry site (IRES) can be inserted between the heavy and light chain coding sequences.

[0078] In some instances, the nucleotide sequences encoding the two chains of an antibody can be cloned into two vectors, which can be introduced into the same or different cells. If the two chains are expressed in different cells, each of them can be isolated from the expressing host cell, and the isolated heavy and light chains can be mixed and incubated under suitable conditions to allow the formation of antibodies.

[0079] In general, a nucleic acid sequence encoding one or all chains of an antibody can be cloned into a suitable expression vector in operable linkage with a suitable promoter using methods known in the art. For example, the nucleotide sequence and the vector can be contacted with a restriction enzyme under suitable conditions to create complementary ends on each molecule, which can pair with each other and be joined together by a ligase. Alternatively, synthetic nucleic acid linkers can be ligated to the ends of the gene. These synthetic linkers contain nucleic acid sequences that correspond to specific restriction sites in the vector. The choice of expression vector / promoter will depend on the type of host cell used to produce the antibody.

[0080] A variety of promoters can be used for expression of the antibodies described herein, including, but not limited to, the cytomegalovirus (CMV) intermediate early promoter, viral LTRs such as Rous sarcoma virus LTR, HIV-LTR, HTLV-1 LTR, simian virus 40 (SV40) early promoter, E. coli lac UV promoter, and herpes simplex tk virus promoter.

[0081] Controllable promoters can also be used, including those that use the lac repressor from E. coli as a transcriptional regulator that controls transcription from a mammalian cell promoter carrying a lac operator [Brown, M. et al., Cell, 49:603-612 (1987)] and those that use the tetracycline repressor (tetR) [Gossen, M., and Bujard, H., Proc. Natl. Acad. Sci. USA 89:5547-555115 (1992); Yao, F. et al., Human Gene Therapy, 9:1939-1950 (1998); Shockelt, P. et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)]. Other systems include FK506 dimers, VP16 or p65 using astradiol, RU486, diphenol murislerone, or rapamycin. Inducible systems are available from Invitrogen, Clontech, and Ariad, among others.

[0082] A controllable promoter containing a repressor can be used with an operon. In one embodiment, the lac repressor from E. coli can function as a transcriptional regulator to control transcription from a mammalian cell promoter carrying a lac operator [M. Brown et al., Cell, 49:603-612 (1987)]; Gossen and Bujard (1992); [M. Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992)] combined the tetracycline repressor (tetR) with a transcriptional activator (VP 16) to create a tetR mammalian cell transcriptional activator fusion protein, tTa (tetR-VP 16), in combination with a minimal promoter carrying tetO from the human cytomegalovirus (hCMV) promoter to create a tetR-tet operator system to control gene expression in mammalian cells. In one embodiment, a tetracycline-inducible switch is used. When a tetracycline operator is appropriately placed downstream of the TATA element of the CMVIE promoter, the tetracycline repressor alone (tetR), rather than a tetR mammalian cell transcription factor fusion derivative, can function as a potent transmodulator in mammalian cells to control gene expression (Yao et al., Human Gene Therapy). One particular advantage of this tetracycline-inducible switch is that it does not require the use of tetracycline repressor-mammalian cell transactivator or repressor fusion proteins, which may be harmful to cells in some cases, to achieve its controllable effect (Gossen et al., Natl. Acad. Sci. USA, 89:5547-5551 (1992); Shockett et al., Proc. Natl. Acad. Sci. USA, 92:6522-6526 (1995)).

[0083] In addition, the vector may contain, for example, some or all of the following: a selectable marker gene, such as a neomycin gene for selecting stable or transient transfectants in mammalian cells; an enhancer / promoter sequence from the immediate early gene of human CMV for high-level transcription; a transcription termination signal and an RNA processing signal from SV40 for mRNA stability; an SV40 polyoma origin of replication and ColE1 for proper episomal replication; an internal ribosome binding site (IRES); a versatile multiple cloning site; and T7 and SP6 RNA promoters for in vitro transcription of sense and antisense RNA. Vectors and methods suitable for producing vectors containing transgenes are well known and available in the art. Examples of polyadenylation signals useful for carrying out the methods described herein include, but are not limited to, human collagen I polyadenylation signal, human collagen II polyadenylation signal, and SV40 polyadenylation signal.

[0084] One or more vectors (e.g., expression vectors) containing nucleic acids encoding any of the antibodies can be introduced into suitable host cells for producing the antibodies. The host cells can be cultured under conditions suitable for expression of the antibodies or any of their polypeptide chains. Such antibodies or their polypeptide chains can be recovered by the cultured cells (e.g., from the cells or culture supernatant) by conventional methods, such as affinity purification. If necessary, the antibody polypeptide chains can be incubated for a suitable period of time under suitable conditions to allow production of the antibody.

[0085] In some embodiments, the method for preparing the antibodies described herein includes a recombinant expression vector encoding both the heavy and light chains of an anti-GM-CSF antibody also described herein. The recombinant expression vector can be introduced into a suitable host cell (e.g., dhfr-CHO cells) by conventional methods, such as calcium phosphate-mediated transfection. Positively transformed host cells can be selected and cultured under suitable conditions that allow the expression of the two polypeptide chains that form the antibody, which can be recovered from the cells or from the culture medium. If necessary, the two chains recovered from the host cells can be incubated under suitable conditions that allow the formation of the antibody.

[0086] In one example, two recombinant expression vectors are provided, one encoding the heavy chain of an anti-GM-CSF antibody and the other encoding the light chain of an anti-GM-CSF antibody, both of which can be introduced into a suitable host cell (e.g., dhfr-CHO cells) by conventional methods, such as calcium phosphate-mediated transfection.

[0087] Alternatively, each expression vector can be introduced into a suitable host cell. The positive transformants can be selected and cultured under suitable conditions that allow the expression of the antibody polypeptide chains. If the two expression vectors are introduced into the same host cell, the antibody produced therefrom can be recovered from the host cell or from the culture medium. If necessary, the polypeptide chains can be recovered from the host cell or from the culture medium and then incubated under suitable conditions that allow the formation of the antibody. If the two expression vectors are introduced into different host cells, they can each be recovered from the corresponding host cell or from the corresponding culture medium. The two polypeptide chains can then be incubated under suitable conditions for the formation of the antibody.

[0088] Standard molecular biology techniques are used to prepare the recombinant expression vector, transfect the host cells, select for transformants, culture the host cells, and recover the antibody from the culture medium. For example, some antibodies can be isolated by affinity chromatography with a Protein A or Protein G-bound matrix.

[0089] Any nucleic acids encoding the heavy chain, light chain, or both, of the anti-GM-CSF antibodies described herein, vectors containing them (e.g., expression vectors), and host cells containing the vectors are within the scope of this disclosure.

[0090] III. Pharmaceutical Compositions The antibodies described herein, as well as the encoding nucleic acids or nucleic acid sets, vectors containing them, or host cells containing the vectors, can be mixed with pharma- ceutically acceptable carriers (excipients) to form pharmaceutical compositions for use in treating target diseases. By "acceptable," it is meant that the carrier must be compatible with the active ingredients of the composition (and preferably be able to stabilize the active ingredients) and not harmful to the subject being treated. Pharmaceutically acceptable excipients (carriers), including buffers, are well known in the art. See, for example, Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover.

[0091] The anti-GM-CSF antibody containing pharmaceutical compositions disclosed herein may further comprise a suitable buffering agent. A buffering agent is a weak acid or base used to maintain the pH of a solution near a selected value after the addition of another acid or base. In some examples, the buffering agent disclosed herein may be a buffering agent capable of maintaining a physiological pH despite changes in carbon dioxide concentration (produced by cellular respiration). Exemplary buffering agents include, but are not limited to, HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid) buffer, Dulbecco's phosphate buffered saline (DPBS) buffer, or phosphate buffered saline (PBS) buffer. Such buffering agents may include disodium hydrogen phosphate and sodium chloride, or potassium dihydrogen phosphate and potassium chloride.

[0092] In some embodiments, the buffering agent in the pharmaceutical compositions described herein can maintain a pH value of about 5 to 8. For example, the pH of the pharmaceutical composition can be about 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0. In other examples, the pharmaceutical composition can have a pH value below 7, such as about 7, 6.8, 6.5, 6.3, 6, 5.8, 5.5, 5.3, or 5.

[0093] The pharmaceutical compositions described herein include one or more suitable salts. Salts are ionic compounds that can be formed by the neutralization reaction of an acid and a base. (Skoog, DA; West, DM; Holler, JF; Crouch, SR (2004). "chapters 14-16". Fundamentals of Analytical Chemistry (8th ed.)). A salt is composed of a related number of cations (positively charged ions) and anions (negative ions), and the product is electrically neutral (has no net charge). An ion, as described herein, is an atom or molecule that has gained or lost one or more valence electrons, giving the ion a net positive or negative charge. If a chemical species has more protons than electrons, it has a net positive charge. If there are more electrons than protons, the species has a negative charge.

[0094] A cation (+), as described herein, is an ion that has fewer electrons than protons, giving it a positive charge. (Douglas W. Haywick, (2007-2008). "Elemental Chemistry"). A cation with one positive charge can be referred to as a monovalent cation; a cation with more than one positive charge can be referred to as a polyvalent cation or multivalent cation. A non-limiting example of a monovalent cation is hydrogen (H + ), Sodium (Na + ), potassium (K + ), Ammonium (NH 4+ ), Lithium (Li + ), monovalent copper (Cu + ), silver (Ag + ) and the like. A non-limiting example of a multivalent cation is magnesium (Mg 2+ ), Calcium (Ca 2+ ), Barium (Ba 2+ ), Beryllium (Be 2+ ), Copper (Cu 2+ ), ferrous iron (Fe 2+ ), ferric iron (Fe 3+ ), lead(II)(Pb 2+ ), lead(IV)(Pb 4+ ), Manganese(II)(Mn 2+ ), Strontium (Sr 2+ ), Tin(IV)(Sn 4+ ), Zinc (Zn 2+ ) etc.

[0095] Anions, as described herein, are ions that have more electrons than protons, giving a net negative charge. A non-limiting example of an anion is azide (N 3- ), bromide (Br - ), chloride (Cl - ), fluoride (F - ), hydrides (H - ), iodide (I - ), nitrides (N - ), oxides (O 2- ), sulfide (S 2- ), Carbonic acid (CO3 2-), hydrogen carbonate (HCO3 - ), hydrogen sulfate (HSO4 - ), hydroxide (OH - ), dihydrogen phosphate (H2PO4 - ), sulfuric acid (SO4 2- ), sulfurous acid (SO3 2- ), silicic acid (SiO3 2- ) etc.

[0096] Suitable salts for use in the pharmaceutical compositions described herein may contain a monovalent cation and a monovalent or polyvalent anion. Alternatively, salts for use in the pharmaceutical compositions described herein may contain a monovalent or polyvalent cation and a monovalent anion. Representative salts include, but are not limited to, potassium chloride (KCl), sodium chloride (NaCl), calcium chloride (CaCl2), magnesium chloride (MgCl2), magnesium sulfate (MgSO4), sodium bicarbonate (NaHCO3), ammonium sulfate ((NH4)2SO4), calcium carbonate (Ca2CO3), or combinations thereof.

[0097] The pharmaceutical compositions described herein include one or more suitable surface active agents, such as surfactants. A surfactant is a compound that reduces the surface tension (or interfacial tension) between two liquids, between a gas and a liquid, or between a liquid and a solid. Surfactants can act as detergents, wetting agents, emulsifiers, foaming agents, and dispersants. Suitable surfactants include, in particular, non-ionic agents, such as polyoxyethylene sorbitan (e.g., Tween™ 20, 40, 60, 80, or 85) and other sorbitans (e.g., Span™ 20, 40, 60, 80, or 85). Compositions with surfactants conveniently contain between 0.05% and 5% surfactant, and may be between 0.1% and 2.5%. Of course, other ingredients, such as mannitol or other pharma- ceutically acceptable vehicles, can be added if necessary.

[0098] Pharmaceutical compositions containing anti-GM-CSF as described herein may contain one or more amino acids. Exemplary amino acids include, but are not limited to, glycine, histidine, or arginine.

[0099] The pharmaceutical composition may also include one or more antioxidants. As used herein, an antioxidant is an agent that prevents or slows the oxidative degradation of an active ingredient contained in the composition. As used herein, an antioxidant may be a phenolic antioxidant (sometimes called a true antioxidant), a reducing agent, or a chelating agent. Phenolic antioxidants are sterically hindered phenols that react with free radicals to interrupt the chain reaction. Reducing agents are compounds that have a low redox potential and are therefore more easily oxidized than the drug they are intended to protect. Reducing agents remove oxygen from the medium, thus slowing or preventing the oxidation of the drug. Chelating agents are sometimes called oxidation collaborators. Metal ions, such as Co 2+ , Cu 2+ , Fe 3+ , Fe 2+ , and Mn 2+ shorten the induction period and increase the rate of oxidation. Trace amounts of these metal ions are frequently introduced into drug products during manufacture. Chelating agents have no such antioxidant activity, but enhance the action of phenolic antioxidants by reacting with catalytic metal ions and inactivating them.

[0100] The pharmaceutical compositions described herein may include a sugar derivative. As used herein, a sugar derivative includes sugars and organic compounds derived from sugars. In some examples, the sugar derivative may be a non-reducing sugar, a sugar alcohol, a polyol, a disaccharide, or a polysaccharide.

[0101] The pharmaceutical compositions used in the present methods may include pharma- ceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions. (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wilkins, Ed. KE Hoover). Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed and include buffers such as phosphate, citrate, and other organic acids; antioxidants including ascorbic acid and methionine; preservatives (e.g., octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl, or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins. for example, 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 dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, and sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or non-ionic surfactants such as TWEEN™, PLURONICS™, or polyethylene glycol (PEG).

[0102] In some examples, the pharmaceutical compositions described herein include liposomes containing antibodies (or encoding nucleic acids) that can be prepared by methods known in the art, such as those described in Epstein et al., Proc. Natl. Acad. Sci. USA 82:3688 (1985); Hwang et al., Proc. Natl. Acad. Sci. USA 77:4030 (1980); and U.S. Patent Nos. 4,485,045 and 4,544,545. Liposomes with enhanced circulation time are disclosed in U.S. Patent No. 5,013,556. Particularly useful liposomes can be generated by the reverse phase evaporation method with a liquid composition containing phosphatidylcholine, cholesterol and PEG-derivatized phosphatidylethanolamine (PEG-PE). Liposomes are filtered through filters of specified pore size to obtain liposomes of the desired diameter.

[0103] The antibodies, or encoding nucleic acids, can also be encapsulated in microcapsules, e.g., hydroxymethylcellulose or gelatin-microcapsules and polymethylmethacrylate microcapsules, respectively, prepared, for example, by coacervation techniques or by interfacial polymerization, in colloidal drug delivery systems (e.g., liposomes, albumin microparticles, microemulsions, nanoparticles and nanocapsules), or in microemulsions. Such techniques are known in the art, see, for example, Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000).

[0104] In other examples, the pharmaceutical compositions described herein can be formulated in sustained release format. Suitable examples of sustained release formulations include semipermeable matrices of solid hydrophobic polymers containing the antibody, where the matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly(2-hydroxyethyl methacrylate), or poly(vinyl alcohol), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and 7-ethyl-L20 glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, such as LUPRON DEPOT™ (injectable microparticles consisting of lactic acid-glycolic acid copolymer and leuprolide acetate), sucrose acetate isobutyrate, and poly-D-(−)-3-hydroxybutyric acid.

[0105] In other examples, the pharmaceutical compositions described herein can be formulated in sustained release formats, e.g., Probody™ or Conditionally Active Biologics™, that affect binding selectivity to tissues or tumors by implementing specific protease biotechnologies, e.g., by peptide masking of the antigen binding site of the antibody that allows selective protease cleavage by one or more proteases in the tumor microenvironment. Activation can be formulated reversibly in the normal microenvironment.

[0106] Pharmaceutical compositions used for in vivo administration must be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes. Therapeutic antibody compositions are generally placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.

[0107] The pharmaceutical compositions described herein may be in unit dosage form, for example, tablets, pills, capsules, powders, granules, solutions or suspensions, or suppositories for oral, parenteral or rectal administration, or for administration by inhalation or insufflation.

[0108] To prepare solid compositions such as tablets, the primary active ingredient can be mixed with pharmaceutical carriers, such as conventional tablet ingredients, such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate or gums, and other pharmaceutical diluents, such as water, to form a solid preformulation composition containing a homogenous mixture of the compound of the present invention, or a non-toxic pharmaceutically acceptable salt thereof. When these preformulation compositions are referred to as homogenous, it is meant that the active ingredient is evenly dispersed throughout the composition, and the composition can be readily subdivided into equally effective unit dosage forms, such as tablets, pills, and capsules. This solid preformulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 to about 500 mg of the active ingredient of the present invention. Tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form that provides the advantage of prolonged action. For example, the tablet or pill can include an inner dosage component and an outer dosage component, the latter being in the form of an envelope over the former. The two components are separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and acetyl cellulose.

[0109] Suitable emulsions can be prepared using commercially available fat emulsions such as Intralipid™, Liposyn™, Infonutrol™, Lipofundin™ and Lipiphysan™. The active ingredient can be dissolved in a premixed emulsion composition or can be dissolved in 10 oils (e.g., soybean oil, safflower oil, cottonseed oil, sesame oil, corn oil or almond oil) and an emulsion formed upon mixing of phospholipids (e.g., egg yolk phospholipids, soybean phospholipids or soybean lecithin) and water. It is understood that other ingredients, such as glycerol or glucose, can be added to adjust the osmolality of the emulsion. Suitable emulsions typically contain up to 20% oil, for example between 5-20% oil. The fat emulsion may contain lipid droplets between 0.1 and 1.0 μm, in particular between 0.1 and 0.5 μm, and have a pH in the range of 5.5 to 8.0. The emulsion composition may be one prepared by mixing the antibody with Intralipid™ or its components (soybean oil, egg yolk phospholipids, glycerol and water).

[0110] Pharmaceutical compositions for inhalation or insufflation include solutions and suspensions in pharma- ceutically acceptable aqueous or organic solvents, or combinations thereof, and powders. Liquid or solid compositions may contain suitable pharma- ceutically acceptable excipients as described above. In some embodiments, the compositions are administered by the oral or nasal respiratory route for local or systemic effect. Compositions, preferably in sterile pharma- ceutically acceptable solvents, may be nebulized by using a gas. Nebulized solutions may be inhaled directly from the nebulizing device, or the nebulizing device may be attached to a face mask, tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions may be administered, preferably orally or nasally, from a device that delivers the formulation in an appropriate manner.

[0111] IV. Therapeutic application Any of the antibodies described herein, as well as the encoding nucleic acid or nucleic acid set, vectors containing them, or host cells containing the vectors, are useful for treating GM-CSF-mediated disorders. As used herein, a GM-CSF-mediated disease refers to any medical condition associated with increased levels of GM-CSF or increased sensitivity to GM-CSF. Non-limiting examples of GM-CSF-mediated diseases are inflammatory diseases, autoimmune diseases, cancer, infectious diseases, or other disorders that require modulation of the immune response associated with GM-CSF.

[0112] To carry out the methods disclosed herein, an effective amount of the pharmaceutical composition described herein can be administered to a subject (e.g., a human) in need of treatment via a suitable route, such as intravenous administration, e.g., as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerebrospinal, subcutaneous, intra-articular, intrasynovial, intrathecal, oral, inhalation or topical routes. Commercially available nebulizers for liquid formulations, including jet nebulizers and ultrasonic nebulizers, are useful for administration. Liquid formulations can be directly nebulized, and lyophilized powders can be nebulized after reconstitution. Alternatively, the antibodies described herein can be aerosolized using fluorocarbon formulations and metered dose inhalers, and can be inhaled as lyophilized and crushed powders.

[0113] The subject treated by the methods described herein may be a mammal, more preferably a human. Mammals include, but are not limited to, livestock animals, sport animals, pets, primates, horses, dogs, cats, mice and rats. The human subject in need of treatment may be a human patient having, at risk of, or suspected of having an inflammatory disease, an autoimmune disease, cancer, an infectious disease, or other disorder that requires modulation of the immune response. A subject having a target disease or disorder can be identified by routine medical tests, such as laboratory tests, organ function tests, CT scans, or ultrasound. A subject suspected of having any such target disease / disorder will exhibit one or more symptoms of the disease / disorder. A subject at risk of a disease / disorder may be a subject having one or more risk factors for such disease / disorder.

[0114] The methods and compositions described herein can be used to treat inflammatory diseases.Non-limiting examples of inflammatory diseases are Kawasaki disease, chimeric antigen receptor T cell (CAR-T)-induced cytokine release syndrome, CAR-T-induced related encephalopathy, diffuse interstitial lung disease (DPLD), chronic obstructive pulmonary disease (COPD), aortic aneurysm, neuropathic pain, graft-versus-host disease (GVHD), glomerulonephritis, epididymitis, atherosclerosis, erythropoietin resistance, graft-versus-host disease, graft rejection, biliary cirrhosis, and alcohol-induced liver injury, including alcoholic cirrhosis.

[0115] As used herein, Kawasaki disease is a disease that involves the skin, mouth, and lymph nodes, most often affecting children under the age of five. The cause is unknown, but if symptoms are detected early, children with Kawasaki disease recover completely within a few days. If left untreated, it can lead to serious complications that can affect the heart.

[0116] The methods and compositions described herein can be used to treat autoimmune disease.Examples of autoimmune disease are rheumatoid arthritis, including juvenile rheumatoid arthritis, Kawasaki disease, spondyloarthropathy, including ankylosing spondylitis, inflammatory bowel disease, including ulcerative colitis and Crohn's disease, multiple sclerosis, Addison's disease, diabetes mellitus (type I), Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, hemolytic anemia, systemic lupus erythematosus (SLE), lupus nephritis, myasthenia gravis, pemphigus, psoriasis, plaque psoriasis, psoriatic arthritis, autoimmune hepatitis-induced liver damage, rheumatic fever, sarcoidosis, scleroderma, and Sjogren's syndrome.

[0117] As used herein, "rheumatoid arthritis" refers to a type of autoimmune disease characterized by synovial joint inflammation throughout the body. Early symptoms of the disease are joint pain, progressing to joint deformation or damage in body organs, such as blood vessels, heart, lungs, skin, and muscles.

[0118] The methods and compositions described herein can be used to treat cancer.Examples of autoimmune diseases are leukemia, gastric cancer, adenocarcinoma, mesothelioma, breast cancer, pancreatic ductal adenocarcinoma, colitis-associated colorectal cancer (CAC), or hypereosinophilic syndrome (HES).In some examples, leukemia can be juvenile myelomonocytic leukemia (JMML), chronic myelomonocytic leukemia (CMML), or chronic eosinophilic leukemia.

[0119] The methods and compositions described herein can be used to treat cancer. Examples of cancers that can be treated by the methods and compositions described herein include, but are not limited to, leukemia, multiple myeloma, gastric cancer, skin cancer, lung cancer, melanoma, kidney cancer, liver cancer, myeloma, prostate cancer, breast cancer, colorectal cancer, gastric cancer, pancreatic cancer, thyroid cancer, blood cancer, lymphoma, leukemia, skin cancer, ovarian cancer, bladder cancer, urothelial cancer, head and neck cancer, metastatic cancer, and all types of cancer diagnosed with high mutation burden. In certain embodiments, the cancer has high mutation burden. Subjects with or at risk of developing various cancers can be identified by routine medical procedures.

[0120] As used herein, "effective amount" refers to the amount of each active agent, either alone or in combination with one or more other active agents, required to confer a therapeutic effect on a subject. In some embodiments, the therapeutic effect is decreasing GM-CSF activity, increasing the number of activated effector T cells, and / or decreasing the number or activity of regulatory T cells.

[0121] Determining whether a certain amount of antibody has achieved a therapeutic effect is evident to one of ordinary skill in the art. As will be recognized by those of ordinary skill in the art, the effective amount will vary depending on the particular condition being treated, the severity of the condition, individual patient parameters including age, health, size, sex and weight, duration of treatment, nature of concurrent therapy (if any), the particular route of administration, and factors within the knowledge and experience of the medical practitioner. These factors are well known to those of ordinary skill in the art and can be addressed with only routine experimentation.

[0122] It is generally preferred that the maximum amount of each component or combination thereof be used, that is, the best safe dose according to sound medical judgment.

[0123] Empirical considerations, such as half-life, generally contribute to the determination of dosage. For example, antibodies compatible with the human immune system, such as humanized antibodies or fully human antibodies, can be used to extend the half-life of the antibody and prevent it from being attacked by the host's immune system. The frequency of administration can be determined and adjusted over the course of treatment, and is generally, but not necessarily, based on the treatment and / or suppression and / or alleviation and / or delay of the target disease / disorder. Alternatively, a sustained release formulation of the antibody may be appropriate. Various formulations and devices for achieving sustained release are well known in the art.

[0124] In one example, the dosage of the antibody described herein can be determined based on experiments in individuals who receive one or more doses of the antibody. Individuals are given increasing doses of the antagonist. To assess the effectiveness of the antagonist, the indication of the disease / disorder can be:

[0125] In general, for administration of any of the antibodies described herein, the initial candidate dosage may be about 2 mg / kg. For purposes of this disclosure, a typical daily, weekly, biweekly, or triweekly dosage may range anywhere from about 0.1 μg / kg to 3 μg / kg to 30 μg / kg to 100 μg / kg to 300 μg / kg to 0.6 mg / kg, 1 mg / kg, 3 mg / kg, to 10 mg / kg, to 30 mg / kg, to 100 mg / kg or more, depending on the factors mentioned above. For repeated administration over several days, weeks, months or more, depending on the condition, treatment is sustained until a desired suppression of symptoms occurs or until sufficient therapeutic levels are achieved to alleviate the target disease or disorder or its symptoms. A typical dosing regimen includes administering an initial dose of about 3 mg / kg every three weeks, followed by a maintenance dose of about 1 mg / kg of the antibody once every six weeks, or a maintenance dose of about 1 mg / kg every three weeks. However, other dosing regimens may be useful depending on the pharmacokinetic decay pattern the medical practitioner wishes to achieve. For example, dosing of 1 mg / kg once every 3 weeks in combination treatment with at least one additional immunotherapeutic agent is contemplated. In some embodiments, dosing in the range of about 3 μg / mg to 3 mg / kg (e.g., about 3 μg / mg, about 10 μg / mg, about 30 μg / mg, about 100 μg / mg, about 300 μg / mg, about 1 mg / kg, and about 3 mg / kg) may be used. In some embodiments, the dosing frequency is once every week, once every 2 weeks, once every 3 weeks, once every 4 weeks, once every 5 weeks, once every 6 weeks, once every 7 weeks, once every 8 weeks, once every 9 weeks, or once every 10 weeks, or once every month, once every 2 months, once every 3 months, or more. The progress of this treatment is easily monitored by conventional techniques and assays. The dosing regimen (including the antibody used) may vary over time.

[0126] In some embodiments, for a normal weight adult patient, a dose ranging from about 0.1 to 5.0 mg / kg may be administered. In some examples, the dose of the anti-GM-CSF antibodies described herein may be 10 mg / kg. The particular dosing regimen, i.e., dose, timing and repetition, will depend on the particular individual and that individual's medical history, as well as the characteristics of the individual agent (e.g., the half-life of the agent, and other considerations known in the art).

[0127] For purposes of this disclosure, appropriate dosages of the antibodies described herein will depend on the particular antibody, antibodies, and / or non-antibody peptides (or compositions thereof) used, the type and severity of the disease / disorder, whether the antibody is administered for prophylactic or therapeutic purposes, previous treatments, clinical history and patient response to antagonists, and the discretion of the attending physician. Typically, the clinician will administer the antibody until a dosage is reached that achieves the desired result. In some embodiments, the desired result is a reduction in tumor size, progression-free survival, and / or an increase in overall survival. Methods for determining whether a dosage will produce the desired result will be apparent to one of skill in the art. Administration of one or more antibodies may be continuous or intermittent, depending, for example, on the health status of the recipient, regardless of whether the purpose of administration is therapeutic or prophylactic, and other factors known to medical practitioners. Administration of the antibody may be essentially continuous over a preselected period of time, or may be a series of interval doses, for example, either before, during, or after the onset of the target disease or disorder.

[0128] As used herein, the term "treat" refers to the application or administration of a composition comprising one or more active agents to a subject having a target disease or disorder, a symptom of a disease / disorder, or a predisposition to a disease / disorder, for the purpose of curing, curing, palliating, ameliorating, altering, treating, improving, ameliorating, or affecting the disorder, a symptom of a disease, or a predisposition to a disease or disorder. Alleviating a target disease / disorder includes delaying the onset or progression of the disease, or reducing the severity of the disease.

[0129] Alleviating a disease does not necessarily require a curative outcome. As used herein, "delaying" the onset of a target disease or disorder means delaying, hindering, slowing, retarding, stabilizing, and / or postponing the progression of the disease. This delay can be of various lengths of time, depending on the history of the disease and / or the individual being treated. A method of "delaying" or alleviating the onset of a disease or delaying the onset of a disease is a method that reduces the likelihood of developing one or more symptoms of the disease in a given time frame and / or reduces the severity of symptoms in a given time frame, compared to not using the method. Such comparisons are typically based on clinical trials using a sufficient number of subjects to produce statistically significant results.

[0130] In some embodiments, the antibodies described herein are administered to a subject in need of treatment in an amount sufficient to inhibit the activity of the target antigen by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more) in vivo. In other embodiments, the antibodies are administered in an amount effective to reduce the activity level of the target antigen by at least 20% (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90% or more).

[0131] The pharmaceutical composition can be administered to a subject using conventional methods known to those skilled in the art of medicine, depending on the type of disease or site of disease to be treated. The composition can also be administered via other conventional routes, for example, parenterally, topically, orally, by inhalation spray, rectally, nasally, orally, vaginally, or via an implanted reservoir. As used herein, the term "parenteral" includes subcutaneous, intradermal, intravenous, intraperitoneal, intratumoral, intraarticular, intraarterial, intrasynovial, intrasternal, intrathecal, intralesional, and intracerebral injection or infusion techniques. Additionally, the pharmaceutical composition can be administered to a subject via an injectable depot administration route, such as using 1-month, 3-month, or 6-month depot injectable or biodegradable materials and methods. In some examples, the pharmaceutical composition is administered intraocularly or intravitreally.

[0132] Injectable compositions may contain a variety of carriers, such as vegetable oils, dimethylactamide, dimethylformamide, ethyl lactate, ethyl carbonate, isopropyl myristate, ethanol, and polyols (glycerol, propylene glycol, liquid polyethylene glycol, etc.). For intravenous injection, water-soluble antibodies may be administered by the drip method, whereby a pharmaceutical composition containing the antibody and a physiologically acceptable excipient is infused. Physiologically acceptable excipients may include, for example, 5% dextrose, 0.9% saline, Ringer's solution, or other suitable excipients. For intramuscular formulations, for example, a sterile formulation of a suitable soluble salt form of the antibody may be dissolved and administered in a pharmaceutical excipient, such as water for injection, 0.9% saline, or 5% glucose solution.

[0133] In one embodiment, the antibody is administered via a site-specific or targeted local delivery technique. Examples of site-specific or targeted local delivery techniques include various implantable depot sources or local delivery catheters of the antibody, such as infusion catheters, indwelling catheters, or needle catheters, artificial vascular grafts, adventitial wraps, shunts and stents, or other implantable devices, site-specific carriers, direct injection, or direct administration. See, e.g., PCT application WO 00 / 53211 and U.S. Patent No. 5,981,568.

[0134] Targeted delivery of therapeutic compositions containing antisense polynucleotides, expression vectors, or subgenomic polynucleotides can also be used.Receptor-mediated DNA delivery techniques are described, for example, in Findeis et al., Trends Biotechnol. (1993) 11:202; Chiou et al., Gene Therapeutics: Methods and Applications of Direct Gene Transfer (JA Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988) 263:621; Wu et al., J. Biol. Chem. (1994) 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA (1990) 87:3655; Wu et al., J. Biol. Chem. (1991) 266:338.

[0135] Therapeutic compositions containing polynucleotides (e.g., encoding an antibody described herein) are administered in gene therapy protocols in the range of about 100 ng to about 200 mg of DNA for local administration. In some embodiments, concentrations ranging from about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA or more can be used during gene therapy protocols.

[0136] The therapeutic polynucleotides and polypeptides described herein can be delivered using gene delivery vehicles. Gene delivery vehicles can be of viral or non-viral origin (see generally Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5:845; Connelly, Human Gene Therapy (1995) 1:185; and Kaplitt, Nature Genetics (1994) 6:148). Expression of such coding sequences can be induced using endogenous mammalian or heterologous promoters and / or enhancers. Expression of the coding sequences can be either constitutive or regulated.

[0137] Viral-based vectors for delivery of a desired polynucleotide and expression in a desired cell are well known in the art. Exemplary viral-based vehicles include, but are not limited to, recombinant retroviruses (see, e.g., PCT Applications WO 90 / 07936; WO 94 / 03622; WO 93 / 25698; WO 93 / 25234; WO 93 / 11230; WO 93 / 10218; WO 91 / 02805; U.S. Pat. Nos. 5,219,740 and 4,777,127; British Patent No. 2,200,651; and European Patent No. 0 345 242), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki Forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246), and Venezuelan equine encephalitis virus (ATCC VR-1247). VR-923; ATCC VR-1250; ATCC VR-1249; ATCC VR-532), and adeno-associated virus (AAV) vectors (see, e.g., PCT applications WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984 and WO 95 / 00655). Administration of DNA linked to killed adenovirus can also be used, as described in Curiel, Hum. Gene Ther. (1992) 3:147.

[0138] Non-viral delivery vehicles and methods can also be used, including, but not limited to, polycation-enriched DNA with or without linkage to killed adenovirus alone (see, e.g., Curiel, Hum. Gene Ther. (1992) 3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem. (1989) 264:16985); eukaryotic cell delivery vehicle cells (see, e.g., U.S. Pat. No. 5,814,482; PCT applications WO 95 / 07994; WO 96 / 17072; WO 95 / 30763; and WO 97 / 42338) and nuclear charge neutralization or fusion with cell membranes. Naked DNA can also be used. Exemplary naked DNA transfer methods are described in PCT application WO 90 / 11092 and U.S. Pat. No. 5,580,859. Liposomes that can serve as gene delivery vehicles are described in U.S. Patent No. 5,422,120; PCT applications WO 95 / 13796; WO 94 / 23697; WO 91 / 14445; and European Patent No. 0524968. Further approaches are described in Philip, Mol. Cell. Biol. (1994) 14:2411, and Woffendin, Proc. Natl. Acad. Sci. (1994) 91:1581.

[0139] The specific administration regimen used in the methods described herein, i.e., dosage, timing and repetition, depends on the specific subject and the subject's medical history. In some embodiments, more than one antibody, or a combination of an antibody and another suitable therapeutic agent, can be administered to a subject in need of treatment. Antibodies can also be used in combination with other agents, contributing to enhance and / or complement the effectiveness of the agent. The effectiveness of treatment for the target disease / disorder can be evaluated by methods well known in the art.

[0140] Anti-GM-CSF antibodies and treatment methods, including those described in this disclosure, can be utilized in combination with other types of treatments for the target disease or disorder disclosed herein. In this context, the term "in combination" means that the antibody composition and the therapeutic agent are given either simultaneously or sequentially. Examples include chemotherapy, immunotherapy (e.g., treatments including anti-inflammatory drugs, immunosuppressants, therapeutic antibodies, antibodies, CAR T cells, or cancer vaccines), surgical procedures, radiation, gene therapy, etc., or anti-infective treatments. Such treatments can be administered simultaneously or sequentially (in any order) with the treatments described in this disclosure.

[0141] For example, the combination therapy may include an anti-GM-CSF antibody and pharmaceutical composition described herein co-formulated and / or co-administered with at least one additional therapeutic agent. In one embodiment, the additional agent is a cancer chemotherapeutic agent, e.g., oxaliplatin, gemcitabine, docetaxel. In another embodiment, the additional agent may be a disease-modifying antirheumatic drug (DMARD), e.g., methotrexate, azathioprine, chloroquine, hydroxychloroquine, cyclosporine A, sulfasalazine for the treatment of RA. Such combination therapy may advantageously utilize therapeutic agents administered at lower dosages, thus preventing possible toxicities or complications associated with various monotherapies. Furthermore, the additional therapeutic agents disclosed herein may act in addition to or differently from the GM-CSF / STAT 5 pathway, thus enhancing and / or synergizing the effects of the anti-GM-CSF antibody.

[0142] When the antibody compositions described herein are used concomitantly with a second therapeutic agent, subtherapeutic doses of either the composition or the second agent, or subtherapeutic doses of both, can be used to treat a subject having or at risk of developing a disease or disorder associated with GM-CSF-mediated cell signaling. As used herein, a "subtherapeutic dose" refers to a dose that is less than the dose that would produce a therapeutic result in a subject when administered in the absence of one or more other agents. Thus, a subtherapeutic dose of an agent is one that would not produce the desired therapeutic result in a subject in the absence of administration of an anti-GM-CSF antibody described herein. Therapeutic doses of many agents in clinical use are well known in the pharmaceutical arts, and additional therapeutic doses can be determined by one of skill in the art without undue experimentation. Therapeutic doses are widely described in the literature, e.g., Remington's Pharmaceutical Sciences, 18th ed., 1990; and many other medical publications relied upon by physicians for guidance in the treatment of diseases and injuries. For additional useful agents, see also Physician's Desk Reference, 59.sup.th edition, (2005), Thomson PDR, Montvale NJ; Gennaro et al. Eds. Remington's The Science and Practice of Pharmacy 20th edition, (2000), Lippincott Williams and Wilkins, Baltimore Md.; Braunwald et al., Eds. Harrison's Principles of Internal Medicine, 15.sup.th edition, (2001), McGraw Hill, NY; Berkow et al., Eds. The Merck Manual of Diagnosis and Therapy, (1992), Merck Research Laboratories, Rahway NJ.

[0143] V. Diagnostic Applications Any of the anti-GM-CSF antibodies disclosed herein can also be used to detect the presence of GM-CSF (e.g., secreted GM-CSF) in vitro or in vivo. Results obtained from such detection methods can be used for diagnostic purposes (e.g., diagnosing diseases associated with secreted GM-CSF) or for scientific research purposes (e.g., identifying new cell types that secrete GM-CSF, studying the biological activity and / or regulation of secreted GM-CSF). For use in assays, e.g., diagnostic use, the anti-GM-CSF antibodies described herein can be conjugated to a detectable label (e.g., an imaging agent such as a contrast agent) to detect the presence of GM-CSF (e.g., secreted GM-CSF) either in vivo or in vitro. As used herein, "conjugated" or "bound" means that two entities are associated, preferably with sufficient affinity that a therapeutic / diagnostic benefit of the association between the two entities is manifested. The association between the two entities can be either direct or via a linker, e.g., a polymeric linker.

[0144] Conjugates or bonds can include covalent or non-covalent bonds, as well as other forms of association, such as the inclusion of one entity on or within the other, or of either or both entities on or within a third entity, such as a micelle.

[0145] In one example, the anti-GM-CSF antibodies described herein can be conjugated to a detectable label, which is a compound capable of emitting a detectable signal, either directly or indirectly, so that the aptamer can be detected, measured, and / or quantified in vitro or in vivo. Examples of such "detectable labels" are intended to include, but are not limited to, fluorescent labels, chemiluminescent labels, colorimetric labels, enzyme markers, radioisotopes, and affinity tags, such as biotin. Such labels can be conjugated to the aptamer directly or indirectly by conventional methods.

[0146] In some embodiments, the detectable label is an agent suitable for detecting GM-CSF-secreting cells in vitro and may be a radioactive molecule, a radiopharmaceutical, or an iron oxide particle. Suitable radioactive molecules for in vivo imaging include, but are not limited to, 122 I, 123 I, 124 I, 125 I, 131 I, 18 F, 75 Br, 76 Br, 77 Br, 211 At, 225 Ac, 177 Lu, 153 Sm, 186 Re, 188 Re, 67 Cu, 213 Bi, 212 Bi, 212 Pb, and 67 Representative radiopharmaceuticals suitable for in vivo imaging include: 111 Inoxyquinoline, 131 I sodium iodide, 99 mTc mebrophenin, and 99 mTc red blood cells, 123 I sodium iodide, 99 mTc exercise gym, 99 mTc macroaggregated albumin, 99 mTc-medronate, 99 mTc mertiatide, 99 mTc oxidronate, 99 mTc pentatate, 99 mTc pertechnetate, 99 mTc-sestamibi, 99 mTc sulfur colloid, 99 Examples include mTc tetrofosmin, thallium-201, or xenon-133.

[0147] The reporting agent may be a dye, such as a fluorophore, useful for detecting diseases mediated by GM-CSF-secreting cells in tissue samples.

[0148] To perform a diagnostic assay in vitro, an anti-GM-CSF antibody can be contacted with a sample suspected of containing GM-CSF, such as GM-CSF-secreting cells or soluble GM-CSF in a disease microenvironment. The antibody and sample can be incubated for a suitable period of time and under suitable conditions to allow binding of the antibody to the GM-CSF antigen. Such an interaction can then be detected by routine methods, such as ELISA, histological staining, or FACS.

[0149] To perform an in vivo diagnostic assay, a suitable amount of an anti-GM-CSF antibody conjugated to a label (e.g., an imaging or contrast agent) can be administered to a subject in need of testing. The presence of the labeled antibody can be detected based on the signal emitted by the label by routine methods.

[0150] To perform scientific research assays, anti-GM-CSF antibodies can be used to test the biological activity of GM-CSF, detect the presence of GM-CSF in cells, and / or control the effect of secreted GM-CSF. For example, a suitable amount of anti-GM-CSF can be contacted with a sample suspected of producing GM-CSF (e.g., a new cell type not previously identified as a GM-CSF producing cell). The cells are permeabilized before contacting with the anti-GM-CSF antibody. The antibody and sample can be incubated for a suitable period of time and under suitable conditions to allow binding of the antibody to the GM-CSF antigen. Such an interaction can then be detected by routine methods, such as ELISA, histological staining, or FACS.

[0151] VI. KITS FOR THERAPEUTIC AND DIAGNOSTIC APPLICATIONS The present disclosure also provides kits for the therapeutic or diagnostic applications disclosed herein. Such kits can include one or more containers containing an anti-GM-CSF antibody, such as any of those described herein.

[0152] In some embodiments, the kit may include instructions for use according to any of the methods described herein. The included instructions may include instructions for administration of an anti-GM-CSF antibody to treat, delay onset, or alleviate a target disease, such as those described herein. The kit may further include instructions for selecting an individual suitable for treatment based on identifying whether the individual has a target disease. In yet other embodiments, the instructions include instructions for administering the antibody to an individual at risk for a target disease.

[0153] Instructions associated with the use of anti-GM-CSF antibodies generally include information regarding dosages, dosing schedules, and routes of administration for the intended treatment. Containers may be unit dose, bulk packages (e.g., multi-dose packages) or sub-unit doses. Instructions accompanying kits of the invention are typically written instructions on a label or insert (e.g., a paper sheet included with the kit), although machine-readable instructions (e.g., instructions recorded on a magnetic or optical storage disk) are also acceptable.

[0154] The label or package insert indicates that the composition is used for treating, delaying the onset of, and / or ameliorating a disease or disorder that can be treated by modulating the immune response, such as an autoimmune disease. Instructions can be provided for practicing any of the methods described herein.

[0155] The kits of the invention are in suitable packaging, including but not limited to vials, bottles, jars, flexible packaging (e.g., enclosed in mylar or plastic bags), and the like.

[0156] Also contemplated are packages for use in combination with a particular device, such as an inhaler, a nasal administration device (e.g., a nebulizer), or an injection device, such as a minipump. The kit may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic needle). The container may also have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic needle). At least one active agent in the composition is an anti-GM-CSF antibody, such as those described herein.

[0157] The kit may optionally provide additional components, such as buffers and interpretive information. Typically, the kit includes a container and a label or package insert on or associated with the container. In some embodiments, the invention provides an article of manufacture comprising the contents of the kit described above.

[0158] Also provided herein is a kit for use in detecting secreted GM-CSF in a sample. Such a kit may include any of the anti-GM-CSF antibodies described herein. In some examples, the anti-GM-CSF antibody may be conjugated to a detectable label, such as those described herein. As used herein, "conjugated" or "bound" means that two entities are associated, preferably with sufficient affinity that the therapeutic / diagnostic benefit of the association between the two entities is manifested. The association between the two entities may be either direct or via a linker, such as a polymeric linker. Conjugation or binding may include covalent or non-covalent binding, as well as other forms of association, such as the inclusion of one entity on or within the other, or either or both entities on or within a third entity, such as a micelle.

[0159] Alternatively, or in addition, the kit may include a second antibody capable of binding to the anti-GM-CSF antibody. The kit may further include instructions for using the anti-GM-CSF antibody to detect secreted GM-CSF.

[0160] IV. General Techniques The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Molecular Cloning: A Laboratory Manual, second edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney, ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al. eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al. eds., 1994); Current Protocols in Immunology (JEColigan et al. eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., ed., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999); The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995). Without further elaboration, it is believed that one skilled in the art can, based on the preceding description, utilize the present invention to its fullest extent. The following specific embodiments are, therefore, to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way. All publications cited herein are incorporated by reference for the purpose or subject matter referenced herein.

[0161] Without further elaboration, it is believed that one skilled in the art can, based on the above description, utilize the present invention to its fullest extent. The following specific embodiments are therefore to be construed as merely illustrative, and not limitative of the remainder of the disclosure in any way. All publications cited herein are incorporated by reference for the purpose or subject matter referenced herein. EXAMPLES

[0162] Example 1 Identification and preparation of recombinant anti-GM-CSF antibodies (i) Isolation of human B cells secreting anti-GM-CSF autoantibodies and identification of anti-GM-CSF antibodies CD3 - / CD19 + B cells were isolated from human subjects by fluorescence-activated cell sorting (FACS). - / CD19 + B cells were further sorted into IgG-producing B cells and subsequently tested for granulocyte-macrophage colony-stimulating factor (GM-CSF) binding activity. B cells showing GM-CSF binding activity were individually sorted and expanded ex vivo (B cell clones). mRNA was extracted from each B cell clone and the heavy chain variable domain (V H ) and the light chain variable domain (V L ) was obtained by reverse transcription.

[0163] (ii) Construction of a vector set for expressing anti-GM-CSF antibody 1D2V H and V L The DNA fragments encoding 1D2 were cloned into a human IgG1κ expression vector containing a signal peptide and a human IgG1 constant region, respectively, by GeneArt® Seamless Cloning and Assembly Enzyme Mix (Invitrogen). Competent E. coli (Biotech) was transformed with 5 μl of each recombinant vector. Transformed E. coli colonies were screened by PCR. PCR products of the expected size (approximately 1,800 bp) were sequenced for confirmation. Plasmid DNA was isolated from 3 mL bacterial cultures of transformed E. coli grown at 37°C for 18 hours in Luria-Bertani broth containing 100 μg / mL ampicillin using QIAprep® Spin columns (Qiagen). To compare 1D2 with other anti-GM-CSF antibodies, the V-terminal region of AMG203 and MOR103, anti-GM-CSF antibodies currently being tested in phase 2 clinical trials, was used. H and VL The DNA fragment encoding was cloned into a human IgG1κ expression vector using the methods described above.

[0164] The variable weight (V H ) chain and variable light (V L The amino acid sequences of the 1D2 chains are listed in Table 3. The complementarity determining regions (CDRs) were identified according to Kabat (Wu, TT and Kabat, EA, 1970 J. Exp. Med. 132: 211-250) or the IMGT system (Lefranc M.-P. et al., 1999 Nucleic Acids Research, 27, 209-212). In Table 1, the CDRs following "K:" were identified according to the Kabat system and the CDRs following "I:" were identified according to the IMGT system. The antibody of the present disclosure is designated 1D2.

[0165] [Table 3]

[0166] AMG203 (namilumab) and MOR103 (otilimab) are therapeutic antibodies that bind to human GM-CSF known in the art, see, e.g., U.S. Patent No. 8,017,748 and U.S. Patent No. 7,867,495.

[0167] (iii) Recombinant antibody production and purification ExpiCHO-S™ cells (Thermo Scientific, A29133) were seeded in 125 mL flasks in 30 mL of ExpiCHO-S™ Expression Medium (Thermo Scientific, A29100-01) and cultured under standard conditions. During the exponential growth phase, 6×10 6ExpiCHO-S™ cells were transiently transfected with 20 μg of vector encoding 1D2, AMG203, or MOR103, respectively, using the ExpiFectamine™ CHO Transfection Kit (Thermo Scientific, A29129). 18-22 hours after transfection, ExpiFectamine™ CHO Enhancer and ExpiCHO™ Feed were added to the flasks. Cells were cultured for 8 days. The supernatant of each culture was centrifuged at 3000 rpm for 10 min to remove cell debris and then filtered through a 0.45 μm filter.

[0168] The recombinant antibodies in the supernatant were then precipitated with Protein A Sepharose® Fast Flow beads (GE Healthcare, 17-1279-03). 30 mL of the supernatant was mixed with 100 μL of Protein A Sepharose® Fast Flow beads, and the mixture was incubated at 4° C. for 2 hours with rotation in a 50 mL tube. The mixture was centrifuged at 3000 rpm for 10 minutes, and the supernatant was removed. The remaining beads were washed with PBS at a 20:1 PBS:beads (v / v) ratio. The precipitated antibodies were eluted with 0.1 M glycine (pH 2.5) at a 5:1 glycine buffer:beads (v / v) ratio. The eluate was collected in a tube containing 1 M Tris buffer (pH 9.0) at a volume 1 / 10 of the elution volume. The final eluate was dialyzed against PBS buffer. Each dialyzed eluate contains purified recombinant antibodies 1D2, AMG203, and MOR103, respectively.

[0169] Example 2 Characterization of recombinant anti-GM-CSF antibodies (i) Binding affinity of 1D2 to human GM-CSF Kinetic binding analysis of anti-GM-CSF antibodies to recombinant human GM-CSF (rhGM-CSF) was determined by surface plasmon resonance (SPR) using a Biacore 3000 instrument (GE Healthcare). Goat anti-human IgG (5 μg / mL) (SouthernBiotech) was stabilized in 10 mM acetate buffer, pH 4.5, and immobilized on an EDC / NHS preactivated carboxymethyl dextran 5 (CM5) sensor chip (GE Healthcare) by an amine coupling kit (GE Healthcare). HBS-EP buffer (HBS; 10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.005% surfactant Tween-20, pH 7.4) (GE Healthcare) was used as the running buffer. 1D2, AMG203, and MOR103 were each captured on a CM5 sensor chip by goat anti-human IgG. A CM5 sensor chip coated only with goat anti-human IgG was used as a reference chip. rhGM-CSF (Biolegend) was injected at different concentrations over the mAb-coated CM5 sensor chip and the reference chip. The flow rate was 30 μl / min. Three blank injections and two 12.5 nM rhGM-CSF injections were included to account for any drift and to check the reproducibility of the assay. Injection and dissociation times were 180 and 600 s. The data were fitted to a 1:1 model using the Biacore 3000 evaluation software. Figures 1A-1C show the binding curves obtained by passing different concentrations of rhGM-CSF protein over anti-GM-CSF mAb immobilized on a CM5 sensor chip. For 1D2, 0, 1.5625, 3.125, 6.25, 12.5, and 25 nM rhGM-CSF were used in the assay. For AMG203 and MOR103, 0, 6.25, 12.5, 25, 50, and 100 nM of rhGM-CSF were used in the assay. The antibody binding kinetic rate constants (Ka and Kd) and the equilibrium dissociation constant (KD=Kd / Ka) are shown in Table 4. The 1D2 antibody has a lower K Dvalues, indicating that the 1D2 antibody has a higher affinity for rhGM-CSF compared to AMG203 and MOR103.

[0170] [Table 4]

[0171] (ii) 1D2 inhibits GM-CSF-induced STAT5 phosphorylation in human PBMCs GM-CSF can induce STAT5 phosphorylation in human peripheral blood mononuclear cells (PBMCs), which can be inhibited by GM-CSF neutralizing antibodies. The ability of 1D2, AMG203, and MOR103 to inhibit GM-CSF-induced STAT5 phosphorylation in PBMCs was measured. Freshly isolated human PBMCs were stimulated with 10 ng / mL rhIL-3 or 10 ng / mL rhGM-CSF in the presence or absence of 2 μg / mL anti-GM-CSF antibodies as indicated. After stimulation, PBMCs were stained for cell surface CD14 with anti-CD14-FITC antibody (BD Bioscience). Cells were then fixed with fixation buffer and permeabilized with permIII buffer (BD Bioscience). After permeabilization, intracellular phospho-STAT5 was stained with anti-phosphorylated Stat5 (pY694) antibody. Cells were analyzed by flow cytometry for quantification of phosphorylated Stat5. Figure 2A-2B show flow cytometry analysis of inhibition of phospho-STAT5 signaling by anti-GM-CSF antibody. In the absence of anti-GM-CSF antibody, both rhGM-CSF and rhIL3 induced strong STAT5 phosphorylation (pY694) in human PBMC. Antibody 1D2 completely abolished rhGM-CSF-induced STAT5 phosphorylation in human PBMC. AMG203 and MOR103 also blocked rhGM-CSF-induced STAT5 phosphorylation in PBMC. In contrast, 1D2 did not neutralize the biological activity of IL-3, indicating that 1D2 specifically inhibited GM-CSF. The above results showed that 1D2 exhibited strong GM-CSF neutralizing activity in human PBMC. Table 11 shows the quantification of MFI in the flow cytometry chart.

[0172] [Table 5]

[0173] (iii) Inhibition of GM-CSF-dependent TF-1 cell proliferation by 1D2 GM-CSF can induce cell proliferation of TF-1, a human erythroleukemia cell line. The proliferation properties of GM-CSF on TF-1 cells can be inhibited by GM-CSF neutralizing antibodies. The ability of 1D2 to block the proliferation effect of rhGM-CSF on TF-1 cells was measured. TF-1 cells (ATCC, CRL-2003) were obtained from Bioresource Collection and Research Center (BCRC, Taiwan). For proliferation assays, TF-1 cells were grown at 1.5 × 10 per well in 100 μl of RPMI. 4 Cells were seeded in 96-well plates. Recombinant human GM-CSF (rhGM-CSF, 0.5 ng / mL, Biolegend) was pre-incubated with various amounts of 1D2 in 100 μl of RPMI for 25 min. The mixture was then added to the seeded TF-1 cells. TF-1 cell proliferation was measured 72 h after stimulation using the CellTiter-Glo assay (Promega). As shown in Figure 3, 1D2 inhibited rhGM-CSF-induced TF-1 proliferation in a dose-dependent manner. Antibody 1D2 was inhibited at subnanomolar concentrations, 1.436 × 10 -10 The results showed that 1D2 could effectively inhibit the biological activity of GM-CSF.

[0174] (iv) Inhibition of GM-CSF-induced CCL17 secretion in human PBMCs by 1D2 One of the various biological activities of GM-CSF is to drive the production of the CCL17 chemokine in human monocytes / macrophages, an important biomarker for the progression of GM-CSF-associated inflammatory diseases. CCL17 has been identified as a major downstream effector chemokine in various GM-CSF-associated inflammatory diseases. We investigated the effect of 1D2 in inhibiting GM-CSF-induced secretion of CCL17 in human PBMCs. Human PBMCs were isolated from healthy volunteers using the Ficoll-Paque method. 2 × 10 5Isolated human PBMCs were stimulated with 1 ng / mL rhGM-CSF pre-incubated with various amounts of antibody 1D2 for 24 hours. The supernatants of PBMC cultures were collected and CCL17 secretion by PBMCs was analyzed by ELISA. As shown in Figure 4, 1D2 potently inhibited CCL17 secretion in rhGM-CSF-stimulated PBMCs in a dose-dependent manner. The IC50 of 1D2 was 1.586 x 10 -10 M, which was similar to the concentration required for half-maximal inhibition of TF-1 cell proliferation. Taken together, these data indicate that 1D2 effectively neutralizes GM-CSF activity in a biological context highly relevant to GM-CSF-associated inflammatory diseases, such as Kawasaki disease.

[0175] Example 3 SDR mutant clones of antibody 1D2 (i) Identification of SDR mutant clones of antibody 1D2 with sufficient binding affinity to human GM-CSF Determining the residues that are important for the molecular activity of an antibody is a challenge that helps fine-tune the antibody's function in terms of binding affinity and epitope specificity. Some residues in the CDRs are essential for epitope binding affinity and / or specificity. Many computational methods have been developed to detect these functional residues, commonly known as "specificity determining residues" (SDRs). In this disclosure, the heavy and light chain sequences of antibody 1D2 were analyzed. A 3D model of antibody 1D2 was computationally constructed and analyzed. Based on the analysis, V H 7 amino acid residues and V L The following three amino acid residues were selected as SDRs. The residues are denoted as X1 to X for ease of reference: 10 Labeled as V H The SDRs of are: heavy chain D31 (HD31) (X1), HK32 (X2), HK54 (X3), HR100 (X4), HY102 (X5), HY103 (X6) and HQ105 (X7). L The SDRs are light chain V32 (LV32)(X8), LP94 (X9) and LV95 (X 10 The SDR was then mutated to other amino acids by site-directed mutagenesis to produce the mutant V HOr V L HEK293 cells were cloned using wild-type V L Mutant V paired with a vector encoding H A vector encoding a mutant V L Wild-type V H The cells were co-transfected with a vector encoding rhGM-CSF. The antibody clones were recovered from the culture medium. The binding affinity of each antibody clone to human GM-CSF was tested by ELISA. ELISA was performed according to the following procedure: ELISA plates were coated with 50 μl of 1 μg / mL rhGM-CSF. The plates were washed three times with 200 μl of PBST, followed by blocking with 1% BSA for 1 h at room temperature. The antibody clones to be tested were diluted 1:12000. 100 μl of diluted antibody was added to the cells at 100 μl / well. The plates were incubated for 1 h at room temperature. The plates were then washed and incubated with a 1:5000 dilution of goat anti-human secondary antibody conjugated with HRP for 1 h at room temperature. The ELISA assay was developed in 100 μl of developing buffer containing H2O2-Amplx Red per well for 5 min in the dark and the fluorescence intensity was read at Ex. 530 / Em. 590 nm with a cutoff of 570 nm. The Unit Binding Activity (UBA) of each sample was calculated as the fluorescence signal / antibody clone dilution factor. The percentage of binding affinity of the mutant antibody clones to GM-CSF was calculated as (UBA of each mutant clone / UBA of 1D2). * Calculated based on 100%.

[0176] A set of antibodies with SDR mutations that retained more than 40% of the binding affinity to human GM-CSF compared to 1D2 was identified, forming a library of antibody 1D2 variants capable of binding to GM-CSF. Table 5 shows the SDRs in the CDRs (SDRs are shown in bold) and the mutations that retained the binding affinity of the antibody to human GM-CSF. The binding affinities of the various mutated antibody clones are shown in Table 2.

[0177] [Table 6]

[0178] The binding affinities of several mutant antibody clones were tested by Biacore, as shown in Tables 6 and 7 below.

[0179] [Table 7]

[0180] [Table 8]

[0181] (ii) Inhibition of GM-CSF-dependent TF-1 cell proliferation by SDR mutant antibody clones of antibody 1D2 The ability of several selected SDR mutant antibody clones to inhibit GM-CSF-induced TF-1 cell proliferation was tested. Figures 5A and 5B show that the SDR mutant antibodies inhibited TF-1 cell proliferation but had high IC 50 The IC values ​​of the antibodies tested in Figures 5A and 5B are shown. 50 is shown in Table 8.

[0182] [Table 9]

[0183] Example 4 Removal of DD motif hindrance from antibody 1D2 heavy chain framework region As a lead antibody for further development, antibody 1D2 needs to be stable in production. The amino acid sequence of 1D2 was analyzed for interferences that would reduce antibody stability. A DD motif was identified in the heavy chain framework region at amino acid residues 89 and 90. Spontaneous isomerization of the Asp residue in the DD motif can occur during production, storage, or processing, resulting in loss of antibody function or potency. To eliminate interferences caused by the DD motif, the aspartic acid can be mutated to glutamic acid, which is functionally similar to aspartic acid. Thus, two isomerization site mutants (D89E and D90E mutations) of antibody 1D2 were constructed, as shown in FIG. 6A. The binding affinities of the D89E (ED mutation) and D90E (ED mutation) mutations to human GM-CSF were measured by Biacore. FIG. 6B-D show the binding curves of WT (1D2), ED mutation, and DE mutation. Table 9 shows the antibody binding kinetic rate constants (Ka and Kd) and equilibrium dissociation constants (KD=Kd / Ka) of 1D2, ED mutations, and DE mutations.

[0184] [Table 10]

[0185] The ability of the ED and DE mutant antibodies to block the proliferative effects of rhGM-CSF on TF-1 cells was measured and compared to the wild type antibody 1D2. For proliferation assays, TF-1 cells were plated at 1.5×10 per well in 100 μl of RPMI. 4 Cells were seeded in a 96-well plate. Recombinant human GM-CSF (rhGM-CSF, 0.5 ng / mL, Biolegend) was pre-incubated with various amounts of antibody 1D2, ED mutant antibody, or DE mutant antibody in 100 μl of RPMI for 25 min. The mixture was then added to TF-1 cells. TF-1 cell proliferation was measured 72 h after stimulation using the CelTiter-Glo assay (Promega). As shown in Figure 6E, antibodies D89E (ED) and D90E (DE) each increased 9.714 × 10 -11 M, 1.231×10 -10The wild-type antibody 1D2 exhibited potent inhibition of GM-CSF-induced TF-1 cell proliferation with an IC50 of 6.312 × 10 -11 The mutant antibody inhibited TF-1 cell proliferation with an IC50 of 1.0 M. The data show that the D89E and D90E mutations did not affect the biological function of the antibody. The mutant antibody is able to exert GM-CSF neutralizing effects at a level comparable to that of the wild-type antibody 1D2.

[0186] Another function of GM-CSF is to induce IL-8 production in U937 cells. U-937 cells were maintained in suspension culture in RPMI-1640 supplemented with 10% heat-inactivated FBS and 1% penicillin / streptomycin at 37°C in a humidified atmosphere of 5% CO2. U-937 cells were plated in 96-well plates (2 × 10 5 rhGM-CSF (1 ng / mL) was then pre-incubated with the indicated antibodies in 100 μl of RPMI assay medium for 25 min, followed by seeding at 2 × 10 5 U-937 cells were added to 100 μl of assay medium. After 24 h of incubation, culture supernatants were collected and IL-8 concentrations were measured using ELISA according to the manufacturer's instructions. Data were fitted for half-maximal inhibition of IL-8 production by nonlinear regression curve fitting using GraphPad Prism7. As shown in Figure 6F, the IC50 of wild-type antibody 1D2, ED mutant, and DE mutant for inhibition of IL-8 secretion in U937 cells was 5.94 × 10, respectively. -11 M, 5.10×10 -11 M, and 5.40 x 10 -11 M. This data indicates that the mutant antibodies are able to exert a GM-CSF neutralizing effect at a level comparable to that of the wild-type antibody 1D2.

[0187] Furthermore, the ability of the ED and DE mutant antibodies to inhibit GM-CSF-induced CCL17 production in PBMCs was measured and compared to the wild-type antibody 1D2. Human PBMCs were isolated from healthy volunteers using the Ficoll-Paque method. 2×10 5Isolated human PBMCs were stimulated with 1 ng / mL rhGM-CSF pre-incubated with various amounts of antibody 1D2, ED mutant antibody or DE mutant antibody and incubated for 24 hours. The supernatants of PBMC cultures were collected and CCL17 secretion by PBMCs was analyzed by ELISA. As shown in Figure 6G, the ED and DE mutant antibodies potently inhibited CCL17 secretion in PBMCs stimulated with rhGM-CSF in a dose-dependent manner. The IC of wild-type antibody 1D2 and the ED mutants in inhibiting CCL17 secretion from PBMCs was 0.01 mg / mL. 50 are 7.14×10 -11 M and 5.43 x 10 -11 M. This data indicates that the ED and DE mutant antibodies are able to exert GM-CSF neutralizing effects at levels comparable to that of the wild-type antibody 1D2.

[0188] The heterogeneity of 1D2, ED and DE mutant antibodies was then analyzed by hydrophobic interaction chromatography (HIC). HIC separates proteins according to the difference in hydrophobicity of their surfaces by exploiting the reversible interaction between proteins and the hydrophobic surface of the HIC resin. The high performance liquid chromatography (UPLC) system consisted of an Acquity UPLC H-class bio system with a quaternary UPLC pump and a quaternary solvent manager (QSM), an automatic sample injector (SM) with a 50 μl sample loop and a photodiode array detector (Waters). Separation was achieved by injecting 10 μg of each sample and using a TSKgel Butyl NPR (TOSOH, 4.6 mm ID×3.5 cm) at 35° C. The mobile phase consisted of two buffers: Buffer A (2 M ammonium sulfate in 25 mM sodium phosphate, pH 7.0) and Buffer B (25 mM sodium phosphate, pH 7.0). The flow rate was 0.5 mL / min and the total running time was approximately 25 min. The absorbance was monitored at 214 and 280 nm. In addition, UV spectra were recorded from 400-210 nm using a diode array detector. The chromatograms were processed and calculated by Empower software (v3.0, Waters). As shown in Figure 6H, 1D2, ED and DE mutant antibodies yielded peaks at similar retention times compared to a different antibody, Herceptin, indicating that the ED and DE mutant antibodies have similar surface hydrophobicity to the wild-type antibody 1D2. Furthermore, the ED mutation had a reduced level of shoulder species compared to 1D2 and the DE mutation, indicating that the purity of the ED mutant antibody is higher. The results indicate that the ED mutation has improved biophysical properties for manufacturing and / or storage.

[0189] Furthermore, the aggregation state of purified 1D2, ED and DE mutant antibodies was analyzed by dynamic light scattering (DLS). DLS analysis was measured using a DynaPro NanoStar (Wyatt) with a laser wavelength of 658 nm. Antibodies were diluted to 2 mg / mL in PBS and filtered through a 0.02 μm pore size filter (alumina-based membrane) before measurement. 10 μl samples were transferred to disposable cuvettes and analyzed at 25°C. DYNAMICS software was used for scheduling and automatic acquisition with an acquisition time of 5 seconds and 10 acquisitions per measurement of each sample. The instruments are equipped with an integration algorithm that automatically determines the laser power (%) and attenuation level (%) of each measurement in real time. The scattered light data was fitted (Dynamics software; Wyatt) assuming the scattered light as a Rayleigh sphere. "Radius" is the diameter of the protein particle, "mass percentage" is the amount of each size fraction in percentage, and "PD percentage" is a measure of antibody polydispersity. The higher the PD percentage, the more polydisperse the sample is. As shown in Table 10 (Table 11) and Figure 6I, the ED mutation has a reduced polydispersity percentage compared to the 1D2 and DE mutations. This result indicates that the ED mutation is less prone to aggregation than the wild type 1D2 and the DE mutation. The ED mutation may have improved biophysical properties during development.

[0190] [Table 11]

[0191] Taken together, the ED and DE mutant antibodies have similar biological functions as the wild-type 1D2 antibody, while the D89E and D90E mutations increase the stability of the antibody for production and shelf life.

[0192] Example 5 Binding specificity of 1D2, ED and DE mutant antibodies Human and mouse GM-CSF share approximately 56% homology and are species-specific. Rhesus GM-CSF shares 96.5% homology with human GM-CSF (Hutchinson et al., J Med Virol 2001, 65:561-565, Genbank Accession No. AY007376). The ability of 1D2, ED and DE mutant antibodies to bind to mouse or rhesus GM-CSF was measured by ELISA. ELISA was performed as follows. Briefly, GM-CSF protein was coated onto an ELISA-absorbed plate and then blocked with 1% BSA in PBS. Subsequently, monoclonal antibody 1D2, 1D2-D89E (ED) or 1D2-D90E (DE) was added to the plate and incubated for 1 hour at room temperature. After incubation, the plate was washed three times with TBST and incubated with HRP-conjugated anti-human kappa light chain secondary antibody. The plates were washed three times, TMB peroxidase substrate was added, and the reaction was stopped by adding 1 N sulfuric acid. Finally, the absorbance at 450 nM was measured using an ELISA reader and plotted using GraphPad Prism7 software to fit the data points to a nonlinear regression curve.

[0193] Figure 7A shows that antibody 1D2 can bind with high affinity to human GM-CSF and rhesus GM-CSF, but cannot bind to mouse GM-CSF. Furthermore, the binding ability of the ED and DE mutant antibodies to human and rhesus GM-CSF was measured by ELISA. Figures 7B-7C show that the ED and DE mutant antibodies can bind to human and rhesus GM-CSF, similar to wild-type antibody 1D2.

[0194] Other embodiments All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0195] From the above description, those skilled in the art can easily ascertain the essential features of the present invention, and can make various changes and modifications to the present invention to suit various applications and conditions without departing from the spirit and scope thereof. Accordingly, other embodiments are within the scope of the appended claims.

[0196] Equivalent While several embodiments of the present invention are described and illustrated herein, those skilled in the art will readily envision various other means and / or structures for performing the functions and / or results and / or obtaining one or more of the advantages described herein, and each such variation and / or modification is considered to be within the scope of the embodiments of the present invention described herein. More generally, those skilled in the art will readily recognize that all parameters, dimensions, materials, and shapes described herein are meant to be exemplary, and that the actual parameters, dimensions, materials, and / or shapes will depend on the particular application or applications in which the teachings of the present invention are used. Those skilled in the art will be able to recognize and ascertain, using no more than routine experimentation, many equivalents to the specific embodiments of the present invention described herein. Thus, it will be understood that the foregoing embodiments are presented by way of example only, and that within the scope of the appended claims and their equivalents, the embodiments of the present invention may be practiced without otherwise being specifically described and claimed. The inventive embodiments of the present disclosure are directed to the individual properties, systems, documents, materials, kits, and / or methods described herein. Furthermore, any combination of two or more of such properties, systems, documents, materials, kits, and / or methods is within the inventive scope of this disclosure, provided that such properties, systems, documents, materials, kits, and / or methods are not mutually inconsistent.

[0197] As provided and used herein, all definitions should be understood to control over dictionary definitions, definitions in documents incorporated by reference, and / or ordinary meanings of the defined terms.

[0198] All references, patents, and patent applications disclosed herein are incorporated by reference with respect to the subject matter cited, which in some cases may include the entire document.

[0199] The indefinite articles "a" and "an," as used herein in the specification and the claims, unless clearly indicated to the contrary, should be understood to mean "at least one."

[0200] The phrase "and / or" as used herein in the specification and claims should be understood to mean "either or both" of the conjoined elements, i.e., elements that are conjunctive in some cases and disjunctive in other cases. Multiple elements listed with "and / or" should be interpreted in the same manner, i.e., "one or more" conjoined elements. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, either related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to "A and / or B," when used in conjunction with open-ended language such as "comprising," may refer in one embodiment to only A (optionally including elements other than B); in another embodiment to only B (optionally including elements other than A); in yet another embodiment to both A and B (optionally including other elements); and so forth.

[0201] As used herein in the specification and claims, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" is to be interpreted as being inclusive, i.e., including at least one, including more than one, of some or the elements of the list, and optionally additional unlisted items. Only terms clearly indicating the contrary, such as "only one of" or "exactly one of," or when used in the claims, "consisting of" refers to the inclusion of exactly one element of some or the elements of the list. In general, the term "or," as used herein, will only be interpreted as indicating exclusive alternatives (i.e., "one or the other, but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of." "Consisting essentially of," when used in the claims, has its ordinary meaning as used in the field of patent law.

[0202] As used herein in the specification and claims, the phrase "at least one," with reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements of the list, but not necessarily including at least one of each and every element specifically listed in the list of elements, and not excluding any combination of elements of the list of elements. This definition also allows for elements, in some cases, to be present other than the specifically identified elements in the list of elements to which the phrase "at least one" refers, either related or unrelated to those specifically identified elements. Thus, as a non-limiting example, "at least one of A and B" (or, equivalently, "at least one of A or B" or, equivalently, "at least one of A and / or B") can refer, in one embodiment, to at least one, and optionally more than one, A in the absence of B (optionally including elements other than B); in another embodiment, to at least one, and optionally more than one, B in the absence of A (optionally including elements other than A); in yet another embodiment, to at least one, and optionally more than one, A, and at least one, and optionally more than one, B (optionally including other elements); etc.

[0203] It should also be understood that, unless expressly stated to the contrary, in any method claimed herein that includes two or more steps or actions, the order of the method steps or actions is not necessarily limited to the order in which the method steps or actions are recited.

Claims

1. 1. An isolated antibody that binds to granulocyte-macrophage colony-stimulating factor (GM-CSF), HC CDR1 of SEQ ID NO:5; HC CDR2 of SEQ ID NO:6; HC CDR3 of SEQ ID NO:7; LC CDR1 of SEQ ID NO:8; LC CDR2 of SEQ ID NO:9; and LC CDR3 of SEQ ID NO: 10 V comprising the amino acid sequence of SEQ ID NO: 1, 3 or 4 H and V comprising the amino acid sequence of SEQ ID NO:2 L 1. An isolated antibody comprising:

2. 2. The isolated antibody of claim 1, which specifically binds to human GM-CSF.

3. 2. The isolated antibody of claim 1, which cross-reacts with human GM-CSF and non-human GM-CSF.

4. 4. The isolated antibody of claim 3, wherein the non-human GM-CSF is Rhesus macaque GM-CSF.

5. An isolated antibody described in any one of claims 1 to 4, which is a humanized antibody.

6. 6. The isolated antibody of any one of claims 1 to 5, which is a full length antibody or an antigen-binding fragment thereof.

7. 7. The isolated antibody of claim 6, which is a full-length antibody that is an IgG molecule.

8. 8. The isolated antibody of any one of claims 1 to 7, conjugated to a detectable label.

9. 9. A pharmaceutical composition comprising an isolated antibody according to any one of claims 1 to 8 and a pharma- ceutically acceptable carrier.

10. 10. The pharmaceutical composition of claim 9, wherein the pharma- ceutically acceptable carrier comprises a buffer, a surfactant, a salt, an amino acid, an antioxidant, a sugar derivative, or a combination thereof.

11. 11. The pharmaceutical composition of claim 10, wherein the sugar derivative is a non-reducing sugar, a sugar alcohol, a polyol, a disaccharide, or a polysaccharide.

12. A nucleic acid or set of nucleic acids that together encodes the isolated antibody of any one of claims 1 to 8.

13. 13. The nucleic acid or nucleic acid set according to claim 12, which is a vector or a vector set.

14. The nucleic acid or nucleic acid set according to claim 13, wherein the vector or vector set is an expression vector or expression vector set.

15. A host cell comprising a vector or a vector set as defined in claim 13 or 14.

16. 16. The host cell of claim 15, selected from the group consisting of a bacterial cell, a yeast cell, an insect cell, a plant cell, and a mammalian cell.

17. 1. A method for producing an antibody that binds human GM-CSF, comprising: (i) culturing a host cell according to claim 15 or claim 16 under conditions allowing expression of an antibody that binds human GM-CSF; (ii) harvesting the cultured host cells or culture medium to collect antibodies that bind to human GM-CSF; The method includes:

18. The method of claim 17, further comprising the step of purifying the antibody that binds to human GM-CSF.

19. 12. A pharmaceutical composition according to any one of claims 9 to 11 for modulating an immune response mediated by GM-CSF in a subject.

20. 20. The composition of claim 19, wherein the subject is a human patient having, suspected of having, or at risk of having a disease associated with GM-CSF.

21. The composition of claim 20, wherein the disease associated with GM-CSF is an inflammatory disease, an autoimmune disease, or cancer.

22. 22. The composition of claim 21, wherein the disease is an autoimmune disease including rheumatoid arthritis, juvenile rheumatoid arthritis, spondyloarthropathy, ankylosing spondylitis, multiple sclerosis, psoriasis, plaque psoriasis, acute gouty arthritis, or osteoarthritis.

23. 22. The composition of claim 21, wherein the disease is an inflammatory disease, including Kawasaki disease, chimeric antigen receptor T cell (CAR-T)-induced cytokine release syndrome, CAR-T-induced associated encephalopathy, diffuse interstitial lung disease (DPLD), chronic obstructive pulmonary disease (COPD), aortic aneurysm, neuropathic pain, or graft-versus-host disease (GVHD).

24. 22. The composition of claim 21, wherein the disease is cancer, including leukemia, gastric cancer, adenocarcinoma, mesothelioma, breast cancer, pancreatic ductal adenocarcinoma, colorectal cancer (CAC), or hypereosinophilic syndrome (HES).

25. 25. The composition of claim 24, wherein the leukemia comprises juvenile myelomonocytic leukemia (JMML), chronic myelomonocytic leukemia (CMML), or chronic eosinophilic leukemia.

26. 26. The composition of any one of claims 19 to 25, wherein the subject has undergone or is undergoing further treatment for the disease.

27. 1. A method for detecting the presence of GM-CSF, comprising: (i) contacting in vitro a biological sample suspected of containing GM-CSF with an isolated antibody according to any one of claims 1 to 8; and (ii) measuring the binding of the antibody to GM-CSF in the sample; The method includes:

28. 28. The method of claim 27, wherein the biological sample is obtained from a human subject suspected of having or at risk of having a disease associated with GM-CSF.

29. 29. The method of claim 27 or claim 28, wherein the contacting step is carried out by administering to the subject an effective amount of an anti-GM-CSF antibody.

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