Isolated antibody or fragment thereof, composition, transgenic microorganism, use of an isolated antibody or fragment thereof, and in vitro method for detecting GM-CSF expression in a sample.

Abstract

Anti-GmCsf antibodies or fragments thereof, including humanized antibodies and fragments, are provided. Uses of antibodies and fragments for therapeutic, diagnostic, and prognostic purposes are also provided. Therapeutic uses of antibodies and fragments, for example, include the treatment of inflammatory and autoimmune diseases and disorders.

Description

75 Isolated antibody or fragment thereof, composition, transgenic microorganism, use of an isolated antibody or fragment thereof, and in vitro method for detecting GM-CSF expression in a sample. FUNDAMENTALS

[001] Granulocyte-macrophage colony-stimulating factor (GMCSF or GM-CSF), also known as colony-stimulating factor 2 (CSF2), is a monomeric glycoprotein secreted by macrophages, T cells, mast cells, NK cells, endothelial cells, and fibroblasts that functions as a cytokine. Naturally occurring pharmaceutical analogs of GM-CSF are also referred to as sargramostim and molgramostim. Unlike granulocyte colony-stimulating factor, which specifically promotes neutrophil proliferation and maturation, GM-CSF affects more cell types, especially macrophages and eosinophils.

[002] GM-CSF is a monomeric glycoprotein that functions as a cytokine. GM-CSF stimulates stem cells to produce granulocytes (neutrophils, eosinophils, and basophils) and monocytes. Monocytes leave the circulation and migrate to the tissue, after which they mature into macrophages and dendritic cells. Thus, it is part of the immune / inflammatory cascade, whereby the activation of a small number of macrophages can rapidly lead to an increase in their numbers, a crucial process for fighting infection. GM-CSF also has some effects on mature immune system cells. These include, for example, inhibiting neutrophil migration and causing an alteration in the receptors expressed on the cell surface.

[003] GM-CSF signals, via signal transducer and transcription activator, STAT5. In macrophages, it has also been shown to signal via STAT3. The cytokine activates macrophages to inhibit fungal survival. It induces deprivation of intracellular free zinc and increases the production of reactive oxygen species that culminate in zinc deprivation by fungi and Petition 870260079734, dated 07 / 08 / 2026, page 11 / 97 / 75 toxicity. Thus, GM-CSF facilitates the development of the immune system and promotes defense against infections. GM-CSF also plays a role in embryonic development by functioning as an embryocin produced by the reproductive tract.

[004] GM-CSF is manufactured using recombinant DNA technology and is marketed as a therapeutic protein called molgramostim or, when the protein is expressed in yeast cells, sargramostim. It is used as a drug to stimulate the production of white blood cells and thus prevent neutropenia after chemotherapy. GM-CSF has also been evaluated in clinical trials for its potential as a vaccine adjuvant in HIV-infected patients.

[005] GM-CSF inhibition, in contrast, may be useful for treating diseases such as inflammatory diseases and autoimmune disorders, including rheumatoid arthritis (OA), multiple sclerosis (MS), and plaque psoriasis. GM-CSF inhibition may also be useful for cancer treatment. SUMMARY

[006] The present description provides an anti-GM-CSF antibody having high binding affinity for human GM-CSF proteins and having potent activities inhibiting the binding of GM-CSF to its receptor. These anti-GM-CSF antibodies are useful for therapeutic purposes such as the treatment of various types of inflammatory diseases, autoimmune disorders and cancers, and can also be used for diagnostic and prognostic purposes.

[007] The present description, in one embodiment, provides an isolated antibody or fragment thereof, wherein the antibody or fragment thereof has specificity for a human GM-CSF protein and comprises a VH CDR1 of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, a VH CDR3 of SEQ ID NO: 3, a VL CDR1 of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 5, and a VL CDR3 of SEQ ID NO: 6. In some embodiments, the antibody or fragment thereof Petition 870260079734, dated 07 / 08 / 2026, p. 12 / 97 / 75 additionally comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. In some embodiments, the light chain constant region is a kappa or lambda chain constant region. In some embodiments, the antibody or fragment thereof is of an IgG, IgM, IgA, IgE, or IgD isotype. In some embodiments, the isotype is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody or fragment thereof is a chimeric antibody, a humanized antibody, or a fully human antibody.

[008] In some embodiments, the antibody or fragment thereof is a humanized antibody. In some embodiments, the antibody or fragment thereof comprises a variable heavy chain region comprising one or more amino acid residues selected from the group consisting of: (a) Glu at position 1, (b) Arg at position 98, (c) Ser at position 72, (d) Ala at position 68, (e) Leu at position 70, (f) Ile at position 48, (g) Asp at position 26, and (h) Leu at position 29, according to Kabat numbering, and combinations thereof.

[009] In some embodiments, the variable heavy chain region comprises a fragment of DYTLT (SEQ ID NO: 42) or GYTFT (SEQ ID NO: 43) starting at position 26 according to Kabat numbering.

[0010] In some embodiments, the antibody or fragment thereof comprises a variable light chain region comprising one or more amino acid residues selected from the group consisting of: (a) Ala at position 46, (b) Asp at position 60, (c) Asp at position 70, (d) Ser at position 43, and (f) Phe at position 87, according to Kabat numbering, and combinations thereof.

[0011] In some embodiments, the antibody or fragment thereof comprises a variable heavy chain region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8-17, or a peptide having at least 90% sequence identity. Petition 870260079734, dated 07 / 08 / 2026, page 13 / 97 / 75 for an amino acid sequence selected from the group consisting of SEQ ID NO: 8-17. In some embodiments, the variable heavy chain region comprises the amino acid sequence of SEQ ID NO: 11, 14 or 17.

[0012] In some embodiments, the antibody or fragment thereof comprises a variable light chain region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 19-22, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 19-22. In some embodiments, the variable light chain region comprises the amino acid sequence of SEQ ID NO: 19 or 22.

[0013] In some embodiments, the variable heavy chain region comprises the amino acid sequence of SEQ ID NO: 14 and the variable light chain region comprises the amino acid sequence of SEQ ID NO: 22. In some embodiments, the antibody or fragment thereof is a bispecific antibody or a variable single-chain fragment.

[0014] The present description, in one embodiment, provides an isolated antibody or fragment thereof, wherein the antibody or fragment thereof has specificity for a human GM-CSF protein and comprises a VH CDR1 of SEQ ID NO: 23, a VH CDR2 of SEQ ID NO: 24, a VH CDR3 of SEQ ID NO: 25, a VL CDR1 of SEQ ID NO: 26, a VL CDR2 of SEQ ID NO: 27, and a VL CDR3 of SEQ ID NO: 28. In some embodiments, the antibody or fragment thereof further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof. In some embodiments, the light chain constant region is a kappa or lambda chain constant region. In some embodiments, the antibody or a fragment thereof is an IgG, IgM, IgA, IgE, or IgD isotype. In some embodiments, the isotype is IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody or a fragment thereof is a chimeric antibody. Petition 870260079734, dated 07 / 08 / 2026, page 14 / 97 / 75 a humanized antibody, or a completely human antibody.

[0015] In some embodiments, the antibody or fragment thereof is a humanized antibody. In some embodiments, the antibody or fragment thereof comprises a variable heavy chain region comprising one or more amino acid residues selected from the group consisting of E1, R84, Y27, I28, I48, T68, L70, or T30, according to Kabat numbering, and combinations thereof.

[0016] In some embodiments, the variable heavy chain region comprises a fragment of GYIFT (SEQ ID NO: 44), GYIFS (SEQ ID NO: 45), or GGTFS (SEQ ID NO: 46) starting at position 26 according to Kabat numbering.

[0017] In some embodiments, the antibody or fragment thereof comprises a variable light chain region comprising one or more amino acid residues selected from the group consisting of: V48, D57, Q70 or S43, according to Kabat numbering, and combinations thereof.

[0018] In some embodiments, the antibody or fragment thereof comprises a variable heavy chain region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 29-35, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 29-35. In some embodiments, the variable heavy chain region comprises the amino acid sequence of SEQ ID NO: 34 or 35.

[0019] In some embodiments, the antibody or fragment thereof comprises a variable light chain region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 3641, or a peptide having at least 90% sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO: 36-41. In some embodiments, the variable light chain region Petition 870260079734, dated 07 / 08 / 2026, page 15 / 97 / 75, includes the amino acid sequence of SEQ ID NO: 38 or 39.

[0020] In some embodiments, the variable heavy chain region comprises the amino acid sequence of SEQ ID NO: 34 and the variable light chain region comprises the amino acid sequence of SEQ ID NO: 38. In some embodiments, the variable heavy chain region comprises the amino acid sequence of SEQ ID NO: 35 and the variable light chain region comprises the amino acid sequence of SEQ ID NO: 39. In some embodiments, the antibody or fragment thereof is a bispecific antibody or a single-chain variable fragment.

[0021] In one embodiment, a composition is provided comprising the antibody or fragment thereof of the present description and a pharmaceutically acceptable carrier. Also provided, in one embodiment, is an isolated cell comprising one or more polynucleotides encoding the antibody or fragment thereof of the present description.

[0022] In one embodiment, the present description provides a method for treating an inflammatory or autoimmune disease or condition in a patient in need thereof, comprising administering to the patient the antibody or a fragment thereof, or a composition of the present description. Uses of the antibody or a fragment thereof, or a composition for the manufacture of a medicament for treating an inflammatory or autoimmune disease or condition are also provided.

[0023] In some modalities, the inflammatory disease or condition is selected from the group consisting of Alzheimer's disease, Addison's disease, atherosclerosis, ankylosing spondylitis, arthritis, osteoarthritis (OA), rheumatoid arthritis (RA), psoriatic arthritis (PA), ankylosing spondylitis, asthma, atherosclerosis, chronic obstructive pulmonary disease (COPD), Crohn's disease, colitis, dermatitis, diverticulitis, fibromyalgia, hepatitis, irritable bowel syndrome (IBS), systemic lupus erythematosus (SLE), nephritis, Parkinson's disease (PD), vasculitis, and ulcerative colitis. Petition 870260079734, dated 07 / 08 / 2026, page 16 / 97 / 75

[0024] Also provided, in one embodiment, is a method for reducing or relieving pain in a patient in need thereof, comprising administering to the patient the antibody or fragment thereof of the present description.

[0025] In some modalities, the autoimmune disease or condition is selected from the group consisting of alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes (type 1), celiac disease, autoimmune juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, autoimmune myocarditis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjögren's syndrome, systemic lupus erythematosus, autoimmune thyroiditis, Hashimoto's thyroiditis, autoimmune uveitis, vitiligo, and granulomatosis with polyangiitis (of Wegener).

[0026] A method is also provided for detecting GMCSF expression in a sample, comprising contacting the sample with the antibody or a fragment thereof under conditions such that the antibody or fragment thereof binds to GM-CSF, and detecting the binding which indicates GMCSF expression in the sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 shows confirmatory ELISA linkage test results for selecting primary hybridoma clones for subcloning.

[0028] Figure 2 shows confirmatory ELISA linkage test results for selecting primary hybridoma clones for subcloning.

[0029] Figure 3 shows dose-dependent binding of test antibodies to human GM-CSF. Petition 870260079734, dated 07 / 08 / 2026, page 17 / 97 / 75

[0030] Figure 4 shows graphically the binding kinetics of antibodies with recombinant human GM-CSF.

[0031] Figure 5 shows dose-dependent inhibition of GMCSF binding to the GM-CSF alpha receptor by antibodies.

[0032] Figure 6 shows that the antibodies significantly decreased the level of GM-CSF-induced pSTAT5 activation.

[0033] Figure 7 shows the GMCSF-dependent inhibition of TF-1 proliferation by antibodies.

[0034] Figure 8 shows that all humanized antibodies demonstrated potent binding power against human GM-CSF.

[0035] Figure 9 shows that some humanized antibodies exhibited stronger inhibition of TF-1 proliferation.

[0036] Figure 10 shows that the tested antibodies effectively blocked pSTAT5 signaling.

[0037] Figure 11 shows dose-dependent binding of antibodies to GM-CSF rhesus.

[0038] Figure 12 shows the pharmacokinetic parameters of Hu23F4-27 in a graph. DETAILED DESCRIPTION Definitions

[0039] It should be noted that the term “an” entity refers to one or more of that entity; for example, “an antibody” is understood as representing one or more antibodies. As such, the terms “an” (or “an”), “one or more,” and “at least one” may be used interchangeably herein.

[0040] As used herein, the term “polypeptide” is intended to encompass a single “polypeptide” as well as “polypeptides” in the plural, and refers to a molecule composed of monomers (amino acids) linearly linked by amide bonds (also known as peptide bonds). The term “polypeptide” refers to any chain or chains of two or more Petition 870260079734, dated 07 / 08 / 2026, page 18 / 97 / 75 amino acids and does not refer to a specific length of the product. Thus, peptides, dipeptides, tripeptides, oligopeptides, “protein”, “amino acid chain” or any other term used to refer to a chain or chains of two or more amino acids are included in the definition of “polypeptide” and the term “polypeptide” may be used instead of, or interchangeably with, any of these terms. The term “polypeptide” is also intended to refer to the products of post-expression modifications of the polypeptide, including, without limitation, glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting / blocking groups, proteolytic cleavage or modification by non-naturally occurring amino acids. A polypeptide may be derived from a natural biological source or produced by recombinant technology, but it is not necessarily translated from a designated nucleic acid sequence.It can be generated in any way, including by chemical synthesis.

[0041] The term “isolate,” as used herein with respect to cells, nucleic acids such as DNA or RNA, refers to molecules separated from other DNAs or RNAs, respectively, that are present in the natural source of the macromolecule. The term “isolate,” as used herein, also refers to a nucleic acid or peptide that is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA techniques, or chemical precursors or other chemicals when chemically synthesized. Furthermore, an “isolated nucleic acid” is intended to include nucleic acid fragments that do not occur naturally as fragments and would not be found in the natural state. The term “isolate” is also used herein to refer to cells or polypeptides that are isolated from other proteins or cellular tissues. Isolated polypeptides are intended to include both purified and recombinant polypeptides.

[0042] As used herein, the term “recombinant,” pertaining either to polypeptides or to polynucleotides, means a form of the polypeptide or Petition 870260079734, dated 07 / 08 / 2026, page 19 / 97 / 75 polynucleotide that does not exist naturally, a non-limiting example of which can be created by combining polynucleotides or polypeptides that would not normally occur together.

[0043] “Homology” or “identity” or “similarity” refers to the similarity of sequences between two peptides or between two nucleic acid molecules. Homology can be determined by comparing a position in each sequence that can be aligned for comparison purposes. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. A degree of homology between sequences is a function of the number of corresponding or homologous positions shared by the sequences. An “unrelated” or “non-homologous” sequence shares less than 40% identity, although preferably less than 25% identity, with one of the sequences in the present description.

[0044] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%) of “sequence identity” to another sequence means that, when aligned, this percentage of bases (or amino acids) are equal when comparing the two sequences. This alignment and percentage homology or sequence identity can be determined using software programs known in the art, for example, those described in Ausubel et al. eds. (2007) Current Protocols in Molecular Biology. Preferably, default parameters are used for alignment. One alignment program is BLAST, using default parameters.Specifically, the programs are BLASTN and BLASTP, using the following default parameters: Genetic code = default; filter = none; strand = both; cutoff value = 60; expected = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sorted by = HIGH SCORE; Databases = . Petition 870260079734, dated 07 / 08 / 2026, page 20 / 97 / 75 non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS + SwissProtein + SPupdate + PIR translations. Biologically equivalent polynucleotides are those having the specific percent homology noted above and encoding a polypeptide having the same or similar biological activity.

[0045] The term “an equivalent nucleic acid or polynucleotide” refers to a nucleic acid having a nucleotide sequence that has a certain degree of homology, or sequence identity, with the nucleotide sequence of the nucleic acid or its complement. A homolog of a double-stranded nucleic acid is intended to include nucleic acids having a nucleotide sequence that has a certain degree of homology with or with its complement. In one aspect, nucleic acid homologs are capable of nucleic acid or complement hybridization. Similarly, “an equivalent polypeptide” refers to a polypeptide having a certain degree of homology, or sequence identity, with the amino acid sequence of a reference polypeptide. In some aspects, the sequence identity is at least about 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99%.In some respects, the equivalent polypeptide or polynucleotide has one, two, three, four, or five additions, deletions, substitutions, and combinations thereof, as compared to the reference polypeptide or polynucleotide. In some respects, the equivalent sequence retains the activity (e.g., epitope binding) or structure (e.g., salt bridge) of the reference sequence.

[0046] Hybridization reactions can be carried out under different “stringency” conditions. In general, a low-stringency hybridization reaction is carried out at about 40°C in about 10 x SSC or an equivalent ionic strength / temperature solution. A moderate-stringency hybridization is typically carried out at about 50°C in about 6 x SSC, and a high-stringency hybridization reaction is generally carried out at about 60°C in about 1 x SSC. Hybridization reactions can also be Petition 870260079734, dated 07 / 08 / 2026, page 21 / 97 / 75, performed under “physiological conditions” that are well known to those skilled in the art. A non-limiting example of a physiological condition is the temperature, ionic strength, pH, and Mg2+ concentration normally found in a cell.

[0047] A polynucleotide is composed of a specific sequence of nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and uracil (U) for thymine when the polynucleotide is RNA. Thus, the term “polynucleotide sequence” is the alphabetical representation of a polynucleotide molecule. This alphabetical representation can be the entry for databases in a computer having a central processing unit and used for applications in bioinformatics, such as functional genomics and homology search. The term “polymorphism” refers to the coexistence of more than one form of a gene or portion thereof. The portion of a gene of which there are at least two different forms, that is, two different nucleotide sequences, is referred to as a “polymorphic region of a gene”. The polymorphic region can be a single nucleotide, whose identity differs in different alleles.

[0048] The terms “polynucleotide” and “oligonucleotide” are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. The following are non-limiting examples of polynucleotides: a gene or gene fragment (e.g., a probe, primer, EST or SAGE tag), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, dsRNA, siRNA, miRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, DNA isolated from any sequence, RNA isolated from any sequence, nucleic acid probes, and primers. A polynucleotide can Petition 870260079734, dated 07 / 08 / 2026, page 22 / 97 / 75, includes modified nucleotides, such as methylated nucleotides and nucleotide analogs. If present, modifications to the nucleotide structure may be conferred before or after the assembly of the polynucleotide. The nucleotide sequence may be interrupted by non-nucleotide components. A polynucleotide may be further modified after polymerization, such as by conjugation with a labeling component. The term also refers to both single-stranded and double-stranded molecules. Unless otherwise specified or required, any embodiment of this description that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms known or provided to complete the double-stranded form.

[0049] The term “encode” as applied to polynucleotides refers to a polynucleotide that is said to “encode” a polypeptide if, in its native state or when manipulated by methods well known to those skilled in the art, it can be transcribed and / or translated to produce mRNA for the polypeptide and / or a fragment thereof. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced from it.

[0050] As used herein, an “antibody” or “antigen-binding polypeptide” refers to a polypeptide or a polypeptide complex that specifically recognizes and binds to an antigen. An antibody may be a complete antibody and any antigen-binding fragment or a single chain thereof. Thus, the term “antibody” includes any protein- or peptide-containing molecule comprising at least a portion of an immunoglobulin molecule having antigen-binding biological activity. Examples of such include, but are not limited to, a complementarity-determining region (CDR) of a heavy or light chain or a ligand-binding portion thereof, a heavy chain, or a variable chain region. Petition 870260079734, dated 07 / 08 / 2026, page 23 / 97 / 75 light, a heavy chain or constant region of light chain, a scaffold region (SR), or any portion thereof, or at least a portion of a binding protein.

[0051] The terms “antibody fragment” or “antigen-binding fragment,” as used herein, refer to a portion of an antibody such as F(ab')2, F(ab)2, Fab', Fab, Fv, scFv, and the like. Regardless of structure, an antibody fragment binds to the same antigen that is recognized by the intact antibody. The term “antibody fragment” includes aptamers, spiegelmers, and diabodies. The term “antibody fragment” also includes any synthetic or genetically engineered protein that acts as an antibody by binding to a specific antigen to form a complex.

[0052] A “single-chain variable fragment” or “scFv” refers to a fusion protein of the variable regions of the heavy (VH) and light (VL) chains of immunoglobulins. In some respects, the regions are connected with a short linker peptide of ten to about 25 amino acids. The linker may be glycine-rich for flexibility, as well as serine or threonine for solubility, and may either connect the N-terminus of the VH with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original immunoglobulin despite the removal of the constant regions and the introduction of the linker. ScFv molecules are known in the art and are described, for example, in US Patent 5,892,019.

[0053] The term antibody encompasses several broad classes of polypeptides that can be distinguished biochemically. Those skilled in the art will appreciate that heavy chains are classified as gamma, mu, alpha, delta, or epsilon (γ, μ, α, δ, ε) with some subclasses within the same (e.g., Yl-y4). It is the nature of this chain that determines the “class” of the antibody such as IgG, IgM, IgA, IgG, or IgE, respectively. Immunoglobulin subclasses (isotypes), for example, IgG1, IgG2, IgGs, IgG4, IgG5, etc., are well distinguished and are known to confer functional specialization. Petition 870260079734, dated 07 / 08 / 2026, p. 24 / 97 / 75 Modified versions of each of these classes and isotypes are readily discernible to those skilled in the art in view of the present description and, consequently, are within the scope of the present description. All immunoglobulin classes are clearly within the scope of the present description; the following discussion will generally be directed to the IgG class of immunoglobulin molecules. With respect to IgG, a standard immunoglobulin molecule comprises two identical light chain polypeptides of approximately 23,000 Daltons molecular weight, and two identical heavy chain polypeptides of 53,000–70,000 Daltons molecular weight. The four chains are typically joined by disulfide bonds in a “Y” configuration in which the light chains encircle the heavy chains starting from the mouth of the “Y” and continuing through the variable region.

[0054] Antibodies, antigen-binding polypeptides, variants, or derivatives thereof of the description include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized, primatized, or chimeric antibodies, single-chain antibodies, epitope-binding fragments, for example, Fab, Fab' and F(ab')2, Fd, Fvs, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv), fragments comprising either a VK or VH domain, fragments produced by a Fab expression library, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to LIGHT antibodies described herein). Immunoglobulin or antibody molecules described may be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass of immunoglobulin molecule.

[0055] Light chains are classified as either kappa or lambda (κ,λ). Each class of heavy chain can be connected to either a kappa or lambda light chain. In general, the light and heavy chains are covalently linked to each other, and the "tail" portions of the two heavy chains are linked to each other by covalent disulfide bonds or non-covalent bonds when the Petition 870260079734, dated 07 / 08 / 2026, p. 25 / 97 / 75 Immunoglobulins are generated either by hybridomas, B cells, or genetically engineered host cells. In the heavy chain, the amino acid sequence runs from an N-terminus at the ends of the Y-configuration fork to the C-terminus at the bottom of each chain.

[0056] Both light and heavy chains are divided into regions of structural and functional homology. The terms “constant” and “variable” are used functionally. In this respect, it will be appreciated that the variable domains of both the light (VK) and heavy (VH) chain portions determine antigen specificity recognition. Conversely, the constant domains of the light chain (CK) and heavy chain (CH1, CH2, or CH3) confer important biological properties such as secretion, transplacental mobility, Fc receptor binding, complement binding, and the like. By convention, the numbering of the constant region domains increases as they become more distal from the antigen-binding site or antibody amino-terminus. The N-terminal portion is a variable region and the C-terminal portion is a constant region; the CH3 and CK domains actually comprise the carboxy terminus of the heavy and light chains, respectively.

[0057] As indicated above, the variable region allows the antibody to selectively and specifically recognize and bind epitopes to antigens. That is, the VK domain and the VH domain, or subset of the complementarity-determining regions (CDRs), of an antibody combine to form the variable region that defines a three-dimensional binding site on the antigen. This quaternary antibody structure forms the antigen-binding site present at the end of each arm of the Y. More specifically, the antigen-binding site is defined by three CDRs in each of the VH and VK chains (i.e., CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3). In some cases, for example, some immunoglobulin molecules derived from camelid species or engineered based on camelid immunoglobulins, a complete immunoglobulin molecule may consist of Petition 870260079734, dated 07 / 08 / 2026, p. 26 / 97 / 75 heavy chains only, without light chains. See, for example, HamersCasterman et al., Nature 363:446-448 (1993).

[0058] In naturally occurring antibodies, the six “complementarity-determining regions” or “CDRs” present in each antigen-binding domain are short, non-contiguous amino acid sequences that are specifically positioned to form the antigen-binding domain as the antibody assumes its three-dimensional configuration in an aqueous environment. The remaining amino acids in the antigen-binding domains, referred to as “scaffold” regions, show less intermolecular variability. The scaffold regions largely adopt a β-sheet conformation, and the CDRs form loops that connect and, in some cases, form part of the β-sheet structure. Thus, the scaffold regions act to form a scaffold that provides the correct orientation of CDRs through non-covalent inter-chain interactions. The antigen-binding domain formed by the positioned CDRs defines a surface complementary to the epitope on the immunoreactive antigen.This complementary surface promotes the non-covalent binding of the antibody to its cognate epitope. The amino acids comprising the CDRs and the scaffold regions, respectively, can be readily identified by any given variable heavy or light chain region by a knowledgeable person, because they have been precisely defined (see “Sequences of Proteins of Immunological Interest,” Kabat, E., et al., US Department of Health and Human Services, (1983); and Chothia and Lesk, J. Mol. Biol., 196:901-917 (1987)).

[0059] In cases where there are two or more definitions of a term that are used and / or accepted in the art, the definition of the term as used here is intended to include all of those meanings, unless explicitly stated otherwise. A specific example is the use of the term “complementarity-determining region” (“CDR”) to describe the non-contiguous antigen combining sites found within the variable region of both Petition 870260079734, dated 07 / 08 / 2026, p. 27 / 97 / 75 heavy and light chain polypeptides. This particular region was described by Kabat et al., US Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983) and by Chothia et al., J. Mol. Biol. 196: 901-917 (1987), which are incorporated herein by reference in their entirety. The definitions of CDR according to Kabat and Chothia include overlapping or subsets of amino acid residues when compared to each other. Even so, application of both definitions to refer to a CDR of an antibody or variants thereof is intended to be within the scope of the term as defined and used herein. The appropriate amino acid residues encompassing CDRs as defined by each of the references cited above are specified in the table below for comparison. The exact number of residues that comprise a particular CDR will vary depending on the sequence and size of the CDR.Those skilled in the art can routinely determine that the residues comprise a particular CDR of the antibody's variable region amino acid sequence. Kabat Chothia CDR-H1 31-35 26-32 CDR-H2 50-65 52-58 CDR-H3 95-102 95-102 CDR-L1 24-34 26-32 CDR-L2 50-56 50-52 CDR-L3 89-97 91-96

[0060] Kabat et al. also defined a numbering system for variable domain sequences that is applicable to any antibody. A knowledgeable person can unambiguously assign this “Kabat numbering” system to any variable domain sequence without relying on any experimental data beyond the sequence itself. As used herein, “Kabat numbering” refers to the numbering system specified by Kabat et al., US Dept. of Health and Human Services, “Sequence of proteins of Immunological Interest” (1983).

[0061] In addition to the table above, the Kabat numbering system describes the CDR regions as follows: CDR-H1 starts at Petition 870260079734, dated 07 / 08 / 2026, page 28 / 97 / 75 approximately amino acid 31 (that is, approximately 9 residues after the first cysteine ​​residue), includes approximately 5-7 amino acids, and terminates at the next tryptophan residue. CDR-H2 begins at the fifteenth residue after the end of CDR-H1, includes approximately 16-19 amino acids, and terminates at the next arginine or lysine residue. CDR-H3 begins at approximately the third third of the amino acid residue after the end of CDR-H2; includes 3-25 amino acids; and terminates in the sequence WG-XG, where X is any amino acid. CDR-L1 begins at approximately residue 24 (that is, after a cysteine ​​residue); includes approximately 1017 residues; and terminates at the next tryptophan residue. CDR-L2 begins at approximately the sixteenth residue after the end of CDR-L1 and includes approximately 7 residues.CDR-L3 begins approximately in the third third of the residue after the end of CDR-L2 (that is, after a cysteine ​​residue); it includes approximately 7-11 residues and terminates in the sequence F or WGXG, where X is any amino acid.

[0062] Antibodies described herein may be of any animal origin, including birds and mammals. Preferably, the antibodies are human, murine, donkey, rabbit, goat, guinea pig, camel, llama, horse, or chicken antibodies. In another embodiment, the variable region may be chondritoid in origin (e.g., from sharks).

[0063] As used herein, the term “heavy chain constant region” includes amino acid sequences derived from an immunoglobulin heavy chain. A polypeptide comprising a heavy chain constant region comprises at least one of: a CH1 domain, a hinge domain (e.g., hinge, top, middle and / or bottom region), a CH2 domain, a CH3 domain, or a variant or fragment thereof. For example, an antigen-binding polypeptide for use in the description may comprise a polypeptide chain comprising a CH1 domain; a polypeptide chain comprising Petition 870260079734, dated 07 / 08 / 2026, page 29 / 97 / 75 a CH1 domain, at least a portion of a hinge domain, and a CH2 domain; a polypeptide chain comprising a CH1 domain and a CH3 domain; a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, and a CH3 domain, or a polypeptide chain comprising a CH1 domain, at least a portion of a hinge domain, a CH2 domain, and a CH3 domain. In another embodiment, a polypeptide of the description comprises a polypeptide chain comprising a CH3 domain. Additionally, an antibody for use in the description may lack at least a portion of a CH2 domain (e.g., all or part of a CH2 domain). As specified above, it will be understood by a person versed in the field that the constant region of the heavy chain can be modified so that they vary in amino acid sequence from that of the naturally occurring immunoglobulin molecule.

[0064] The constant heavy chain region of an antibody described herein may be derived from different immunoglobulin molecules. For example, a constant heavy chain region of a polypeptide may comprise a CH1 domain derived from an IgG1 molecule and a hinge region derived from an IgG3 molecule. In another example, a constant heavy chain region may comprise a hinge region derived partly from an IgG1 molecule and partly from an IgG3 molecule. In yet another example, a heavy chain portion may comprise a chimeric hinge derived partly from an IgG1 molecule and partly from an IgG4 molecule.

[0065] As used herein, the term “light chain constant region” includes amino acid sequences derived from antibody light chains. Preferably, the light chain constant region comprises at least one of either a kappa constant domain or a lambda constant domain.

[0066] A “light-heavy chain pair” refers to the collection of a Petition 870260079734, dated 07 / 08 / 2026, page 30 / 97 / 75 light chain and heavy chain that can form a dimer through the disulfide bond between the CL domain of the light chain and the CH1 domain of the heavy chain.

[0067] As previously indicated, the subunit structures and three-dimensional configuration of the constant regions of the various immunoglobulin classes are well known. As used herein, the term “VH domain” includes the amino-terminal variable domain of an immunoglobulin heavy chain and the term “CH1 domain” includes the first (most amino-terminal) constant region domain of an immunoglobulin heavy chain. The CH1 domain is adjacent to the VH domain and is amino-terminal to the hinge region of an immunoglobulin heavy chain molecule.

[0068] As used herein, the term “CH2 domain” includes the portion of a heavy chain molecule that extends, for example, from about residue 244 to residue 360 ​​of an antibody using conventional numbering schemes (residues 244 to 360, Kabat numbering system; and residues 231-340, EU numbering system; see Kabat et al., US Dept. of Health and Human Services, “Sequences of Proteins of Immunological Interest” (1983). The CH2 domain is unique in that it is not closely paired with another domain. Instead, two branched N-linked carbohydrate chains are interposed between the two CH2 domains of an intact native IgG molecule. It has also been well documented that the CH3 domain extends from the CH2 domain to the C-terminus of the IgG molecule and comprises approximately 108 residues.

[0069] As used herein, the term “hinge region” includes the portion of a heavy chain molecule that links the CH1 domain to the CH2 domain. This hinge region comprises approximately 25 residues and is flexible, thus allowing two N-terminal antigen-binding regions to move independently. Hinge regions can be Petition 870260079734, dated 07 / 08 / 2026, page 31 / 97 / 75 subdivided into three distinct domains: superior, middle and inferior hinge domains (Roux et al., J. Immunol 161:4083 (1998)).

[0070] As used herein, the term “disulfide bond” includes the covalent bond formed between two sulfur atoms. The amino acid cysteine ​​comprises a thiol group that can form a disulfide bond or bridge with a second thiol group. In most naturally occurring IgG molecules, the CH1 and CK regions are linked by a disulfide bond and the two heavy chains are linked by two disulfide bonds at positions corresponding to 239 and 242 using the Kabat numbering system (position 226 or 229 in the EU numbering system).

[0071] As used herein, the term “chimeric antibody” will be retained to mean any antibody in which the immunoreactive region or site is obtained from or derived from a first species and the constant region (which may be intact, partial, or modified according to the present description) is obtained from a second species. In some embodiments, the target binding region or site will be from a non-human source (e.g., mouse or primate) and the constant region is human.

[0072] As used herein, “percentage of humanization” is calculated by determining the number of amino acid differences in the framework (i.e., non-CDR difference) between the humanized domain and the germline domain, subtracting this number from the total number of amino acids, and then dividing this by the total number of amino acids and multiplying by 100.

[0073] By “specifically binds” or “has specificity for,” it is generally understood that an antibody binds to an epitope via its antigen-binding domain, and that the binding entails some complementarity between the antigen-binding domain and the epitope. According to this definition, an antibody is said to “specifically bind” to an epitope when it binds to this epitope via its antigen-binding domain more readily than it would bind to a random, unrelated epitope. The term “specificity” Petition 870260079734, dated 07 / 08 / 2026, p. 32 / 97 / 75 is used here to qualify the relative affinity with which a certain antibody binds to a certain epitope. For example, antibody “A” may be considered to have a higher specificity for a given epitope than antibody “B,” or antibody “A” may be considered to bind to epitope “C” with a higher specificity than it would for the related epitope “D.”

[0074] As used herein, the terms “treat” or “treatment” refer to both therapeutic treatment and prophylactic or preventive measures, where the objective is to prevent or slow down (reduce) an undesirable physiological change or disorder, such as the progression of an autoimmune disease. Beneficial or desired clinical outcomes include, but are not limited to, symptom relief, reduction in disease extent, stabilized disease status (i.e., no worsening), delay or retardation of disease progression, improvement or palliative improvement of disease status, and detectable or undetectable remission (partial or total). “Treatment” may also mean prolonging survival relative to expected survival if not receiving treatment. Those who need treatment include those who already have the condition or disorder, as well as those likely to have the condition or disorder or those in whom the condition or disorder should be prevented.

[0075] By “subject” or “individual” or “animal” or “patient” or “mammal” is meant any subject, particularly a mammal, for whom diagnosis, prognosis or therapy is desired. Mammals include humans, domestic animals, farm and zoo animals, sporting animals or pets such as dogs, cats, guinea pigs, rabbits, rats, mice, horses, cattle, cows and so forth.

[0076] As used herein, phrases such as “for a patient requiring treatment” or “an individual requiring treatment” include individuals, such as mammals, who would benefit from the administration of an antibody or composition described herein, for example, used for Petition 870260079734, dated 07 / 08 / 2026, page 33 / 97 / 75 detection, for a diagnostic procedure and / or for treatment. Anti-GM-CSF antibodies

[0077] This description provides anti-GM-CSF antibodies with high affinity for the human GM-CSF protein. The tested antibodies exhibited potent binding and inhibitory activities and are usable for therapeutic and diagnostic applications. In addition to the original murine antibodies, the humanized antibodies also showed strong binding affinity for rhesus GM-CSF and human GM-CSF, whose binding blocked GM-CSF binding to the GM-CSF alpha receptor and blocked GM-CSF-induced pSTAT5 signaling, and inhibited GM-CSF-dependent proliferation of TF-1.

[0078] According to an embodiment of the present description, an antibody is provided that includes variable heavy chain and light chain domains with CDR regions as defined and, SEQ ID NO: 1-6 or SEQ ID NO: 23-28, as shown below. Table 1a. Sequences of CDR regions of 23F4 _____________ Name Sequences SEQ ID NO: CDR1 of VH SHYLH 1 CDR2 of VH WIFPGDDKTKYNEKFKG 2 CDR3 of VH GTKYLNWNFDV 3 CDR1 of VL KANQNVGTTLA 4 CDR2 of VL SASYRYS 5 CDR3 of VL HQYTTYPLT 6 Table 1b. Sequences of CDR regions of 50C5 Name Sequences SEQ ID NO: CDR1 of VH PYSIH 23 CDR2 of VH YINPSTGYIEYNQHFKD 24 CDR3 of VH GGDYEGYFDY 25 CDR1 of VL RLNENIYSFLA 26 CDR2 of VL NAETLAE 27 CDR3 of VL QQHYGTPYT 28

[0079] As demonstrated in the experimental examples, antibodies containing these CDR regions, whether mouse, humanized, or chimeric, had potent GM-CSF binding and inhibitory activities. In some embodiments, an anti-GM-CSF antibody of the present description includes VH and VL CDRs as listed in Table 1a-b, with one, two, or three additional modifications. Such modifications may be addition, deletion, or Petition 870260079734, dated 07 / 08 / 2026, p. 34 / 97 / 75 amino acid replacement.

[0080] In some embodiments, the modification is a substitution in no more than one of the residues of each of the RDCs. In some embodiments, the modification is a substitution in one, two, or three residues. In one embodiment, the modification is a substitution in one of the residues. Such substitutions, in some embodiments, are conservative substitutions.

[0081] A “conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine).Thus, a non-essential amino acid residue in an immunoglobulin polypeptide is preferably replaced with another amino acid residue from the same side chain family. In another embodiment, an amino acid chain can be replaced with a structurally similar chain that differs in the order and / or composition of side chain family members.

[0082] Non-limiting examples of conservative amino acid substitutions are given in the table below, where a similarity score of 0 or higher indicates conservative substitution between the two amino acids. Table 2. Amino Acid Similarity Matrix CGPSATDENQHKRVMILFYWW -8 -7 -6 -2 -6 -5 -7 -7 -4 -5 -3 -3 2 -6 -4 -5 -2 0 0 17 Y 0 -5 -5 -3 -3 -3 -4 -4 -2 -4 0 -4 -5 -2 -2 -1 -1 7 10 F -4 -5 -5 -3 -4 -3 -6 -5 -4 -5 -2 -5 -4 -1 0 1 2 9 L -6 -4 -3 -3 -2 -2 -4 -3 -3 -2 -2 -3 -3 2 4 2 6 I -2 -3 -2 -1 -1 0 -2 -2 -2 -2 -2 -2 -2 4 2 5 M -5 -3 -2 -2 -1 -1 -3 -2 0 -1 -2 0 0 2 6 Petition 870260079734, dated 07 / 08 / 2026, p. 35 / 97 / 75 V -2 -1 -1 -1 0 0 -2 -2 -2 -2 -2 -2 -2 4 R -4 -3 0 0 -2 -1 -1 -1 0 1 2 3 6 K -5 -2 -1 0 -1 0 0 0 1 1 0 5 H -3 -2 0 -1 -1 -1 1 1 2 3 6 Q -5 -1 0 -1 0 -1 2 2 1 4 N -4 0 -1 1 0 0 2 1 2 E -5 0 -1 0 0 0 3 4 D -5 1 -1 0 0 0 4 T -2 0 0 1 1 3 A -2 1 1 1 2 S 0 1 1 1 P -3 -1 6 G -3 5 C 12 Table 3. Conservative Amino Acid Substitutions For Amino Acid Substitution with Alanine D-Ala, Gly, Aib, β-Ala, L-Cys, D-Cys Arginine D-Arg, Lys, D-Lys, Orn D-Orn Asparagine D-Asn, Asp, D-Asp, Glu, D-Glu Gln, D-Gl Acid, D-Asppartic, D-Asn Asn, Glu, D-Glu, Gln, D-Gln Cysteine ​​D-Cys, S-Me-Cys, Met, D-Met, Thr, D-Thr, L-Ser, D-Ser Glutamine D-Gln, Asn, D-Asn, Glu, D-Glu, Asp, D-Asp acid, D-Glu, D-Asp, D-Glu, D-Asp D-Asn, Gln, D-Gln Glycine Ala, D-Ala, Pro, D-Pro, Aib, β-Ala Isoleucine D-Ile, Val, D-Val, Leu, D-Leu, Met, D-Met Leucine Val, D-Val, Met, D-Met, D-Ile, D-Leu, Ile-Lys, Arg, D-Lys, Arg, Arg D-Orn Methionine D-Met, S-Me-Cys, Ile, D-Ile, Leu, D-Leu, Val, D-Val Phenylalanine D-Phe, Tyr, D-Tyr, His, D-His, Trp, D-Trp Proline D-Pro Serine D-Serine, Thr, D-Thr, allo-Thr, D-Cys, L-Cys D-Thr, Ser, D-Ser, allo-Thr, Met, D-Met, Val, D-Val Tyrosine D-Tyr, Phe, D-Phe, His, D-His, Trp, D-Trp Valine D-Val, Leu, D-Leu, Ile, D-Ile, Met, D-Met

[0083] In some embodiments, an antibody or fragment thereof includes no more than one, no more than two, or no more than three of the above substitutions.

[0084] In some embodiments, the antibody or fragment thereof has specificity for a human GM-CSF protein and comprises a VH CDR1 from SEQ ID NO: 1, a VH CDR2 from SEQ ID NO: 2, a VH CDR3 from SEQ ID NO: 3, a VL CDR1 from SEQ ID NO: 4, a VL CDR2 from SEQ ID NO: 5, and a VL CDR3 from SEQ ID NO: 6. Non-limiting examples of VH are presented in SEQ ID NO: 7-17, of which SEQ ID NO: 7 is mouse VH, and SEQ ID NO: 8-17 are the Petition 870260079734, dated 07 / 08 / 2026, p. 36 / 97 / 75 humanized. Additionally, these humanized VH include one or more retromutations of the mouse version. Similarly, non-limiting examples of VL (VK) are presented in SEQ ID NO: 18-22. SEQ ID NO: 18 is a mouse sequence, and SEQ ID NO: 19-22 are humanized sequences, among which SEQ ID NO: 20-22 include one or more retromutations, as shown in the examples.

[0085] Retromutations are shown to be usable for retaining some characteristics of anti-GM-CSF antibodies. Consequently, in some embodiments, the anti-GM-CSF antibodies of the present description, in particular human or humanized antibodies, include one or more retromutations. In some embodiments, the VH retromutation (i.e., amino acid included at the specified position) is one or more selected from (a) Glu at position 1 (E1), (b) Arg at position 98 (R98), (c) Ser at position 72 (S72), (d) Ala at position 68 (A68), (e) Leu at position 70 (L70), (f) Ile at position 48 (I48), (g) Asp at position 26 (D26), and (h) Leu at position 29 (L29), according to Kabat numbering, and combinations thereof.

[0086] In some embodiments, the humanized antibody includes at least an E1 retromutation of HV. In some embodiments, the humanized antibody includes at least E1 and R98 retromutations of HV. In some embodiments, the humanized antibody includes at least E1 retromutations of HV and another as striped above. In some embodiments, the humanized antibody includes at least one HV retromutation group (E1, R98 and S72), (E1, R98, S72 and A68), (E1, R98, S72, A68, L70 and I48), (E1, R98, S72, A68, L70, I48, D26 and L29), (E1 and S72), (E1, S72 and L70), (E1, S72, L70, I48 and A68), (E1, S72, L70, I48, A68, D26 and L29).

[0087] In some embodiments, the variable heavy chain region comprises a fragment of DYTLT (SEQ ID NO: 42) or GYTFT (SEQ ID NO: 43) at the N-terminal end of a CDR1, that is, starting at position 26 according to Kabat numbering. In one embodiment, the region Petition 870260079734, dated 07 / 08 / 2026, page 37 / 97 / 75 heavy chain variable comprises DYTLT (SEQ ID NO: 42). In one embodiment, the heavy chain variable region comprises GYTFT (SEQ ID NO: 43).

[0088] In some embodiments, the humanized antibody includes one or more of the retromutations. In some embodiments, the VL retromutation is one or more selected from (a) Ala at position 46 (A46), (b) Asp at position 60 (D60), (c) Asp at position 70 (D70), (d) Ser at position 43 (S43), and (f) Phe at position 87 (F87), according to Kabat numbering, and combinations thereof.

[0089] In some embodiments, the humanized antibody includes at least two, three, or four A46, D60, D70, S43, or F87 VL retromutations. In some embodiments, the humanized antibody includes at least A46 VL retromutation. In some embodiments, the humanized antibody includes at least A46 and D60 VL retromutations and another, as listed above. In some embodiments, the humanized antibody includes at least VL retromutation groups (A46, D60, and D70) or (A46, D60, D70, S43, and F87).

[0090] In some embodiments, the humanized antibody includes at least VH retromutations (E1, R98, S72, A68, L70, and I48) and no VL retromutations. In some embodiments, the humanized antibody includes at least VH retromutations (E1, S72, L70, I48, A68, D26, and L29) and no VL retromutations. In some embodiments, the humanized antibody includes at least VH retromutations (E1 and S72) and VL retromutations (A46, D60, D70, S43, and F87).

[0091] In some embodiments, the anti-GM-CSF antibody of the present description includes a VH of SEQ ID NO: 8-17, and a VL of SEQ ID NO: 19-22, or their respective biological equivalents. A biological equivalent of a VH or VL is a sequence that includes the designated amino acids while having a total of 80%, 85%, 90%, 95%, 98%, or 99% sequence identity. A biological equivalent of SEQ ID NO: 10, of this Petition 870260079734, dated 07 / 08 / 2026, p. 38 / 97 / 75 mode, it may be a VH that has a total of 80%, 85%, 90%, 95%, 98% or 99% sequence identity for SEQ ID NO: 10 but retains CDRs (SEQ ID NO: 1-3 or their variants), and optionally retains one or more, or all of the retromutations.

[0092] In one embodiment, VH has the amino acid sequence SEQ ID NO: 11 and VL has the amino acid sequence SEQ ID NO: 19. In one embodiment, VH has the amino acid sequence SEQ ID NO: 17 and VL has the amino acid sequence SEQ ID NO: 19. In one embodiment, VH has the amino acid sequence SEQ ID NO: 11 and VL has the amino acid sequence SEQ ID NO: 22. Each of the sequences mentioned, as noted, can also be replaced with its biological equivalents.

[0093] In some embodiments, the antibody or fragment thereof has specificity for a human GM-CSF protein and comprises a VH CDR1 from SEQ ID NO: 23, a VH CDR2 from SEQ ID NO: 24, a VH CDR3 from SEQ ID NO: 25, a VL CDR1 from SEQ ID NO: 26, a VL CDR2 from SEQ ID NO: 27, and a VL CDR3 from SEQ ID NO: 28. Non-limiting examples of VH are presented in SEQ ID NO: 29-35, among which SEQ ID NO: 29 is mouse VH, and SEQ ID NO: 30-35 are humanized. Additionally, these humanized VH include one or more retromutations to the mouse version. Similarly, non-limiting examples of VL (VK) are presented in SEQ ID NO: 36-41. SEQ ID NO: 36 is a mouse sequence, and SEQ ID NO: 37-41 are humanized sequences, among which SEQ ID NO: 38-41 include one or more retromutations, as shown in the examples.

[0094] Retromutations are shown to be usable for some features of anti-GM-CSF antibodies. Consequently, in some embodiments, the anti-GM-CSF antibodies in the present description, particularly human or humanized ones, include one or more of the Petition 870260079734, dated 07 / 08 / 2026, p. 39 / 97 / 75 retromutations. In some embodiments, a VH retromutation (i.e., amino acid included at the specified position) is one or more selected from E1, R84, Y27, I28, I48, T68, L70, or T30, according to Kabat numbering, and combinations thereof.

[0095] In some embodiments, the humanized antibody includes at least HV E1 retromutation. In some embodiments, the humanized antibody includes at least HV E1 and R84 retromutations. In some embodiments, the humanized antibody includes at least HV E1 retromutations and another, as listed above. In some embodiments, the humanized antibody includes at least HV retromutation group (E1), (E1 and R84), (E1, R84, Y27 and I28), (E1, R84, Y27, I28 and I48), (E1, R84, Y27, I28, I48, T68 and L70), or (E1, R84, Y27, I28, I48, T68, L70 and T30).

[0096] In some embodiments, the variable heavy chain region comprises a fragment of GYIFT (SEQ ID NO: 44), GYIFS (SEQ ID NO: 45), or GGTFS (SEQ ID NO: 46) at the N-terminal end of a CDR1, that is, starting at position 26 according to Kabat numbering. In one embodiment, the variable heavy chain region comprises GYIFT (SEQ ID NO: 44). In one embodiment, the variable heavy chain region comprises GYIFS (SEQ ID NO: 45). In one embodiment, the variable heavy chain region comprises GGTFS (SEQ ID NO: 46).

[0097] In some embodiments, the humanized antibody includes one or more of the retromutations. In some embodiments, the VL retromutation is one or more selected from V48, D57, Q70, or S43, according to Kabat numbering, and combinations thereof.

[0098] In some embodiments, the humanized antibody includes at least two, three, or four of the V48, D57, Q70, or S43 retromutations of VL. In some embodiments, the humanized antibody includes at least the V48 retromutation of VL. In some embodiments, the humanized antibody includes at least the V48 and D57 retromutations of VL and another, as listed above. Petition 870260079734, dated 07 / 08 / 2026, p. 40 / 97 / 75 In some embodiments, the humanized antibody includes at least the VL retromutation group (V48), (V48 and D57), (V48, D57 and Q70) or (V48, D57, Q70 and S43).

[0099] In some embodiments, the humanized antibody includes at least VH retromutations (E1, R84, Y27, I28, I48, T68, and L70) and VL retromutation V48. In some embodiments, the humanized antibody includes at least VH retromutations (E1, R84, Y27, I28, I48, T68, L70, and T30) and VL retromutations (V48 and D57).

[00100] In some embodiments, the anti-GM-CSF antibody of the present description includes a VH of SEQ ID NO: 30-35, and a VL of SEQ ID NO: 37-41, or their respective biological equivalents. A biological equivalent of a VH or VL is a sequence that includes the designated amino acids while having a total of 80%, 85%, 90%, 95%, 98%, or 99% sequence identity. A biological equivalent of SEQ ID NO: 35, therefore, may be a VH that has a total of 80%, 85%, 90%, 95%, 98%, or 99% sequence identity to SEQ ID NO: 35 but retains the CDRs (SEQ ID NO: 23-25 ​​or their variants), and optionally retains one or more, or all of the retromutations.

[00101] In one embodiment, VH has the amino acid sequence SEQ ID NO: 34 and VL has the amino acid sequence SEQ ID NO: 38. In another embodiment, VH has the amino acid sequence SEQ ID NO: 35 and VL has the amino acid sequence SEQ ID NO: 39. Each of the sequences mentioned, as noted, can also be replaced with its biological equivalents.

[00102] It should also be understood by a person versed in knowledge that antibodies as described herein may be modified so that they vary in amino acid sequence from the naturally occurring binding polypeptide from which they were derived. For example, a polypeptide or amino acid sequence derived from a protein called Petition 870260079734, dated 07 / 08 / 2026, p. 41 / 97 / 75 may be similar, for example, having a determined percentage identity for the starting sequence, for example, it may be 60%, 70%, 75%, 80%, 85%, 90%, 95%, 98%, or 99% identical to the starting sequence.

[00103] The antibodies and fragments of the present description may be monospecific or bispecific antibodies or fragments, in some embodiments. For a bispecific antibody, the specificity may be for a different target epitope of GM-CSF or a different target protein that is usable for a particular use, for example, therapeutic use. In one aspect, the target protein is a cytokine such as TNF-alpha, IL-6, IL-1, and IL-17. In another aspect, the target protein is a chemokine, such as CCL2, CXCL12, and CXCL13. In another aspect, the target protein is a cell surface protein, such as CD3, CSF-1R, CD20, and CD73.

[00104] In some embodiments, the antibody comprises an amino acid sequence or one or more moieties not normally associated with an antibody. Exemplary modifications are described in greater detail below. For example, an antibody of the description may comprise a flexible linker sequence, or it may be modified to add a functional moiety (e.g., PEG, a drug, a toxin, or a label).

[00105] Antibodies, variants, or derivatives thereof of the description include derivatives that are modified, i.e., by the covalent attachment of any type of molecule to the antibody, such that the covalent attachment does not prevent the antibody from binding to the epitope.For example, but not limited to, antibodies can be modified, for instance, by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protective / blocking groups, proteolytic cleavage, binding to a cellular ligand or other protein, etc. Any of the numerous chemical modifications can be carried out by known techniques, including but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, antibodies can... Petition 870260079734, dated 07 / 08 / 2026, page 42 / 97 / 75 containing one or more non-classical amino acids.

[00106] In some embodiments, antibodies can be conjugated or otherwise linked to another molecule to form a bifunctional molecule. The second molecule can be one of therapeutic agents, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceutical agents, or PEGs. Some non-limiting examples are cytokines or other soluble factors such as IL-10, IL-25, IL-27, IL-33, IL-35, and IL-36. Also provided in some embodiments are antibody-drug conjugates that include an antibody or fragment of the present description and a small molecule drug.

[00107] Antibodies may be conjugated or fused to a therapeutic agent, which may include detectable labels such as radioactive labels, an immunomodulator, a hormone, an enzyme, an oligonucleotide, a therapeutic or diagnostic photoactive agent, a cytotoxic agent, which may be a drug or a toxin, an ultrasound-enhancing agent, a non-radioactive label, a combination thereof, and other such agents known in the art.

[00108] Antibodies can be detectably labeled by attaching them to a chemiluminescent compound. The presence of the chemiluminescently labeled antigen-binding polypeptide is then determined by detecting the presence of luminescence that arises during the course of a chemical reaction. Examples of particularly useful chemiluminescent labeling compounds are luminol, isoluminol, acridinium ester, imidazole, acridinium salt, and oxalate ester.

[00109] Antibodies can also be labeled detectably using fluorescent metals, such as 152Eu, or others from the lanthanide series. These metals can be fixed to the antibody using metal chelating groups such as diethylenetriaminepentaacetic acid (DTPA) or ethylenediaminetetraacetic acid (EDTA). Techniques for conjugating various moieties to Petição 870260079734, de 07 / 08 / 2026, pág. 43 / 97 / 75 um anticorpo são bem conhecidos, ver, por exemplo, Arnon et al., “Monoclonal Antibodies For Imunotargeting Of Drugs In Cancer Therapy”, em Monoclonal Antibodies and Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. (1985); Hellstrom et al., “Antibodies For Drug Delivery”, em Controlled Drug Delivery (2a. Ed.), Robinson et al., (eds.), Marcel Dekker, Inc., pp. 62353 (1987); Thorpe, “Antibody Carriers Of Cytotoxic Agents In Cancer Therapy: A Review”, em Monoclonal Antibodies ‘84: Biological and Clinical Applications, Pinchera et al. (eds.), pp. 475-506 (1985); “Analysis, Results, E Future Prospective Of The Therapeutic Use Of Radiolabeled Antibody In Cancer Therapy”, em Monoclonal Antibodies For Cancer Detection E Therapy, Baldwin et al. (eds.), Academic Press pp. 303-16 (1985), e Thorpe et al., “The Preparation E Cytotoxic Properties Of Antibody-Toxin Conjugates ”, Immunol. Rev. (52:119-58 (1982)). Polynucleotides encoding antibodies and methods of preparing antibodies.

[00110] This description also provides isolated polynucleotide or nucleic acid molecules encoding the antibodies, variants, or derivatives thereof described. The polynucleotides described herein may encode the complete variable regions of the heavy and light chains of antigen-binding polypeptides, variants, or derivatives thereof in the same polynucleotide molecule or in separate polynucleotide molecules. Additionally, the polynucleotides described herein may encode portions of the variable regions of the heavy and light chains of antigen-binding polypeptides, variants, or derivatives thereof in the same oligonucleotide molecule or in separate polynucleotide molecules.

[00111] Methods of producing antibodies are well known in the art and described herein. In some embodiments, both the variable and constant regions of the antigen-binding polypeptides of the present description are Petition 870260079734, dated 07 / 08 / 2026, pp. 44 / 97 / 75 completely human. Completely human antibodies can be produced using techniques described in the art and as described herein. For example, completely human antibodies against a specific antigen can be prepared by administering the antigen to a transgenic animal that has been modified to produce such antibodies in response to antigenic challenge, but whose endogenous loci have been deactivated. Exemplary techniques that can be used to obtain such antibodies are described in US Patents 6,150,584; 6,458,592; 6,420,140, ​​which are incorporated by reference in their entirety.

[00112] In some embodiments, the prepared antibodies will not elicit a deleterious immune response in the animal being treated, for example, in a human. In one embodiment, antigen-binding polypeptides, variants, or derivatives thereof of the description are modified to reduce their immunogenicity using techniques recognized in the art. For example, antibodies can be humanized, primatized, deimmunized, or chimeric antibodies can be made. These types of antibodies are derived from a non-human antibody, typically a murine or primate antibody, which retains or substantially retains the antigen-binding properties of the parent antibody, but which is less immunogenic in humans.This can be achieved by several methods, including (a) grafting complete non-human variable domains onto human constant regions to generate chimeric antibodies; (b) grafting at least a portion of one or more non-human complementarity-determining regions (CDRs) onto a human scaffold and constant regions with or without retention of critical scaffold residues; or (c) transplanting complete non-human variable domains but “camouflaging” them with a human-like section by replacing residues on the surface. Such methods are described in Morrison et al., Proc. Natl. Acad. Sci. USA 57:6851-6855 (1984); Morrison et al., Adv. Immunol. 44:65-92 (1988); Verhoeyen et al., Science 239:1534-1536 (1988); Padlan,. Petition 870260079734, dated 07 / 08 / 2026, page 45 / 97 / 75 Molec. Immun. 25:489-498 (1991); Padlan, Molec. Immun. 31:169-217 (1994), and US patent numbers: 5,585,089, 5,693,761, 5,693,762, and 6,190,370, all being incorporated herein by reference in their entirety.

[00113] Deimmunization can also be used to decrease the immunogenicity of an antibody. As used herein, the term “deimmunization” includes altering an antibody to modify T-cell epitopes (see, for example, International Publication Application Nos.: WO / 9852976 A1 and WO / 0034317 A2). For example, variable heavy chain and variable light chain sequences of the starting antibody are analyzed, and a human T-cell epitope “map” of each V region showing the location of epitopes relative to complementarity-determining regions (CDRs) and key residues within the sequence is created. The individual T-cell epitopes from the T-cell epitope map are analyzed in order to identify alternative amino acid substitutions with a low risk of altering the activity of the final antibody.A range of alternative sequences, both heavy and light, is designed, comprising combinations of amino acid substitutions, and these sequences are subsequently incorporated into a binding polypeptide band. Typically, between 12 and 24 variant antibodies are generated and tested for binding and / or function. The complete heavy and light chain genes, comprising constant variable and modified human regions, are then cloned into expression vectors, and the subsequent plasmids are introduced into cell lines for the production of the complete antibody. The antibodies are then compared in appropriate biochemical and biological assays, and the optimal variant is identified.

[00114] The specificity of antigen-binding polypeptides described herein can be determined by in vitro tests such as immunoprecipitation, radioimmunoassay (RIA) and enzyme-linked immunosorbent assay (ELISA).

[00115] Alternatively, techniques described for the production of Petition 870260079734, dated 07 / 08 / 2026, pp. 46 / 97 / 75 single-chain units (US Patent No. 4,694,778; Bird, Science 242:423442 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 55:5879-5883 (1988); and Ward et al., Nature 334:544-554 (1989)) can be adapted to produce single-chain units of the present description. The single-chain units are formed by linking heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single-chain fusion peptide. Techniques for assembling functional Fv fragments in E. coli can also be used (Skerra et al., Science 242: 1038-1041 (1988)).

[00116] Examples of techniques that can be used to produce single-chain Fvs (scFvs) and antibodies include those described in U.S. Patents numbers 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology 203:46-88 (1991); Shu et al., Proc. Natl. Sci. USA 90:1995-1999 (1993); and Skerra et al., Science 240:1038-1040 (1988). For some uses, including in vivo use of antibodies in humans and in vitro detection tests, it may be preferable to use chimeric, humanized, or human antibodies. A chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a constant region from human immunoglobulin. Methods for producing chimeric antibodies are known in the art. See, for example, Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., J. Immunol.Methods 125:191-202 (1989); U.S. Patents numbers 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entirety.

[00117] Humanized antibodies are antibody molecules derived from a non-human species antibody that bind to the desired antigen by having one or more complementarity-determining regions (CDRs) from the non-human and non-human species framework regions from a human immunoglobulin molecule. Frequently, framework residues Petition 870260079734, dated 07 / 08 / 2026, p. 47 / 97 / 75, states that regions of the human scaffold will be replaced with the corresponding residue of a CDR donor antibody to alter, preferably improve, antigen binding. These scaffold substitutions are identified by methods well known in the art, for example, by modeling the interactions of CDR and scaffold residues to identify scaffold residues important for antigen binding and sequence comparison to identify unusual scaffold residues at particular positions. (See, for example, Queen et al., U.S. Patent No. 5,585,089; Riechmann et al., Nature 332:323 (1988), which are incorporated herein by reference in their entirety.) Antibodies can be humanized using various techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91 / 09967; U.S. Patents Nos. 5,225,539; 5,530,101; and 5,585.089), 'varnish' or surface coating (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28(4 / 5):489-498 (1991); Studnicka et al., Protein Engineering 7(6):805-814 (1994); Roguska et al., Proc. Natl. Sci. USA 91:969-973 (1994)), and chain scrambling (US Patent No. 5,565,332, which is incorporated by reference in its entirety).

[00118] Completely human antibodies are particularly desirable for the therapeutic treatment of human patients. Human antibodies can be obtained by various methods known in the art, including phage display methods using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Patents numbers 4,444,887 and 4,716,111; and PCT publications WO 98 / 46645, WO 98 / 50433, WO 98 / 24893, WO 98 / 16654, WO 96 / 34096, WO 96 / 33735, and WO 91 / 10741; each being incorporated herein by reference in its entirety.

[00119] Human antibodies can also be produced using transgenic mice that are unable to express functional endogenous immunoglobulins, but which can express human immunoglobulin genes. For example, immunoglobulin gene complexes of chains Petition 870260079734, dated 07 / 08 / 2026, page 48 / 97 / 75: Human heavy and light chain genes can be introduced randomly or by homologous recombination into mouse embryonic stem cells. Alternatively, the human variable region, the constant region, and the diversity region can be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes. Mouse heavy and light chain immunoglobulin genes can be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents the production of endogenous antibodies. The modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies.Transgenic mice are immunized in the normal way with a selected antigen, for example, with all or a portion of the desired target polypeptide. Monoclonal antibodies directed against the antigen can be obtained from the immunized transgenic mice using conventional hybridoma technology. The human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation and subsequently undergo class switching and somatic mutation. Thus, using such a technique, it is possible to produce therapeutically usable IgG, IgA, IgM, and IgE antibodies. For an overview of this technology for producing human antibodies, see Lonberg and Huszar Int. Rev. Immunol. 73: 65-93 (1995). For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see Lonberg and Huszar Int. Rev. Immunol. 73: 65-93 (1995).For a detailed discussion of this technology for producing human antibodies and human monoclonal antibodies and protocols for producing such antibodies, see, for example, PCT publications WO 98 / 24893; WO 96 / 34096; WO 96 / 33735; US Patents. Petition 870260079734, dated 07 / 08 / 2026, page 49 / 97 / 75 numbers. 5,413,923; 5,625,126; 5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318 and 5,939,598, which are incorporated herein by reference in their entirety. In addition, companies such as Abgenix, Inc. (Freemont, Calif.) and GenPharm (San Jose, Calif.) may be engaged to provide human antibodies directed against a selected antigen using technology similar to that described above.

[00120] Fully human antibodies that recognize a selected epitope can also be generated using a technique referred to as “guided selection.” In this approach, a selected non-human monoclonal antibody, for example, a mouse antibody, is used to guide the selection of a fully human antibody recognizing the same epitope. (Jespers et al., Bio / Technology 72:899-903 (1988). See also, U.S. Patent No. 5,565,332, which is incorporated by reference in its entirety.)

[00121] In another embodiment, DNA encoding the desired monoclonal antibodies can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of specific binding to the genes encoding the heavy and light chains of murine antibodies). Isolated and subcloned hybridoma cells serve as a preferred source of such DNAs. Once isolated, the DNA can be placed into expression vectors, which are then transfected into prokaryotic or eukaryotic host cells such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, and myeloma cells that do not otherwise produce immunoglobulins. More particularly, the isolated DNA (which may be synthetic, as described herein) can be used to clone constant and variable region sequences for antibody fabrication as described in Newman et al., U.S. Patent No. 5,658.570, filed on January 25, 1995, which is incorporated herein by reference. Essentially, this entails... Petition 870260079734, dated 07 / 08 / 2026, pp. 50 / 97 / 75: RNA extraction from selected cells, conversion to cDNA, and amplification by PCR using specific Ig primers. The appropriate primers for this purpose are also described in US Patent No. 5,658,570. As will be discussed in greater detail below, the transformed cells expressing the desired antibody can be cultured in relatively large quantities to provide clinical and commercial supplies of immunoglobulin.

[00122] Additionally, using routine recombinant DNA techniques, one or more of the CDRs of the antigen-binding polypeptides of the present description can be inserted within the scaffold regions, for example, into human scaffold regions to humanize a non-human antibody. The scaffold regions can be naturally occurring or consensus scaffold regions and, preferably, human scaffold regions (see, for example, Chothia et al., J. Mol. Biol. 278:457-479 (1998) for a listing of human scaffold regions). Preferably, the polynucleotide generated by combining the scaffold regions and CDRs encodes an antibody that specifically binds to at least one epitope of a desired polypeptide, for example, LIGHT. Preferably, one or more amino acid substitutions can be made within the scaffold regions, and preferably, the amino acid substitutions improve the binding of the antibody to its antigen.Additionally, such methods can be used to obtain amino acid substitutions or deletions of one or more cysteine ​​residues from variable regions participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present description and within the specialties of the art.

[00123] In addition, techniques have been developed for the production of “chimeric antibodies” (Morrison et al., Proc. Natl. Acad. Sci. USA:851-855 (1984); Neuberger et al., Nature 372:604-608 (1984); Takeda et al., Nature Petition 870260079734, dated 07 / 08 / 2026, page 51 / 97 / 75 314:452-454 (1985)) by splicing genes from a mouse antibody molecule, of appropriate specificity for the antigen, together with genes from a human antibody molecule of appropriate biological activity, can be used. As used herein, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as having a variable region derived from a murine monoclonal antibody and a constant region from human immunoglobulin.

[00124] Yet another highly efficient means of generating recombinant antibodies is disclosed by Newman, Biotechnology 10: 1455-1460 (1992). Specifically, this technique results in the generation of primate-derived antibodies containing monkey variable domains and human constant sequences. This reference is incorporated by reference in its entirety herein. In addition, this technique is also described in U.S. Patents 5,658,570, 5,693,780 and 5,756,096, each being incorporated herein by reference.

[00125] Alternatively, antibody-producing cell lines can be selected and cultured using techniques well known to those skilled in the art. These techniques are described in several primary publications and laboratory manuals. In this respect, techniques appropriate for use in the description as presented below are described in Current Protocols in Immunology, Coligan et al., Eds., Green Publishing Associates and WileyInterscience, John Wiley and Sons, New York (1991), which is incorporated herein by reference in its entirety, including the appendices.

[00126] Additionally, standard techniques known to those skilled in the art may be used to introduce mutations into the nucleotide sequence encoding an antibody of the present description, including, but not limited to, site-directed mutagenesis and PCR-mediated mutagenesis resulting in amino acid substitutions. Preferably, the variants (including derivatives) encode fewer than 50 amino acid substitutions, fewer than 40 amino acid substitutions, fewer than 30 Petition 870260079734, dated 07 / 08 / 2026, p. 52 / 97 / 75 amino acid substitutions, less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions with respect to the reference variable heavy chain region, CDR-H1, CDR-H2, CDR-H3, variable light chain region, CDR-L1, CDRL2, or CDR-L3. Alternatively, mutations may be randomly introduced along with all or part of the coding sequence, such as by saturation metagenesis, and the resulting mutants may be screened for biological activity to identify mutants that retain activity. Treatment methods and use

[00127] As described herein, the antibodies, variants or derivatives of the present description may be used in some methods of treatment and diagnosis.

[00128] This description is further directed to antibody-based therapies involving the administration of the antibodies described to a patient such as an animal, a mammal, and a human to treat one or more of the disorders or conditions described herein. Therapeutic compounds described include, but are not limited to, antibodies described (including variants and derivatives thereof as described herein) and nucleic acids or polynucleotides encoding antibodies described (including variants and derivatives thereof as described herein).

[00129] The antibodies and fragments of the present description, in some embodiments, can be used for the manufacture of a medicament to treat an inflammatory or autoimmune disease or disorder, or cancer. It is also believed that the antibodies and fragments of the present description are usable for the treatment of the mechanisms underlying pain or pain itself.

[00130] Granulocyte-macrophage colony-stimulating factor Petition 870260079734, dated 07 / 08 / 2026, pp. 53 / 97 / 75 (GM-CSF) was originally known for its ability to generate granulocyte and macrophage colonies from bone marrow precursors. It has also been shown to act on mature myeloid cells as pro-survival, activation, and differentiation factors. Recent studies suggest that GMCSF also has many pro-inflammatory functions and plays a critical role in the development of autoimmune and inflammatory diseases.

[00131] GM-CSF promotes the survival and activation of macrophages, neutrophils, and eosinophils, as well as the maturation of dendritic cells (DCs). GM-CSF can polarize macrophages into M1-type inflammatory macrophages, which produce a variety of inflammatory cytokines, such as TNF, IL-6, IL-12p70, IL-23, or IL-1e, and thus promote Th1-Th1 responses. On the other hand, the association of GM-CSF and Th2 immunity is also reported in allergic airway inflammation.

[00132] The GM-CSF receptor consists of a low-affinity GM-CSF-binding α-subunit (GMRa) and a signal transduction ec subunit that is shared with IL-3 and IL-5 receptors. The binary complex of GM-CSF and GMRa interacts with a free ec-subunit and forms the high-affinity hexamer complex. Dodecamer complexes formed by lateral aggregation of two hexamer complexes allow Jak2 associated with an ec-subunit to dimerize and transphosphorylate, but hexamer complexes do not allow this. This structure leads to dose-dependent responses of GM-CSF receptor activation. Low GM-CSF concentrations, as in normal conditions, cause ec phosphorylation and activate the 14-3-3 / PI-3 kinase pathway, which only leads to cell survival.A higher concentration of GM-CSF, such as in an inflammatory condition, disables ec Ser585 phosphorylation and ec Tyr577 mediated phosphorylation and activates the Jak2 / STAT5 pathway, the mitogen-activated protein kinase / Ras pathway, and the PI-3 kinase pathway, resulting in the promotion of cell survival, proliferation, and activation. Petition 870260079734, dated 07 / 08 / 2026, pages 54 / 97 / 75

[00133] A wide variety of cells can produce GM-CSF. The main sources of GM-CSF are T and B cells, monocyte / macrophage endothelial cells, and fibroblasts. Neutrophils, eosinophils, epithelial cells, mesothelial cells, Paneth cells, chondrocytes, and tumor cells can also produce GM-CSF. GM-CSF production is stimulated by several factors, including TNF, IL-1, Toll-like receptor agonists, and prostaglandin E2. Recently, the pathogenicity of GM-CSF-producing CD4 T cells in autoimmune and inflammatory diseases has been elucidated and is gaining increasing attention.

[00134] Recent evidence has revealed that GM-CSF plays critical roles in the development of many autoimmune diseases. Depletion or neutralization of GM-CSF suppresses many models of autoimmune diseases, including experimental autoimmune encephalomyelitis (EAE), arthritis, arthritis-related interstitial lung disease, nephritis, or psoriasis. It is also suggested that inhibition of GM-CSF may be useful for cancer treatment.

[00135] In some embodiments, the inflammatory disease or condition to be treated by the antibodies, fragments and compositions described includes one or more of the following: Alzheimer's disease, Addison's disease, atherosclerosis, ankylosing spondylitis, arthritis, osteoarthritis (OA), rheumatoid arthritis (RA), psoriatic arthritis (PA), ankylosing spondylitis, asthma, atherosclerosis, chronic obstructive pulmonary disease (COPD), Crohn's disease, colitis, dermatitis, diverticulitis, fibromyalgia, hepatitis, irritable bowel syndrome (IBS), systemic lupus erythematosus (SLE), nephritis, Parkinson's disease (PD), vasculitis, and ulcerative colitis.

[00136] In some embodiments, the autoimmune disease or condition to be treated by the antibodies, fragments and compositions described includes one or more of the following: alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes (type 1), celiac disease, autoimmune juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, Petition 870260079734, dated 07 / 08 / 2026, page 55 / 97 / 75 idiopathic thrombocytopenic purpura, myasthenia gravis, autoimmune myocarditis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjögren's syndrome, systemic lupus erythematosus, autoimmune thyroiditis, Hashimoto's thyroiditis, autoimmune uveitis, vitiligo, and granulomatosis with polyangiitis (of Wegener).

[00137] Rheumatoid arthritis (RA) is a long-term autoimmune disorder that primarily affects the joints. It typically results in warm, swollen, and painful joints. Pain and stiffness often worsen following rest. Most commonly, the wrists and hands are involved, with the same joints typically affected on both sides of the body. The disease can also affect other parts of the body. Although the cause of rheumatoid arthritis is unclear, it is believed to involve a combination of genetic and environmental factors. The underlying mechanism involves the body's immune system attacking the joints. This results in inflammation and thickening of the joint capsule. The goals of treatment are to reduce pain, decrease inflammation, and improve the person's overall functioning. Pain medications, steroids, and NSAIDs are often used to help with symptoms.A group of medications called disease-modifying antirheumatic drugs (DMARDs), such as hydroxychloroquine and methotrexate, can be used to try to slow the progression of the disease.

[00138] Osteoarthritis (OA) is a type of joint disease that results from the deterioration of the cartilage in the joints and the underlying bone. The most common symptoms are pain and stiffness in the joints. Initially, symptoms may only occur following exercise, but over time they can become constant. Other symptoms may include joint swelling, decreased range of motion, and, when the back is affected, weakness or numbness in the arms and legs. Causes include damage Petition 870260079734, dated 07 / 08 / 2026, pp. 56 / 97 / 75. Previous joint involvement, abnormal joint or limb development, and hereditary factors are risk factors. The risk is higher in those who are overweight, have one leg of a different length, and have jobs that result in high levels of stress on the joints. Osteoarthritis is believed to be caused by mechanical stress on the joint and low-grade inflammatory processes. Treatment includes exercise, efforts to reduce joint stress, support groups, and pain medication.

[00139] Multiple sclerosis (MS) is a demyelinating disease in which the insulating coverings of nerve cells in the brain and spinal cord are damaged. This damage disrupts the ability of parts of the nervous system to communicate, resulting in a range of signs and symptoms, including physical, mental, and sometimes psychiatric problems. Specific symptoms may include double vision, blindness in one eye, muscle weakness, problems with sensation, or coordination problems. Although the cause is unclear, the underlying mechanism is thought to be either destruction by the immune system or failure of the myelin-producing cells. There is no known cure for multiple sclerosis. Treatments attempt to improve function after an attack and prevent new attacks.

[00140] Asthma is a common inflammatory disease of the airways in the lungs. It is distinguished by variable and recurring symptoms, reversible airflow obstruction, and bronchospasm. Symptoms include episodes of wheezing, coughing, chest tightness, and shortness of breath. Asthma is believed to be caused by a combination of genetic and environmental factors. Environmental factors include exposure to air pollution and allergens. Asthma is classified according to symptom frequency, forced expiratory volume in one second (FEV1), and peak expiratory flow rate. It can also be classified as atopic or non-atopic, where atopy refers to a predisposition to developing a type 1 hypersensitivity reaction. There is no cure for asthma. Symptoms can be avoided by avoiding triggers. Petition 870260079734, dated 07 / 08 / 2026, page 57 / 97 / 75 triggers, such as allergens and irritants, and the use of inhaled corticosteroids. Long-acting beta-agonists (LABAs) or leukotriene antagonists may be used in addition to inhaled corticosteroids if asthma symptoms remain uncontrolled. Treatment of rapidly worsening symptoms is usually with a short-acting beta-2 agonist such as salbutamol and oral corticosteroids. In very severe cases, intravenous corticosteroids, magnesium sulfate, and hospitalization may be required.

[00141] Chronic obstructive pulmonary disease (COPD) is a type of obstructive lung disease distinguished by long-term poor airflow. COPD can include two main conditions, emphysema and chronic bronchitis. In emphysema, the walls between many of the air sacs are damaged. As a result, the air sacs lose their shape and become flaccid. This damage can also destroy the walls of the air sacs, leading to fewer and larger air sacs instead of many small ones. If this happens, the amount of gas exchange in the lungs is reduced. In chronic bronchitis, the lining of the airways becomes constantly irritated and inflamed, and this causes the lining to swell. Too much thick mucus forms in the airways, making breathing difficult. There is no known cure for COPD, but the symptoms are treatable and its progression can be slowed.

[00142] Pain is a painful sensation often caused by intense or harmful stimuli, such as stubbing your toe, burning a finger, putting alcohol on a cut, or hitting your elbow. Pain is a complex, subjective phenomenon; defining pain has been a challenge. Pain is also referred to as an unpleasant sensation and emotional experience associated with potential and actual tissue damage. Pain is sometimes considered a symptom of an underlying condition, such as inflammation.

[00143] In some forms, methods are provided for the Petition 870260079734, dated 07 / 08 / 2026, pp. 58 / 97 / 75, describes the treatment of cancer in a patient in need of it. The method, in one embodiment, involves administering to the patient an effective quantity of an antibody as described herein.

[00144] Non-limiting examples of cancers include bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer.

[00145] Additional diseases or conditions associated with increased cell survival that can be treated, prevented, diagnosed, and / or predicted with the antibodies or variants thereof, or derivatives thereof, described include, but are not limited to, progression and / or metastasis of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas, such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, duct carcinoma, Petition 870260079734, dated 07 / 08 / 2026, page 59 / 97 / 75 biliary, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testicular tumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, menangioma, melanoma, neuroblastoma and retinoblastoma.

[00146] A specific dosage and treatment regimen for any particular patient will depend on a variety of factors, including the particular antibodies, variants or derivatives thereof used, the patient's age, body weight, general health, sex, and diet, and the timing of administration, rate of excretion, drug combination, and the severity of the particular disease being treated. Judgment of such factors by medical caregivers is within the usual capacity of those skilled in the art. The amount will also depend on the individual patient being treated, the route of administration, the type of formulation, the characteristics of the compound used, the severity of the disease, and the desired effect. The amount used can be determined by pharmacological and pharmacokinetic principles well known in the art.

[00147] Methods of administering antibodies, variants include, but are not limited to, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. Antigen-binding polypeptides or compositions may be administered by any convenient route, for example by bolus infusion or injection, by absorption through epithelial or mucocutaneous linings (e.g., oral, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Thus, pharmaceutical compositions containing the antigen-binding polypeptides of the description may be administered orally, rectally, parenterally, intracisternally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal adhesive dressings), buccally, or as Petition 870260079734, dated 07 / 08 / 2026, pp. 60 / 97 / 75, an oral or nasal spray.

[00148] The term “parenteral” as used here refers to modes of administration that include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intra-articular injection and infusion.

[00149] Administration may be systemic or local. In addition, it may be desirable to introduce the antibodies described into the central nervous system by any appropriate route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration may also be employed, for example, by the use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

[00150] It may be desirable to administer the antigen-binding polypeptides or compositions of the description locally in the area requiring treatment; this can be achieved, for example, and not as a limitation, by local infusion during surgery, topical application, for example, in conjunction with a wound dressing after surgery, by injection, via a catheter, via a suppository, or via an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers. Preferably, when administering a protein, including an antibody, of the description, care should be taken to use materials that the protein does not absorb.

[00151] In another embodiment, the antigen-binding polypeptide or compound may be released into a vesicle, in particular a liposome (see Langer, 1990, Science 249:1527-1533; Treat et al., in Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler (eds.), Liss, New York, pp. 353-365 (1989); Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

[00152] In yet another embodiment, the polypeptide binding to Petition 870260079734, dated 07 / 08 / 2026, page 61 / 97 / 75 antigen or composition may be released in a controlled delivery system. In one embodiment, a pump may be used (see Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201; Buchwald et al., 1980, Surgery 88:507; Saudek et al., 1989, N. Engl. J. Med. 321:574). In another embodiment, polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy et al., 1985, Science 228:190; During et al., 1989, Ann. Howard et al., 1989, J. Neurosurg.In yet another modality, a controlled-release system can be placed in close proximity to the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (see, for example, Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)). Other controlled-release systems are discussed in Langer's review (1990, Science 249:1527-1533).

[00153] In a specific embodiment, where the composition of the description comprises a nucleic acid or polynucleotide encoding a protein, the nucleic acid may be administered in vivo to promote the expression of its encoded protein, by constructing the same as part of an appropriate nucleic acid expression vector and administering the same in such a way that it becomes intracellular, for example, by use of a retroviral vector (see U.S. Patent No. 4,980,286), or by direct injection or by use of microparticle bombardment (e.g., a gene gun; Biolistic, DuPont), or coating with lipids or cell surface receptors or transfecting agents, or by administering the same in connection with a homeobox-type peptide that is known as entering the nucleus (see, for example, Joliot et al., 1991, Proc. Natl. Acad. Sci. USA 88:1864-1868), etc. Alternatively, a nucleic acid can be introduced intracellularly and Petition 870260079734, dated 07 / 08 / 2026, pp. 62 / 97 / 75 incorporated into the host cell's DNA by expression, through homologous recombination.

[00154] The amount of antibodies described that will be effective in treating, inhibiting, and preventing an inflammatory, immune, or malignant disease, disorder, or condition can be determined by standard clinical techniques. In addition, in vitro tests may optionally be employed to help identify ideal dosage ranges. The precise dose to be used in the formulation will also depend on the route of administration and the severity of the disease, disorder, or condition, and should be decided according to the judgment of healthcare personnel and the circumstances of each patient. Effective doses can be extrapolated from dose-response curves derived from in vitro or animal model testing systems.

[00155] As a general guideline, the dosage administered to a patient of the antigen-binding polypeptides described herein is typically 0.1 mg / kg to 100 mg / kg of the patient's body weight, between 0.1 mg / kg and 20 mg / kg of the patient's body weight, or 1 mg / kg to 10 mg / kg of the patient's body weight. Generally, antibodies have a longer half-life within the human body than antibodies of other species due to the immune response to foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration are often possible. Additionally, the dosage and frequency of administration of the described antibodies can be reduced by enhancing the absorption and tissue penetration (e.g., brain) of the antibodies through modifications such as, for example, lipidation.

[00156] Methods for treating an infectious or malignant disease, condition, or disorder comprising administering an antibody, variant, or derivative thereof as described are typically tested in vitro and then in vivo in an acceptable animal model for the desired therapeutic treatment or prophylactic activity prior to use in Petition 870260079734, dated 07 / 08 / 2026, pp. 63 / 97 / 75 humans. Suitable animal models, including transgenic animals, are well known to those skilled in the art. For example, in vitro tests to demonstrate the therapeutic utility of the antigen-binding polypeptide described herein include the effect of an antigen-binding polypeptide on a cell line or on a patient tissue sample. The effect of the antigen-binding polypeptide on the cell line and / or tissue sample can be determined using techniques known to those skilled in the art, such as the tests disclosed elsewhere herein.According to the description, in vitro tests that can be used to determine whether administration of a specific antigen-binding polypeptide is indicated include in vitro cell culture tests in which a patient tissue sample is grown in culture and exposed to, or otherwise administered with, a compound, and the effect of such compound on the tissue sample is observed.

[00157] Several delivery systems are known and can be used to administer an antibody of the description or a polynucleotide encoding an antibody of the description, for example, encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor-mediated endocytosis (see, for example, Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432), construction of a nucleic acid as part of a retroviral vector or other vector, etc.

[00158] In another embodiment, the compositions of the description are administered in combination with an antineoplastic agent, an antiviral agent, an antibacterial agent, or an antibiotic or antifungal agent. Any of these agents known in the art may be administered in the compositions of the present description.

[00159] In another embodiment, compositions of the description are administered in combination with a chemotherapeutic agent. Chemotherapeutic agents that may be administered with compositions of the description include, but are not limited to, antibiotic derivatives (by Petition 870260079734, dated 07 / 08 / 2026, page 64 / 97 / 75 example, doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g., tamoxifen); antimetabolites (e.g., fluorouracil, 5-FU, methotrexate, floxuridine, interferon alfa-2b, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine); cytotoxic agents (e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cisplatin, and vincristine sulfate); hormones (e.g., medroxyprogesterone, estramustine sodium phosphate, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and testolactone); nitrogen mustard derivatives (e.g., mephalene, corambucil, mechlorethamine (nitrogen mustard), and thiotepa); steroids and combinations (e.g., betamethasone sodium phosphate);and others (for example, dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide). Combination compositions and therapies

[00160] The anti-GM-CSF antibodies described herein may be used, in some embodiments, together with another therapeutic agent.

[00161] In some embodiments, the second therapeutic agent is an anti-inflammatory agent. Non-limiting examples include aspirin, ibuprofen, naproxen, celecoxib (Celebrex), piroxicam (Feldene), indomethacin (Indocin), meloxicam (Mobic Vivlodex), ketoprofen (Orudis, Ketoprofen ER, Oruvail, Actron), sulindac (Clinoril), diflunisal (Dolobid), nabumetone (Relafen), oxaprozin (Daypro), tolmetin (Tolmetin sodium, Tolectin), salsalate (Disalcid), etodolac (Lodine), fenoprofen (Nalfon), flurbiprofen (Ansaid), ketorolac (Toradol), meclofenamate, and mefenamic acid (Ponstel).

[00162] In some modalities, the second therapeutic agent is appropriate for the treatment of an autoimmune disease. Non-limiting examples include glucocorticoids, an anti-Cd3 antibody such as Petition 870260079734, dated 07 / 08 / 2026, pages 65 / 97 / 75 Muromonab-CD3, IL-2a inhibitors such as basiliximab (Simulect) and daclizumab (Zenapax), calcineurin inhibitors such as tacrolimus and cyclosporine, sirolimus, everolimus, interferons, opioids, TNF-binding proteins or antibodies, and mycophenolate.

[00163] In some modalities, the second therapeutic agent is a cancer chemotherapeutic agent. Chemotherapeutic agents can be categorized by their mechanisms of action into, for example, the following groups: - antimetabolites / anticancer agents such as pyrimidine analogues floxuridine, capecitabine, and cytarabine; - purine analogs, folate antagonists, and related inhibitors; - Antiproliferative / antimitotic agents including natural products such as vinca alkaloids (vinblastine, vincristine) and microtubules such as taxanes (paclitaxel, docetaxel), vinblastine, nocodazole, epotilones, vinorelbine (NAVELBINE®), and epipodophyllotoxins (etoposide, teniposide); - DNA-damaging agents such as actinomycin, ansacrine, busulfan, carboplatin, chlorambucil, cisplatin, cyclophosphamide (CITOXAN®), dactinomycin, daunorubicin, doxorubicin, epirubicin, ifosfamide, melphalan, merchlorethamine, mitomycin, mitoxantrone, nitrosourea, procarbazine, taxol, taxotero, teniposide, etoposide, and triethylenethiophosphoramide; - antibiotics such as dactinomycin, daunorubicin, doxorubicin, idarubicin, anthracyclines, mitoxantrone, bleomycins, plicamycin (mithramycin), and mitomycin; - enzymes such as L-asparaginase that systemically metabolize L-asparagine and deprive cells that do not have the ability to synthesize their own asparagine; - antiplatelet agents; Petition 870260079734, dated 07 / 08 / 2026, pages 66 / 97 / 75 - Antiproliferative / antimitotic alkylating agents such as nitrogen mustards, cyclophosphamide and analogues (melphalan, chlorambucil, hexamethylmelamine, and thiotepa), alkyl nitrosoureas (carmustine) and analogues, streptozocin and triazenes (dacarbazine); - antiproliferative / antimitotic antimetabolites such as folic acid analogues (methotrexate); - platinum coordination complexes (cisplatin, oxyloplatinum, and carboplatin), procarbazine, hydroxyurea, mitotane, and aminoglutethimide; - hormones, hormone analogues (estrogen, tamoxifen, goserelin, bicalutamide, and nilutamide), and aromatase inhibitors (letrozole and anastrozole); - anticoagulants such as heparin, synthetic heparin salts, and other thrombin inhibitors; - fibrinolytic agents such as tissue plasminogen activator, streptokinase, urokinase, aspirin, dipyridamole, ticlopidine, and clopidogrel; - anti-immigration agents; - antisecretory agents (breveldine); - immunosuppressants tacrolimus, sirolimus, azathioprine, and mycophenolate; - compounds (TNP-470, genistein) and growth factor inhibitors (vascular endothelial growth factor inhibitors and fibroblast growth factor inhibitors); - angiotensin receptor blockers, nitric oxide donors; - antisense oligonucleotides; - antibodies such as trastuzumab and rituximab; - cell cycle inhibitors and differentiation inducers such as Petition 870260079734, dated 07 / 08 / 2026, pp. 67 / 97 / 75 tretinoin; - inhibitors, topoisomerase inhibitors (doxorubicin, daunorubicin, dactinomycin, eniposide, epirubicin, etoposide, idarubicin, irinotecan, mitoxantrone, topotecan, and irinotecan), and corticosteroids (cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisone, and prednisolone); - kinase inhibitors of growth factor signal transduction - inducers of dysfunction; - toxins such as cholera toxin, ricin, Pseudomonas exotoxin, Bordetella pertussis adenylate cyclase toxin, diphtheria toxin and caspase activators - and chromatin. Compositions

[00164] The present description also provides pharmaceutical compositions. Such compositions comprise an effective amount of an antibody and an acceptable carrier. In some embodiments, the composition additionally includes a second therapeutic agent.

[00165] In a specific embodiment, the term “pharmaceutically acceptable” means approved by a federal or state government regulatory agency or listed in the US pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans. Furthermore, a “pharmaceutically acceptable carrier” will generally be a non-toxic solid, semi-solid, or liquid filler, diluent, encapsulation material, or auxiliary formulation of any kind.

[00166] The term “carrier” refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as Petition 870260079734, dated 07 / 08 / 2026, pp. 68 / 97 / 75. Peanut oil, soybean oil, mineral oil, sesame oil and similar products. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous solutions of dextrose and glycerol may also be used as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dry skimmed milk, glycerol, propylene glycol, water, ethanol and similar products. The composition may also contain smaller amounts of wetting agents or emulsifiers, or pH buffering agents such as acetates, citrates or phosphates, if desired.Antibacterial agents such as benzyl alcohol or methylparabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and tonicity adjusters such as sodium chloride or dextrose are also considered. These compositions may take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations, and the like. The composition may be formulated as a suppository, with traditional binders and carriers such as triglycerides. The oral formulation may include standard carriers such as pharmaceutical types of mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences by E.W. Martin, incorporated herein by reference.These compositions will contain a therapeutically effective amount of the antigen-binding polypeptide, preferably in purified form, along with an appropriate amount of carrier to provide the appropriate dosage form for the patient. The formulation must be suitable for the route of administration. The parenteral preparation may be enclosed in ampoules, disposable syringes, or multi-dose vials made of glass. Petition 870260079734, dated 07 / 08 / 2026, pages 69 / 97 / 75 or plastic.

[00167] In one embodiment, the composition is formulated according to routine procedures as a pharmaceutical composition adapted for intravenous administration in humans. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to relieve pain at the injection site. Generally, the ingredients are supplied either separately or mixed together in a unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the amount of active agent. Where the composition is to be administered by infusion, it may be dispensed with an infusion bottle containing sterile pharmaceutical-grade saline or water.Where the composition is administered by injection, an ampoule of sterile water for injection or saline solution may be provided so that the ingredients can be mixed before administration.

[00168] The compounds described may be formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. EXAMPLES Example 1. Generation of murine antibodies

[00169] This example describes the process of preparing anti-human GM-CSF mouse monoclonal antibodies using hybridoma technology. Recombinant human GM-CSF protein was used as the antigen. To generate mouse monoclonal antibodies to human GM-CSF, different strains of 6-8 week old mice were used, including mice Petition 870260079734, dated 07 / 08 / 2026, pp. 70 / 97 / 75 BALB / c, C57 / BL6, or SJL mice were first immunized with 20 μg of recombinant human GM-CSF. On days 14, 28, and 42 after the first immunization, the immunized mice were re-immunized with 5 μg of recombinant protein. To select mice producing antibodies that bind the GM-CSF protein, sera from immunized mice were tested by ELISA. Briefly, microtiter plates were coated with human GM-CSF protein at 1 μg / ml in PBS, 100 μl / well at room temperature (RT) overnight, then blocked with 100 μl / well of 5% BSA. Plasma dilutions from immunized mice were added to each well and incubated for 1–2 hours at RT. The plates were washed with PBS / Tween and then incubated with horseradish peroxidase (HRP)-conjugated anti-mouse IgG antibody for 1 hour at RT. After washing, the plates were revealed with ABTS substrate and analyzed by spectrophotometer at OD 405nm.Mice with sufficient anti-GM-CSF IgG titers were boosted with 25 μg of recombinant human GM-CSF protein on day 60 post-immunization. The resulting mice were used for fusions.

[00170] Hybridoma supernatants were tested for anti-GM-CSF IgGs by ELISA screening. Primary ELISA-positive hybridoma clones were selected for subcloning using the limited dilution method and further tested by confirmatory ELISA binding assay (Figure 1) and TF-1 proliferation assay (Figure 2). Prior to GM-CSF stimulation, TF-1 cells were washed with RPMI1640 basal medium and left serum-free overnight. On day 2, these depleted cells were collected and then seeded at a concentration of 3x10⁵ cells / ml in 50 μl per well of a 96-well flat-bottom cell culture plate.

[00171] Recombinant human GM-CSF (Genscript) at a concentration of 0.2 ng / ml (4X) was mixed 1:1 with culture supernatant. Petition 870260079734, dated 07 / 08 / 2026, page 71 / 97 / 75. 20% (4X) hybridoma and 50 μL of the mixture were added to TF1 cells, thus the final concentration for GM-CSF was 0.05 ng / ml and for hybridoma supernatant was 5%. Maximum cell proliferation (0% inhibition) was measured by incubating TF-1 cells at a final GM-CSF concentration of 0.05 ng / ml, without the addition of hybridoma supernatant. 100% inhibition of TF-1 proliferation was measured by omitting GM-CSF from the test and maintaining the cells only in complete RPMI1640 medium. TF-1 cells were then incubated for 72 ha at 37°C. Cell viability was measured using the CellTiter-Glo® luminescent cell viability assay according to the manufacturer's protocol.

[00172] Overall, there were three hybridoma monoclones, 23F4, 32C4, and 50C5, that showed significant inhibition of TF-1 proliferation, while 23F4 and 50C5 were selected for further characterization. Example 2. Binding of murine antibodies to human or Rhesus GM-CSF

[00173] This example tests the dose response to ELISA binding of mouse anti-GM-CSF mAb to recombinant human or rhesus GM-CSF protein (1 μg / ml @ 100 μl).

[00174] Human or recombinant rhesus GM-CSF protein (Genscript) was coated with 1 μg / ml in PBS in microtiter plates for 2 h at room temperature (RT). After antigen coating, the wells were blocked with PBS / 0.05% Tween (PBST) with 1% BSA for 1 h RT. After washing the wells with PBST, different concentrations of anti-GM-CSF antibodies were added to the well and incubated for 1 h RT. For detection of binding antibodies, HRP-conjugated secondary antibodies against Fc mice (Jackson Immuno Research) were added, followed by the addition of fluorogenic substrates (Roche). Between all incubation steps, the plate wells were washed with PBST three times. Fluorescence was measured using a TECAN Spectrafluor plate reader. Petition 870260079734, dated 07 / 08 / 2026, pages 72 / 97 / 75

[00175] As shown in Figure 3, both antibodies 23F4 and 50C5 showed dose-dependent binding to human GM-CSF with EC50s of 11.8 ng / ml and 14.6 ng / ml, respectively, and rhesus GM-CSF with EC50s of 10.2 ng / ml and 21.7 ng / ml, respectively. Example 3. Binding kinetics of murine antibodies to human GM-CSF

[00176] Figure 4 graphically shows the binding kinetics of 23F4 and 50C5 with recombinant human GM-CSF. Recombinant human GM-CSF was defined as an analyte with serial concentrations (100, 50, 25, 12.5, 6.25, 3.125 nM). The antibody-antigen binding kinetics assay was performed using the Biacore T200 system via a mouse antibody capture approach. Anti-mouse Fc IgG was immobilized on a chip with a CM5 sensor, according to the manufacturer's instructions. The test antibody was injected and captured by immobilized anti-mouse Fc IgG. Then, serial concentrations of the antigen were injected individually, and the binding profile was recorded for each antigen analyte concentration, respectively. The test system was regenerated by injection of 10 mM Glycine-HCl, pH 1.5, for 30 seconds.The cycle buffer was HBSEP+ (10mM HEPES, pH 7.4, 150mM NaCl, 3mM EDTA and 0.05% P20). The test temperature was 25 °C, and the association and dissociation times were 180 and 600 seconds, respectively.

[00177] The Biacore data were fitted using the Biacore T200 1.0 evaluation software according to the 1:1 linkage model, to calculate the association rate constants (ka) and dissociation rate constants (kd), as well as the equilibrium constant (KD). In addition to Figure 4, some summary data are presented in the table below. Sample ka (1 / Ms) kd (1 / s) KD (M) 23F4 8.723E+05 3.635E-05 4.168E-11 50C5 2.718E+06 1.204E-04 4.431E-11 Example 4. Blocking the binding of GM-CSF to the alpha GMCSF receptor by murine antibodies. Petition 870260079734, dated 07 / 08 / 2026, pages 73 / 97 / 75

[00178] In order to test the potency of antibodies in blocking GM-CSF binding to the alpha chain of the GM-CSF receptor, recombinant human GMCSF receptor alpha protein (CD116) was coated at 2 μg / ml in PBS on microtiter plates overnight at 4°C. After antigen coating, the wells were blocked with PBS / 0.05% Tween (PBST) with 1% BSA for 1 ha RT. After washing the wells with PBST, different concentrations of anti-GM-CSF antibodies were added to the well in the presence of biotinylated human GM-CSF protein (0.05 μg / ml) and incubated for 1 ha RT.

[00179] For detection of biotinylated GM-CSF binding to the coated receptor, HRP-conjugated streptavidin was added, followed by the addition of fluorogenic substrates (Roche). Between all incubation steps, the plate wells were washed with PBST three times. Fluorescence was measured on a TECAN Spectrafluor plate reader. As shown in Figure 5, both antibodies showed dose-dependent inhibition of GMCSF binding to the alpha GM-CSF receptor. Example 5. GM-CSF blockade by murine antibody-induced pSTAT5 signaling.

[00180] CD14+ monocytes were purified from human peripheral blood using CD14-positive microbeads (Miltenyi Biotec). The purified monocytes were stimulated with human GM-CSF (0.2 ng / ml) for 30 minutes at 37°C in the presence of different concentrations of 23F4 antibody. After incubation, the cells were collected and washed with FACS buffer (1x PBS + 2% FBS) and permeabilized with 2% PFA, followed by cell fixation using ice-cold methanol. Then, PE-conjugated antipSTAT5 antibody was added to the cells for another 30-minute incubation at 4°C and analyzed by flow cytometry. The % inhibition was calculated by [1-(MFI test sample / MFI control)] x 100%. The addition of antibodies would significantly decrease the activation level of pSTAT5. Petition 870260079734, dated 07 / 08 / 2026, pp. 74 / 97 / 75 induced by GM-CSF at a dose of 0.1 or 1 pg / ml (Figure 6). Example 6. GM-CSF-dependent inhibition of TF-1 proliferation by murine antibodies.

[00181] Prior to stimulation with GM-CSF, TF-1 cells were washed with RPMI1640 basal medium and left without medium overnight. On day 2, these depleted cells were collected and then seeded at a concentration of 3x10⁵ cells / ml in 50 µl per well of a 96-well flat-bottom cell culture plate. Recombinant human GM-CSF (Genscript) at a concentration of 0.2 ng / ml (4X) was mixed 1:1 with murine anti-GM-CSF antibodies (0.01 ng / ml - 1000 ng / ml diluted in complete medium) and 50 µl of the mixture were added to TF-1 cells. Maximum cell proliferation (0% inhibition) was measured by incubating TF-1 cells at a final GM-CSF concentration of 0.05 ng / ml, without the addition of antibody. 100% inhibition of TF-1 proliferation was measured by omitting GM-CSF from the test and maintaining the cells only in complete RPMI1640 medium. TF-1 cells were then incubated for 72 ha at 37°C.Cell viability was measured by the CellTiter-Glo® luminescent cell viability assay according to the manufacturer's protocol. The IC50 values ​​for 23F4 and 50C5 in inhibiting TF-1 proliferation were both approximately 10.9 ng / ml (Figure 7). Example 7. Humanization of murine antibodies

[00182] Genes from the variable region of murine antibodies were used to create humanized MAb. In the first step of this process, the amino acid sequence of the VH and VK of the antibodies was compared against the available database of human Ig gene sequences to find all the human germline Ig gene sequences that best match.

[00183] Humanized variable domain sequences were then designed where the CDR1, 2, and 3 of the antibody light and heavy chains were grafted onto the scaffold sequences of human Ig genes. A 3D model was then generated to determine if any scaffold positions would exist. Petition 870260079734, dated 07 / 08 / 2026, p. 75 / 97 / 75, where the substitution of a mouse amino acid for a human amino acid (reverse mutations) could affect the linkage and / or conformation of the CDR. Relevant sequences and reverse mutations are shown in the tables below. CDR sequences of 23F4 VH CDR1 SHYLH (SEQ ID NO. 1) CDR2 WIFPGDDKTKYNEKFKG (SEQ ID NO. 2) CDR3 GTKYLNWNFDV (SEQ ID NO. 3) 23F4 VL CDR sequences CDR1 KANQNVGTTLA (SEQ ID NO. 4) CDR2 SASYRYS (SEQ ID NO. 5) CDR3 HQYTTYPLT (SEQ ID NO. 6) Humanization project for 23F4 Project VH I: VH1-f / JH6 Construct Mutation 23F4 VH Chimera 23F4 VH.1 grafted with CDR, Q1E, T98R 23F4 VH.1a Based on 23F4 VH.1, A72S 23F4 VH.1b Based on 23F4VH.1, A72S, V68A 23F4 VH.1c Based on 23F4VH.1, A72S, V68A, I70L, M48I 23F4 VH.1d Based on 23F4VH.1, A72S, V68A, I70L, M48I, G26D, F29L Project VH II: VH1-f / JH6 23F4 VH.2 grafted with CDR, Q1E 23F4 VH.2a Based on 23F4 VH.2, R72S 23F4 VH.2b Based on 23F4 VH.2, R72S, M70L 23F4 VH.2c Based on 23F4 VH.2, R72S, M70L, M48I, V68A 23F4 VH.2d Based on 23F4 VH.2, R72S, M70L, M48I, V68A, G26D, F29L VK Project: L8 / Jk4 Mutation Construct 23F4Vk Chimera 23F4 Vk.1 grafted with CDR 23F4 Vk.1a Based on 23F4 Vk.1, L46A 23F4 Vk.1b Based on 23F4 Vk.1, L46A, S60D, E70D 23F4 Vk.1c Based on 23F4 Vk.1, L46A, S60D, E70D, A43S, Y87F

[00184] The amino acid and nucleotide sequences of some of the humanized 23F4 antibodies are listed in the table below. Humanized antibody sequences (CDR residues are underlined and retromutations are indicated in boxes) 23F4 VH QVQLQQSGPELVKPGTSMKISCKTSDYTLTSHYLHWVKQR PGQGLEWIGW IFPGDDKTKYNEKFKGKATLTSDKTSNTAYMQLSSLTSEES AVYFCARGT KYLNWNFDV WGTGTTVTVS S SEQ ID NO.7 Petition 870260079734, dated 07 / 08 / 2026, pages 76 / 97 / 75 23F4 VH.1 23F4 VH. Evqlvqsgaevkkpgatvkisckvsgytftshylhwvqqa PGKGLEWMGW ifpgddktkynekfkgrvtitSdtstdtaymelsslrsedta vyycaRgt KYLNUNFDVWGQGTTVTVS S SEQ ID NO.9 23F4 VH.1b Evqlvqsgaevkkpgatvkisckvsgytftshylhwvqqa PGKGLEWMGW ifpgddktkynekfkgRATltSdtstdtaymelsslrsedt avyycaRgt KYLNWNFDVWGQGTTVTVS S SEQ ID NO.10 23F4 VH.1c Evqlvqsgaevkkpgatvkisc PGKGLEW IFPGDDKTKYNEKFKGR[AtLTÍS AVYYCA KYLNWNFDVWG KVSGYTFTSHYLHWVQQA 1 |gw DTSTDTAYMELS SLRSEDT Rgt QGTTVTVSS SEQ ID NO.11 23F4 VH.1d Evqlvqsgaevkkpgatvkisc APGKGLE3 IFPGDDKTKYNEKFKGR[At|lTÍS AVYYCA KYLNWNFDVWG kvsdytLtshylhwvqq y |gw DTSTDTAYMELS SLRSEDT Rgt QGTTVTVSS SEQ ID NO.12 23F4 VH.2 Evqlvqsgaevkkpgasvkvsckasgytftshylhwvrq APGQGLEWMGW ifpgddktkynekfkgrvtmtrdtsistaymelsrlrsddt avyycaRgt KYLNWNFDVWGQGTTVTVS S SEQ ID NO.13 23F4 VH.2a Evqlvqsgaevkkpgasvkvsckasgytftshylhwvrq APGQGLEWMGW ifpgddktkynekfkgrvtmtSdtsistaymelsrlrsddt avyycARgt KYLNWNFDVWGQGTTVTVS S SEQ ID NO.14 23F4 VH.bkvMGW ifpgddktkynekfkgrvtmtSdtsistaymelsrlrsddt Evqlvqsgaevkkpgasvkvs APGQGLEV IFFGDDKTKYNEKFKGRVtILTÍS AVYYCA KYLNWNFDVWG ckasgytftshylhwvrq C MGW dtsistaymelsrlrsddt Rgt QGTTVSS SEQ ID NO.13 2F4 VHc. Evqlvqsgaevkkpgasvkvsckasgytftshylhwvrq apgqglew|gw ifpgddctkynekfkgRÃ|tE|tSdtsistaymelsrlrsddt avyycARgt KYLNWNFDVWGQGTTVTVS S SEQ ID NO.16 23F4 VH.22dktvTVS. EvqlvqsgaevkkpgasvkvsckasdytEtshylhwvrq apgqglew|gw ifpgddktkynekfkgRÃ|tE|tSdtsistaymelsrlrsddt avyycARgt KYLNWNFDVWGQGTTVTVS S SEQ ID NO.17 23F4Vk divltqpqkflstsvgdrvswtckanqnvgttlawyqqkp GQSPKALIYS asyrysgvpdrftgsgsgtdftltisnvqsedlaeyfchqy TTYPLTFGG GTKLEIK SEQ ID NO.18. Petition 870260079734, of 07 / 08 / 2026, p. 77 / 97 / 75 23F4 Vk.1 DIQLTQSPSFLSASVGDRVTITCKANQNVGTTLAWYQQKPG KAPKLLIYS ASYRYSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCHQYT TYPLTFGG GTKVEIK SEQ ID NO.19 23F4 Vk.1a DIQLTQSPSFLSASVGDRVTITCKANQNVGTTLAWYQQKPG KAPKALIYS ASYRYSGVPSRFSGSGSGTEFTLTISSLQPEDFATYYCHQYT TYPLTFGG GTKVEIK SEQ ID NO.20 23F4 Vk.1b DIQLTQSPSFLSASVGDRVTITCKANQNVGTTLAWYQQKPG KAPKALIYS ASYRYSGVPDRFSGSGSGTDFTLTISSLQPEDFATYYCHQY TTYPLTFGG GTKVEIK SEQ ID NO.21 23F4 Vk.1c DIQLTQSPSFLSASVGDRVTITCKANQNVGTTLAWYQQKPG KAPKALIYS asyrysgvpdrfsgsgsgtdftltisslqpedfaty|chqyt TYPLTFGG GTKVEIK SEQ ID NO.22 Combining VH / VK for human protection 23F4 Vk VH 23F4 Vk.1 23F4 Vk.1a 23F4 Vk.1b 23F4 Vk.1c 23F4 Vk 23F4 VH.1 Hu23F4-1 Hu23F4-2 Hu23F4-3 Hu23F4-4 23F4 VH.1a Hu23F4-5 Hu23F4-6 Hu23F4-7 Hu23F4-8 23F4 VH.1b Hu23F4-9 Hu23F4-10 Hu23F4-11 Hu23F4-12 23F4 VH.1c Hu23F4-13 Hu23F4-14 Hu23F4-15 Hu23F4-16 23F4 VH.1d Hu23F4-17 Hu23F4-18 Hu23F4-19 23F4 VH.2 Hu23F4-20 Hu23F4-21 Hu23F4-22 Hu23F4-23 23F4 VH.2a Hu23F4-24 Hu23F4-25 Hu23F4-26 Hu23F4-27 23F4 VH.2b Hu23F4-28 Hu23F4-29 Hu23F4-30 Hu23F4-31 23F4 VH.2c Hu23F4-32 Hu23F4-33 Hu23F4-34 Hu23F4-35 23F4 VH.2d Hu23F4-36 Hu23F4-37 Hu23F4-38 23F4 VH quimera 23F4 Sequências CDR de 50C5 VH CDR1 PYSIH (SEQ ID NO.23) CDR2 YINPSTGYIEYNQHFKD (SEQ ID NO.24) CDR3 GGDYEGYFDY (SEQ ID NO.25) Sequências CDR de 50C5 VL CDR1 RLNENIYSFLA (SEQ ID NO.26) CDR2 NAETLAE (SEQ ID NO.27) CDR3 QQHYGTPYT (SEQ ID NO.28) Projection for humanity at 50C5 Project VH I: VH1-69 / JH6 Construct Mutation 50C5 VH Chimera 50C5 VH.1 grafted with CDR, Q1E 50C5 VH.1a Based on 50C5 VH.1, S84R 50C5 VH.1b Based on 50C5 VH.1, S84R, G27Y, T28I 50C5 VH.1c Based on 50C5 VH.1, S84R, G27Y, T28I, M48I 50C5 VH.1d Based on 50C5 VH.1, S84R, G27Y, T28I, M48I, V68T, I70L 50C5 VH.1e Based on 50C5 VH.1, S84R, G27Y, T28I, M48I, V68T, I70L, S30T Petition 870260079734, dated 07 / 08 / 2026, pages 78 / 97 / 75 VK Project: O12 / Jk4 Construct Mutation 50C5Vk Chimera 50C5 Vk.1 grafted with CDR 50C5 Vk.1a Based on 50C5 Vk.1, I48V 50C5 Vk.1b Based on 50C5 Vk.1, I48V, G57D 50C5 Vk.1c Based on 50C5 Vk.1, I48V, G57D, D70Q 50C5 Vk.1d Based on 50C5 Vk.1, I48V, G57D, D70Q, A43S Humanized antibody sequences (CDR residues are underlined and retromutations are indicated in boxes) 50C5 VH QVQLQQSAAELVRPGASVKMSCKASGYIFTPYSIHWIKQR PGQGLEWIGY INPSTGYIEYNQHFKDRTTLTADKSSSTAYMQLRSLTSEDS AVYYCARGG DYEGYFDYWGQGTTLTVS S SEQ ID NO.29 50C5 VH.1 Evqlvqsgaevkkpgssvkvsckasggtfspysihwvrq APGQGLEWMGY INPSTGYIEYNQHFKDRVTITADKSTSTAYMELSSLRSEDT AVYYCARGG DYEGYFDY WGQGTTVTVS S SEQ ID NO.30 50C5 VH.1a Evqlvqsgaevkkpgssvkvsckasggtfspysihwvrq APGQGLEWMGY inpstgyieynqhfkdrvtitadkststaymelR]slrsedt AVYYCARGG DYEGYFDY WGQGTTVTVS S SEQ ID NO.31 50C5 VH.1b E|VQLVQSGAEVKKPGSSVKVSCKASGYIFSPYSIHWVRQA PGQGLEWMGY INPSTGYIEYNQHFKDRVTITADKSTSTAYMELRSLRSEDT AVYYCARGG DYEGYFDY WGQGTTVTVS S SEQ ID NO.32 50C5 VH.1c E|VQLVQSGAEVKKPGSSVKVSCKASGYIFSPYSIHWVRQA pgqglew|gy INPSTGYIEYNQHFKDRVTITADKSTSTAYMELRSLRSEDT AVYYCARGG DYEGYFDY WGQGTTVTVS S SEQ ID NO.33 50C5 VH.1d e|vqlvqsgaevkkpgssvkvsckasg|yi|fspysihwvrqa pgqglew|gy [NPSTGYIEYNQHFKDRTTLTADKSTSTAYMELRSLRSEDT AVYYCARGG DYEGYFDY WGQGTTVTVS S SEQ ID NO.34 50C5 VH.1e EVQLVQSGAEVKKPGSSVKVSCKASGYIFTPYSIHWVRQ APGQGLEWMGY [NPSTGYIEYNQHFKDRTTLTADKSTSTAYMELRSLRSEDT AVYYCARGG DYEGYFDY WGQGTTVTVS S SEQ ID NO.35 50C5 Vk DIQMTQSPDSLSASVGETVTITCRLNENIYSFLAWYQQRQG KSPQLLVYNA ETLAEDVPSRFSGSGSGTQFSLKISSLQTDDFGTYYCQQHY GTPYTFGGGT NLEIE SEQ ID NO.36 50C5 Vk.1 DIQMTQSPSSLSASVGDRVTITCRLNENIYSFLAWYQQKPG KAPKLLIYNA ETLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHY GTPYTFGGGT KVEIK SEQ ID NO.37. Petition 870260079734, on 08 / 07 / 2026, page. 79 / 97 / 75 50C5 Vk.1a DIQMTQSPSSLSASVGDRVTITCRLNENIYSFLAWYQQKPG kapkllVyna ETLAEGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQHY GTPYTFGGGT KVEIK SEQ ID NO.38 50C5 Vk.1b DIQMTQSPSSLSASVGDRVTITCRLNENIYSFLAWYQQKPG KAPKLLVYNA etlaedvpsrfsgsgsgtdftltisslqpedfatyycqqhy GTPYTFGGGT KVEIK SEQ ID NO.39 50C5 Vk.1c DIQMTQSPSSLSASVGDRVTITCRLNENIYSFLAWYQQKPG kapkllVyna etlaeDVpsrfsgsgsgtQftltisslqpedfatyycqqhy GTPYTFGGGT KVEIK SEQ ID NO.40 50C5 Vk.1d DIQMTQSPSSLSASVGDRVTITCRLNENIYSFLAWYQQKPG k|s]pkllVyna etlaeDVpsrfsgsgsgtQftltisslqpedfatyycqqhy GTPYTFGGGT KVEIK SEQ ID NO.41 Combinação de VH / VK para antico de humanização 50C5 Vk VH 50C5 Vk.1 50C5 Vk.1a 50C5 Vk.1b 50C5 Vk.1c 50C5 Vk.1d 50C5 Vk 50C5 VH.1 50C5 VH.1a Hu50C5-1 Hu50C5-2 Hu50C5-3 Hu50C5-4 Hu50C5-5 50C5 VH.1b Hu50C5-6 Hu50C5-7 Hu50C5-8 Hu50C5-9 Hu50C5-10 50C5 VH.1c Hu50C5-11 Hu50C5-12 Hu50C5-13 Hu50C5-14 Hu50C5-15 50C5 VH.1d Hu50C5-16 Hu50C5-17 Hu50C5-18 Hu50C5-19 Hu50C5-20 50C5 VH.1e Hu50C5-21 Hu50C5-22 Hu50C5-23 Hu50C5-24 50C5 VH Chimera 50C5 Example 8. Binding of humanized antibodies to human GM-CSF

[00185] The humanized variants were tested for binding to recombinant human GMCSF as previously described. Recombinant human GM-CSF protein (Genscript) was coated at 1 µg / ml in PBS on microtiter plates for 2 ha at room temperature (RT). After antigen coating, the wells were blocked with PBS / 0.05% Tween (PBST) with 1% BSA for 1 ha RT. After washing the wells with PBST, different concentrations of humanized anti-GM-CSF antibodies were added to the well and incubated for 1 ha RT.

[00186] For the detection of binding antibodies, HRP-conjugated secondary antibodies against mouse Fc (Jackson Immuno Research) were added, followed by the addition of fluorogenic substrates (Roche). Between all incubation steps, the plate wells were washed with PBST three times. Fluorescence was measured on a reader of Petition 870260079734, dated 07 / 08 / 2026, pp. 80 / 97 / 75 TECAN Spectrafluor plate. As shown in Figure 8, all humanized variants demonstrated similar binding potency against human GM-CSF as compared to chimeric antibody. Example 9. Binding kinetics of humanized antibodies

[00187] The binding kinetics of humanized antibodies were measured by Biacore as previously described. Recombinant human GM-CSF was configured as an assay with serial concentrations (100, 50, 25, 12.5, 6.25, 3.125 nM). The antibody-antigen binding kinetics assay was performed using the Biacore T200 system via a human antibody capture approach. Anti-human Fc IgG was immobilized on a CM5 sensor chip according to the manufacturer's instructions. The test antibody was injected and captured by immobilized anti-human Fc IgG, and then serial concentrations of the antigen were individually injected, and the binding profile was recorded for each antigen analyte concentration, respectively.

[00188] The test system was regenerated by injection of 10 mM GlycineHCl pH 1.5 for 30 seconds. The cycle buffer was HBS-EP+ (10mM HEPES, pH 7.4, 150mM NaCl, 3mM EDTA and 0.05% P20). The test temperature was 25 °C, and the association and dissociation times were 180 and 600 seconds, respectively. Biacore data were configured using Biacore T200 1.0 evaluation software according to a 1:1 binding model to calculate the association rate constant (ka) and dissociation rate constant (kd) as well as the equilibrium constant (KD). As shown in the tables below, 23F4, Hu23F4-13, Hu23F4-27 and Hu23F4-36 demonstrated the strongest binding affinity, as compared to chimeric antibody; 50C5, Hu50C5-8, Hu50C5-17, Hu50C5-18, Hu50C5-19, Hu50C5-21 and Hu50C5-23 demonstrated the strongest binding affinity, as compared to chimeric antibodies. Antigen Antibody ka (1 / Ms) kd (1 / s) KD (M) GM-CSF 3523-Hu23F4-5 1.188E+6 1.239E-4 1.043E-10 Petição 870260079734, de 07 / 08 / 2026, pág. 81 / 97 / 75 3523-Hu23F4 -6 8,76SE+S 1,353E-4 1,544E-10 3523-Hu23F4-13 1,279E+6 1,195E-4 9,349E-11 3523-Hu23F4-20 8,007E+5 1,137E-4 1,420E-10 3523-Hu23 F4-23 8,994E+5 1,164E-4 1,295E-10 3523-Hu23 F4-25 6,354E+5 1,091E-4 1,718E-10 3523-Hu23F4-27 9,232E+5 9,461E-5 1,025E-10 3523-Hu23F4-29 6,673E+S 1,013E-4 1,518E-10 3523-Hu23F4-36 1,271E+6 1,026E-4 8,070E-11 3523-Hu23F4quimera 1,229E+6 3,873E-5 3,150E-11 Antígeno Anticorpo ka (1 / Ms) kd (1 / s) KD (M) GM-CSF 35230Hu50C5-6 4,418E+6 1,375E-4 3,112E-11 35230Hu50C5-8 4,911E+6 1,386E-4 2,822E-11 35230Hu50C5-11 4,368E+6 1,363E-4 3,121E-11 35230Hu50C5-17 4,466E+6 1,112E-4 2,491E-11 35230Hu50C5-19 4,724E+6 1,089E-4 2,306E-11 35230Hu50C5-19 4,622E+6 1,061E-4 2,295E-11 35230Hu50C5-21 4,592E+6 1,082E-4 2,356E-11 35230Hu50C5-23 4,535E+6 1,251E-4 2,760E-11 Quimera 35230Hu50C5- 4,433E+6 1,419E-4 3,202E-11 Example 10. Inhibition of TF-1 proliferation by humanized antibodies

[00189] Before stimulation with GM-CSF, TF-1 cells were washed with RPMI1640 basal medium and left without medium overnight. On day 2, these depleted cells were collected and then seeded at a concentration of 3x10⁵ cells / ml in 50 µl per well of a 96-well flat-bottom cell culture plate. Recombinant human GM-CSF (Genscript) at a concentration of 0.2 ng / ml (4X) was mixed 1:1 with humanized anti-GM-CSF antibodies (0.01 ng / ml - 1000 ng / ml diluted in complete medium) and 50 µl of the mixture were added to TF-1 cells. Maximum cell proliferation (0% inhibition) was measured by incubating TF-1 cells at a final GM-CSF concentration of 0.05 ng / ml, without the addition of antibody. 100% inhibition of TF-1 proliferation was measured by omitting GM-CSF from the test and maintaining the cells only in complete RPMI1640 medium. TF-1 cells were then incubated for 72 ha at 37°C.Cell viability was measured using the CellTiter-Glo® luminescent cell viability assay according to the manufacturer's protocol. Among the humanized 23F4 antibodies tested, Hu23F4-13, Hu23F4-27, and Hu23F4-36 showed the strongest inhibition with IC50 values ​​of 4.95 ng / ml, 3.95 ng / ml, and 3.30 ng / ml, respectively (Figure 9). Among the humanized 50C5 antibodies tested, Hu50C5-23 showed... Petition 870260079734, dated 07 / 08 / 2026, page 82 / 97 / 75 stronger inhibition with an IC50 of 14.31 ng / ml (Figure 9). Antibody name IC50 for TF-1 proliferation Hu23F4-chimera 8.55 ng / ml Hu23F4-5 15.58 ng / ml Hu23F4-6 7.87 ng / ml Hu23F4-13 4.95 ng / ml Hu23F4-20 8.77 ng / ml Hu23F4-23 9.08 ng / ml Hu23F4-25 12.22 ng / ml Hu23F4-27 3.95 ng / ml Hu23F4-29 27.44 ng / ml Hu23F4-36 3.30 ng / ml Antibody name IC50 for TF-1 proliferation Hu50C5-chimera 86.47 ng / ml Hu50C5-6 23.93 ng / ml Hu50C5-8 45.90 ng / ml Hu50C5-11 111.1 ng / ml Hu50C5-17 22.06 ng / ml Hu50C5-18 41.27 ng / ml Hu50C5-19 16.99 ng / ml Hu50C5-21 21.64 ng / ml Hu50C5-23 14.31 ng / ml Example 11. Blocking pSTAT5 signaling by humanized antibodies

[00190] CD14+ monocytes were purified from human peripheral blood using CD14-positive microbeads (Miltenyi Biotec). The purified monocytes were stimulated with human GM-CSF (0.2 ng / ml) for 30 minutes at 37°C in the presence of different concentrations of humanized antibodies. After incubation, the cells were collected and washed with FACS buffer (1x PBS + 2% FBS) and permeabilized with 2% PFA followed by cell fixation using ice-cooled methanol. Then, the anti-pSTAT5 PE-conjugated antibody was added to the cells for another 30-minute incubation at 4°C and analyzed by flow cytometry. % inhibition was calculated by [1-(MFI test sample / control MFI)] x 100%. The addition of Hu23F4-13, Hu23F4-27, Hu23F4-36, Hu50C5-17, and Hu50C5-23 could significantly decrease the level of GMCSF-induced pSTAT5 activation at a dose of 0.1 or 1 pg / ml (Figure 10). Example 12. Binding of humanized antibodies to rhesus GM-CSF

[00191] Recombinant rhesus GM-CSF protein (Genscript) was Petition 870260079734, dated 07 / 08 / 2026, pp. 83 / 97 / 75 coated at 1 ug / ml in PBS on microtiter plates for 2 ha at room temperature (RT). After antigen coating, the wells were blocked with PBS / 0.05% Tween (PBST) with 1% BSA for 1 ha RT. After washing the wells with PBST, different concentrations of humanized anti-GM-CSF antibodies were added to the well and incubated for 1 ha RT. For detection of binding antibodies, HRP-conjugated secondary antibodies against mouse Fc (Jackson Immuno Research) were added, followed by the addition of fluorogenic substrates (Roche). Between all incubation steps, the plate wells were washed with PBST three times. Fluorescence was measured on a TECAN Spectrafluor plate reader.As shown in Figure 11, Hu23F413, Hu23F4-27, and Hu23F4-36 showed dose-dependent binding to GM-CSF rhesus with an EC50 of 7m44 ng / ml, 6m25 ng / ml, and 7m75 ng / ml, respectively; Hu50C5-17 and Hu50C5-23 showed dose-dependent binding to GM-CSF rhesus with an EC50 of 18.86 ng / ml and 21.63 ng / ml, respectively. EC50 Antibody Name for GM-CSF Binding rhesus Hu23F4-13 7.44 ng / ml Hu23F4-27 6.25 ng / ml Hu23F4-36 7.75 ng / ml Hu50C5-17 18.86 ng / ml Hu50C5-23 21.63 ng / ml Example 13. Pharmacokinetics of Hu23F4-27 in cynomolgus monkeys

[00192] Based on previous results from in vitro bioactivity tests, Hu23F4-27 was selected to determine its pharmacokinetic properties in native cynomolgus monkeys. Hu23F4-27 antibody was administered by intravenous bolus injection to native cynomolgus monkeys at different doses of 0.4 mg / kg, 2 mg / kg, and 10 mg / kg, respectively. Plasma samples were collected at selected time points 28 days after dosing, and the respective protein concentration was determined by ELISA. Pharmacokinetic parameters were then calculated using a non-behavioral approach with WinNonlin (Certara, CA) and Petition 870260079734, dated 07 / 08 / 2026, pages 84 / 97 / 75 shown in Figure 12. Group (n=2) T1 / 2 (h) Cmax (gg / ml) AUCc-t (Day*gg / ml) AUCinf (day*gg / ml) CL (ml / h / kg) 10 mg / kg 121.4±11.4 432.2±31.7 1254.5±38.3 1276.1±48.6 0.327±0.012 2 mg / kg 220.7±94.8 74.1±8.3 280.3±46.3 311.4±76.6 0.276±0.068 0.4 mg / kg 178.2±4.8 11.1±4.6 49.4±1.9 52.9±1.7 0.315±0.010

[00193] The present description should not be limited in scope by the specific embodiments described, which are intended as unique illustrations of individual aspects of the description, and any compositions or methods that are functionally equivalent are within the scope of this description. It will be evident to those skilled in the art that various modifications and variations can be made to the methods and compositions of the present description without departing from the spirit or scope of the description. Therefore, this description is intended to cover modifications and variations of this description, provided they are within the scope of the appended claims and their equivalents.

[00194] All publications and patent applications mentioned in this descriptive report are incorporated herein by reference to the same extent as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Petition 870260079734, dated 07 / 08 / 2026, pages 85 / 97

Claims

1 / 5 CLAIMS 1. Isolated antibody or fragment thereof, characterized in that the antibody or fragment thereof has specificity for a human GM-CSF protein and comprises a VH CDR1 of SEQ ID NO: 1, a VH CDR2 of SEQ ID NO: 2, a VH CDR3 of SEQ ID NO: 3, a VL CDR1 of SEQ ID NO: 4, a VL CDR2 of SEQ ID NO: 5, and a VL CDR3 of SEQ ID NO:

6.

2. An antibody or fragment thereof according to claim 1, characterized in that it further comprises a heavy chain constant region, a light chain constant region, an Fc region, or a combination thereof.

3. An antibody or fragment thereof according to claim 1, characterized in that the light chain constant region is a kappa or lambda chain constant region.

4. Antibody or fragment thereof according to claim 1, characterized in that the antibody or fragment thereof is of an IgG, IgM, IgA, IgE or IgD isotype.

5. Antibody or fragment thereof according to claim 4, characterized in that the isotype is IgG1, IgG2, IgG3 or IgG4.

6. Antibody or fragment thereof any of claims 1 to 5, characterized in that the antibody or fragment thereof is a chimeric antibody, a humanized antibody, or a completely human antibody.

7. Antibody or fragment thereof according to claim 6, characterized in that the antibody or fragment thereof is a humanized antibody.

8. Antibody or fragment thereof according to claim 7, characterized in that it comprises a variable heavy chain region comprising one or more amino acid residues selected from the group consisting of: (a) Glu at position 1, (b) Arg at position 98, (c) Ser at position 72, (d) Ala at position 68, (e) Leu at position 70, (f) Ile at position 48, (g) Asp at position 26, and (h) Leu at position 29, according to Kabat numbering, and combinations thereof.

9. Antibody or fragment thereof according to claim 8, characterized in that the variable heavy chain region comprises at least (a) Glu at position 1.

10. Antibody or fragment thereof according to claim 8, characterized in that the variable heavy chain region comprises a fragment of DYTLT (SEQ ID NO: 42) or GYTFT (SEQ ID NO: 43) starting at position 26 according to Kabat numbering.

11. Antibody or fragment thereof according to claim 7, characterized in that it comprises a variable light chain region comprising one or more amino acid residues selected from the group consisting of: (a) Ala at position 46, (b) Asp at position 60, (c) Asp at position 70, (d) Ser at position 43, and (f) Phe at position 87, according to Kabat numbering, and combinations thereof.

12. Antibody or fragment thereof any of the Petition 870260079734, dated 07 / 08 / 2026, page 87 / 97 3 / 5 claims 1 to 11, characterized in that it comprises a variable heavy chain region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 8-17.

13. Antibody or fragment thereof according to claim 1, characterized in that the variable heavy chain region comprises the amino acid sequence of SEQ ID NO: 11, 14 or 17.

14. Antibody or fragment thereof according to any one of claims 1 to 11, characterized in that it comprises a variable light chain region comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 19-22.

15. Antibody or fragment thereof according to claim 1, characterized in that the variable light chain region comprises the amino acid sequence of SEQ ID NO: 19 or 22.

16. Antibody or fragment thereof according to claim 1, characterized in that the variable heavy chain region comprises the amino acid sequence of SEQ ID NO: 14 and the variable light chain region comprises the amino acid sequence of SEQ ID NO:

22.

17. Antibody or fragment thereof according to claim 1, characterized in that the antibody or fragment thereof is a bispecific antibody or variable single-chain fragment.

18. Composition, characterized in that it comprises the antibody or a fragment thereof as defined in any one of claims 1 to 17, and a pharmaceutically acceptable carrier.

19. Transgenic microorganism, characterized in that it comprises one or more polynucleotides encoding the antibody or a fragment thereof as defined in any one of claims 1 to 17.

20. Use of an antibody or fragment thereof as defined in Petition 870260079734, dated 07 / 08 / 2026, pp. 88 / 97 4 / 5 in any of claims 1 to 17, characterized by being in the manufacture of a medicament to treat an inflammatory or autoimmune disease or condition.

21. Use according to claim 20, characterized in that the inflammatory disease or condition is selected from the group consisting of Alzheimer's disease, Addison's disease, atherosclerosis, ankylosing spondylitis, arthritis, osteoarthritis (OA), rheumatoid arthritis (RA), psoriatic arthritis (PA), ankylosing spondylitis, asthma, atherosclerosis, chronic obstructive pulmonary disease (COPD), Crohn's disease, colitis, dermatitis, diverticulitis, fibromyalgia, hepatitis, irritable bowel syndrome (IBS), systemic lupus erythematosus (SLE), nephritis, Parkinson's disease (PD), vasculitis, and ulcerative colitis.

22. Use according to claim 20, characterized in that the autoimmune disease or condition is selected from the group consisting of alopecia areata, autoimmune hemolytic anemia, autoimmune hepatitis, dermatomyositis, diabetes (type 1), celiac disease, autoimmune juvenile idiopathic arthritis, glomerulonephritis, Graves' disease, Guillain-Barré syndrome, idiopathic thrombocytopenic purpura, myasthenia gravis, autoimmune myocarditis, multiple sclerosis, pemphigus / pemphigoid, pernicious anemia, polyarteritis nodosa, polymyositis, primary biliary cirrhosis, psoriasis, rheumatoid arthritis, scleroderma / systemic sclerosis, Sjögren's syndrome, systemic lupus erythematosus, autoimmune thyroiditis, Hashimoto's thyroiditis, autoimmune uveitis, vitiligo, and granulomatosis with polyangiitis. (by Wegener).

23. Use of an antibody or fragment thereof as defined in any of claims 1 to 17, characterized in that it is used in the manufacture of a medicament for treating cancer.

24. Use according to claim 23, characterized in that the cancer is selected from the group consisting of bladder cancer, breast cancer, colorectal cancer, endometrial cancer, esophageal cancer, head and neck cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, pancreatic cancer, prostate cancer, and thyroid cancer.

25. Use of an antibody or fragment thereof as defined in any of claims 1 to 17, characterized in that it is used in the manufacture of a medicament to reduce or alleviate pain.

26. In vitro method for detecting GM-CSF expression in a sample, characterized in that it comprises contacting the sample with the antibody or fragment thereof as defined in any of claims 1 to 17, under conditions for the antibody or fragment thereof to bind to GM-CSF, and detecting the binding which indicates GM-CSF expression in the sample. Petition 870260079734, dated 07 / 08 / 2026, pp. 90 / 97

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