Antibodies targeting IL3

Abstract

The present disclosure relates to antibodies and antibody fragments specific for IL3, as well as nucleic acids encoding such antibodies and pharmaceutical compositions comprising such antibodies. The antibodies of the present invention can block IL-3 activity in target cells and are useful for the prevention and treatment of diseases or dysfunctions associated with elevated levels of IL3, such as inflammatory diseases, autoimmune diseases, fibrotic diseases, hematological malignancies, and other diseases.

Description

[Technical Field]

[0001] The present disclosure relates to antibodies and antibody fragments specific for IL3, as well as nucleic acids encoding such antibodies and pharmaceutical compositions comprising such antibodies. The antibodies of the present invention can block IL-3 activity in target cells and are useful for the prevention and treatment of diseases or dysfunctions associated with elevated levels of IL3, such as inflammatory diseases, autoimmune diseases, fibrotic diseases, hematological malignancies, and other diseases. [Background technology]

[0002] Interleukins belong to a large family of proteins called cytokines. Cytokines are polypeptides that affect the function of certain cells when they bind to specific cell receptors and are divided into subclasses: interleukins, interferons, colony-stimulating factors (CSFs), lymphokines, growth factors, and monokines. It is well known that cytokines also play a major role in cell proliferation, for example, in inflammatory diseases.

[0003] Cell proliferation is a complex process in which growth factors bind to specific receptors on the cell surface, followed by endocytosis, where the cytokine-receptor complex is internalized and triggers a cellular response. Such cellular responses include specific gene transcriptional activities such as DNA synthesis and cell replication. When tested at relatively high concentrations, most cytokines have several distinct biological effects. Because of these effects, there is great interest in investigating the potential therapeutic uses of these proteins.

[0004] Interleukins are mediators of the immune system that are produced in low concentrations primarily in white blood cells. They affect the growth, differentiation, and activity of cells of the immune system and therefore belong to the category of immunoregulatory factors. They also exert their effects by binding to receptors on the surface of target cells, thus altering the transcription rate of specific genes. They play an important role in triggering numerous cellular responses.

[0005] Interleukins, for example, are involved in the immunological cell activation cascade and subsequent inflammatory changes. Irregular and / or abnormal inflammation is a major component and factor in a wide range of human diseases, one of which is the immunological disorder rheumatoid arthritis (RA). However, other immunological diseases are also affected by interleukins.

[0006] IL3, also known as Multi-CSF, is a well-known member of the interleukin family. It has growth-stimulating and differentiation effects on various hematopoietic progenitor cells and acts as a growth factor for mast cells and basophils. Together with IL5 and GM-CSF, IL3 belongs to a family of hematopoietic cytokines with four short alpha-helical bundles. GM-CSF and IL3 stimulate the formation of neutrophilic and eosinophilic granulocyte colonies and macrophages. It further stimulates the formation of mast, megakaryocyte, and pure mixed erythroid colonies (D. Metcalf, "The hematopoietic colony-stimulating factors," 1984, Elsevier, Amsterdam).

[0007] Mature IL-3 consists of 133 amino acids (excluding the 19-amino acid signal sequence) and is known to stimulate colony formation by human hematopoietic progenitor cells and DNA synthesis by human acute myeloid leukemia (AML) blasts. IL-3 binds to a unique receptor, also known as the CD123 antigen. The receptor belongs to the type I cytokine receptor family and is a heterodimer with a unique α chain paired with a common β subunit (β or CDW131). IL-3 binds to a unique α receptor subunit. However, signal transduction is mediated by the common β receptor subunit (β) via the JAK2-STAT5 pathway.

[0008] Human IL-3 has two potential N-glycosylation sites at positions 15 and 70 of mature IL-3. It is well established that IL-3 expressed by eukaryotic cells is N-glycosylated (see, e.g., Biomol and Protein Expr Purif (2003) 31:34-41). Glycosylated and non-glycosylated versions of IL-3 have similar biological activity as measured by IL-3-induced proliferation of tumor cells. In the case of murine IL-3, it has also been shown that glycosylation does not affect the biological activity of IL-3 (Cytokine (1993) 5:291-7; J Biol Chem (1988), 263:14511-7).

[0009] To date, there is no suggestion in the literature that glycosylation affects the ability of monoclonal antibodies to block IL3 bioactivity. The inhibitory activity of monoclonal antibodies was characterized using either E. coli-derived IL3 (J Biol. Chem (1991) 266:10624-31, J Immunol (1991) 146:893-8) or nonglycosylated IL3 (J Biol. Chem (1991) 266:21310-7). Antibody binding to IL3 was tested using both glycosylated and nonglycosylated IL3 and was found to be glycosylation-independent. IL3 is primarily produced by activated CD4+ T cells and contributes to the growth, differentiation, and survival of CD34+ hematopoietic progenitor cells in particular. In vitro, IL3 has been observed to promote the differentiation of basophils and mast cells from bone marrow cells. It has also been observed to induce IL-6 release by murine basophils and upregulate MHC-II expression and IL-1 secretion in monocytes / macrophages. Furthermore, IL-3 supports the differentiation of monocytes into dendritic cells and osteoclasts.

[0010] Since the initial detection of IL3 in a human genomic library, determining its role in healthy humans and its possible role in the development of disease has been the focus of investigation. The ability of cytokines to initiate or regulate hematopoiesis has been of interest, particularly in relation to immune system dysfunction or disease. Such disorders appear to be associated with disorders of the hematopoietic system, and it was hypothesized that such diseases could be treated by providing viable progenitor cells to the hematopoietic system. Inducing such progenitor cells to differentiate was viewed as a means to treat the respective diseases.

[0011] Until a few years ago, little was known about the role of IL3 in autoimmune diseases, particularly rheumatoid arthritis (RA). RA is the most common inflammatory disease of the joints. The initial disease stage often develops gradually, but may also present with brief outbursts. Pain occurs primarily in the joints of the fingers or toes, but other joints may also be affected. Affected joints exhibit swelling and are usually warm. The disease primarily progresses in episodes, which usually last for weeks to months. Between attacks, there is generally improvement in symptoms.

[0012] The etiology of RA is still unknown. An autoimmune cause is strongly suspected, with viral and bacterial causes also being discussed. Genetic influences have been reported by several authors (Arthritis Rheum (2009) 60:661-8, Arthritis Rheum (2004);50 3085-92). It is assumed that misdirected immune cells invade the affected joint and trigger the production of pro-inflammatory cytokines. According to one theory, the balance between cytokines is disrupted in RA. It has been reported that IL1, IL6, and TNFα are present in excess in RA and are thought to be involved in harmful inflammatory processes in cartilage tissue and osteoclast activation.

[0013] Treatment of rheumatoid arthritis remains difficult and burdensome for patients due to the need for medications with a high risk of adverse side effects. One approach to treating the disease is symptomatic treatment, primarily using nonsteroidal anti-inflammatory drugs (NSAIDs). These drugs act as anti-inflammatory and analgesic agents, often only achieving pain relief. The drugs further interfere with certain steps in the inflammatory cascade, leading to the production of prostaglandins by cyclooxygenase. However, NSAIDs do not affect the underlying inflammatory process and therefore cannot slow joint destruction, the most detrimental effect of RA.

[0014] To prevent joint destruction and disease activity, a further current approach to treating RA is the use of disease-modifying antirheumatic drugs (DMARDs). These medications actually modify the disease process. An example of a DMARD is methotrexate, the most commonly used antirheumatic drug, whose effect is based on the reversible inhibition of the enzyme dihydrofolate reductase. Another substance commonly used to treat RA is leflunomide, which works by intervening in pyrimidine metabolism. Both medications are long-acting and therefore must be administered for a longer period (usually 12–16 weeks) to achieve the desired effect. To bridge the time until the DMARD improves the disease, most patients are given steroids.

[0015] A further approach to treating RA is the development of "biologics," which block cytokines such as TNF, IL6, and IL1 or costimulatory molecules such as B7 or deplete leukocyte subsets (e.g., B cells). Biologics (e.g., the TNF antibody infliximab) are mostly used for severe disease processes, and DMARDs have not been able to adequately control disease activity. Biologics affect multiple signaling pathways in the immune system, have a variety of serious side effects, including bacterial and viral infections, and carry a higher risk of neoplasia. All known treatments have significant drawbacks and side effects. Therefore, the goal was to develop a new drug for the treatment of RA that is effective, selectively expressed over other cytokines in patients with autoimmune diseases, particularly RA, and has fewer side effects than currently used treatment regimens.

[0016] More recently, the involvement of IL3 in autoimmune diseases, particularly RA, has been described. WO 2010 / 063488 describes that IL3 inhibitors can be used to treat the early stage of rheumatoid arthritis. The above patent application mentions that IL-3 mRNA was not detected in the synovium of RA patients, and the effect of IL3 was not observed in cultured fibroblasts, but genetic analysis found a correlation between a single nucleotide polymorphism in the IL3 promoter gene and RA. Based on this finding and further research showing the existence of significantly elevated levels of IL3 in RA patients, WO 2010 / 063488 proposes the use of inhibitors, mainly antibodies or antibody fragments, antibody variants, or antibody multimers, in preventive RA treatment, therapeutic treatment in the early stage of the disease, or maintenance treatment. IL3 has also been linked to other diseases, such as systemic lupus (Kidney Int (2015) 88: 1088-98), encephalitis (JCI Insight (2016) 1: e87157, myocarditis (J Exp Med (2019) 216: 369-83), sepsis (Science (2015) 347: 1260-5), pneumonia (JCI Insight (2020) 5: e133652), renal fibrosis (Kidney Int (2015) 88: 1088-98), myocardial fibrosis (J Exp Med (2019) 216: 369-83), and allograft fibrosis (J Immunol (2019) 202: 3514-23).

[0017] However, there remains a need for effective antibodies specific for IL3 with high affinity and avidity. Because the in vivo effectiveness of antibodies for use in treating diseases or dysfunctions in a patient's body is essential, there is also an urgent need for antibodies that are effective in an in vivo setting. Therefore, it is highly desirable to provide antibodies that can efficiently and specifically inhibit the activity of IL3 in vivo, and thus serve as useful agents for treating diseases in patients diagnosed with elevated levels of IL3.

[0018] IL3 antibodies that meet some of these criteria are described in WO 2017 / 081218, e.g., antibody P8C11 (DSM ACC3281). However, antibody P8C11 is a murine antibody. Humanization of antibody P8C11 has not been successful to date. The present invention successfully humanizes P8C11. The humanization process described herein is highly cumbersome, involving six steps to obtain a humanized derivative of antibody P8C11, designated GRT002-H16L2. Surprisingly, this humanized variant not only retains the full functional activity of the parent antibody GRT002-H16L2, but also has improved biophysical properties. For example, compared to P8C11, GRT002-H16L2 exhibits stability over a wider pH range and at higher temperatures.

[0019] Therefore, GRT002-H16L2 and its fragments, variants, or conjugates are ideally suited for clinical development for the treatment of patients requiring anti-IL3 antibody blockade. The antibody can also be used to detect human IL-3 expressed in human cells. [Prior art documents] [Patent documents]

[0020] [Patent Document 1] International Publication No. 2010 / 063488 Brochure [Patent Document 2] International Publication No. 2017 / 081218 Brochure [Non-patent literature]

[0021] [Non-Patent Document 1] D. Metcalf, “The hematopoietic colony-stimulating factors”, 1984, Elsevier, Amsterdam. [Non-patent document 2] Biomol and Protein Expr Purif(2003)31:34-41

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Summary of the Invention

Problems to be Solved by the Invention

[0022] The present disclosure relates to novel humanized antibodies and antibody fragments specific for human IL3. The humanized antibodies and antibody fragments are derived from the parent antibody P8C11, which is difficult to humanize by conventional means. Surprisingly, the inventors not only succeeded in humanizing the antibody P8C11, but also showed increased stability at higher temperatures and over a wider pH range, especially at acidic pH.

[0023] The present disclosure relates to humanized antibodies and antibody fragments specific for human IL3 that block IL3 activity in TF1 cells and human basophils.

[0024] The present disclosure also relates to humanized antibodies and antibody fragments specific for human IL3 that bind to amino acids 41 to 67 of human IL3 (SEQ ID NO: 1).

[0025] The present disclosure also relates to humanized antibodies and antibody fragments specific for human IL3 that do not bind to human IL-5 and human GM-CSF.

[0026] Preferred antibodies and antibody fragments of the present disclosure are of the human IgG class. Even more preferred antibodies or antibody fragments of the present disclosure are of the IgG1 class.

[0027] Preferred antibodies and antibody fragments of the present disclosure are monoclonal antibodies or antibody fragments.

[0028] The present disclosure also relates to antibodies and antibody fragments specific for human IL3 that have superior biophysical properties, including high temperature stability and stability over a wide pH range when compared to the parent antibody P8C11. [Means for solving the problem]

[0029] Preferred humanized antibodies and antibody fragments of the present disclosure comprise an HCDR1 region of SEQ ID NO:3, an HCDR2 region of SEQ ID NO:4, an HCDR3 region of SEQ ID NO:5, an LCDR1 region of SEQ ID NO:10, an LCDR2 region of SEQ ID NO:11, and an LCDR3 region of SEQ ID NO:12.

[0030] Preferred antibodies and antibody fragments of the present disclosure comprise a VH of SEQ ID NO:36 and a VL of SEQ ID NO:16.

[0031] The present disclosure also relates to vectors comprising such nucleic acids or nucleic acid compositions.

[0032] The present disclosure also relates to host cells containing such nucleic acids, nucleic acid compositions or vectors.

[0033] The antibodies and antibody fragments of the present disclosure are for use in medicine. Preferred are the antibodies and antibody fragments of the present disclosure for use in the treatment of inflammatory diseases, autoimmune diseases, fibrotic diseases, hematological malignancies, and potentially other diseases.

[0034] The present disclosure also relates to pharmaceutical compositions comprising the antibodies and antibody fragments and pharmaceutically acceptable antibodies and antibody fragments of the present disclosure. [Brief explanation of the drawings]

[0035] [Figure 1] FIG. 1 shows an analysis of the binding properties of antibody GRT002-H16L2 compared to the parent antibody P8C11. [Figure 2] FIG. 1 shows the results of epitope mapping of antibody GRT002-H16L2. [Figure 3] FIG. 1 shows that antibodies P8C11 and GRT002-H16L2 block IL-3 activity in TF1 cells to a similar extent. [Figure 4] FIG. 1 shows that antibodies P8C11 and GRT002-H16L2 block IL-3 activity to a similar extent in human basophils. [Figure 5] FIG. 1 shows the determination of the melting temperature of antibody P8C11 as determined by differential scanning calorimetry. [Figure 6] FIG. 1 shows the determination of the melting temperature of antibody GRT002-H16L2 as determined by differential scanning calorimetry. [Figure 7]Figure 1 shows that repeated freezing and thawing does not affect the functional activity of GRT002-H16L2 as measured by the level of CD131 expression in basophils. FTC = freeze-thaw cycles. [Figure 8] 1 shows the effect of exposure of GRT002-H16L2 and chimeric P8C11 to higher temperatures as measured by the level of CD131 expression on basophils. Antibody GRT002-H16L2 has higher temperature stability than chimeric antibody P8C11. [Figure 9] Figure 1 shows the effect of incubation of GRT002-H16L2 and chimeric P8C11 at pH 2, as measured by CD131 expression levels in basophils. Antibody GRT002-H16L2 has higher pH stability than antibody P8C11. [Figure 10] FIG. 1 shows that incubation of antibody GRT002-H16L2 in human plasma for at least 14 days does not affect functional activity, as measured by the level of CD131 expression on basophils. DETAILED DESCRIPTION OF THE INVENTION

[0036] definition The present disclosure relates to antibodies that specifically bind to IL3.

[0037] The term "IL3" or "IL-3" refers to a member of the interleukin protein family having the following amino acid sequence (Uniprot: P08700): MSRLPVLLLLQLLVRPGLQ APMTQTTPLKTSWV N CSNMIDEIITHLKQPPLPLLDFNNLNGEDQDILMENNLRRPNLEAFNRAVKSLQ N ASAIESILKNLLPCLPLATAAPTRHPIHIKDGDWNEFRRKLTFYLKTLENAQAQQTTLSLAIF (SEQ ID NO: 1)

[0038] The signal sequence of IL3 is underlined in the above sequence. Potential N-glycosylation sites are shown in bold and underlined.

[0039] The term "antibody" as used herein refers to a protein comprising at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds that interact with an antigen. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains: CH1, CH2, and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain, CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity-determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxy terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant region of an antibody can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (C1q) of the classical complement system. The term "antibody" includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, and chimeric antibodies. Antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., Igd, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. Both the light and heavy chains are divided into regions of structural and functional homology.

[0040] The term "antibody fragment" as used herein refers to one or more parts of an antibody that retains the ability to specifically interact with antigen (for example, by binding, steric hindrance, stabilizing spatial distribution).Examples of binding fragments include but are not limited to: Fab fragment, monovalent fragment consisting of VL, VH, CL and CH1 domain, F(ab)2 fragment, bivalent fragment comprising two Fab fragments linked by disulfide bridge at hinge region; Fd fragment consisting of VH domain and CH1 domain; Fv fragment consisting of VL domain and VH domain of single arm of antibody; dAb fragment consisting of VH domain (Ward et al., (1989) Nature 341:544-546); And isolated complementarity-determining region (CDR). Furthermore, although the two domains of an Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker that allows them to be produced using recombinant methods as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as a single-chain Fv (scFv); see, e.g., Bird et al., (1988) Science 242:423-426; and Huston et al., (1988) Proc. Natl. Acad. Sci. 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "antibody fragment." These antibody fragments are obtained using conventional techniques known to those of skill in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antibody fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs (see, e.g., Hollinger and Hudson, (2005) Nature Biotechnology 23:1 126-1 136). Antibody fragments can be grafted onto scaffolds based on polypeptides such as fibronectin type III (Fn3) (see U.S. Pat. No. 6,703,199, which describes fibronectin polypeptide monobodies).Antibody fragments can be assembled into single-chain molecules comprising a pair of tandem Fv segments (VH-CH1-VH-CH1) which, together with complementary light chain polypeptides, form a pair of antigen-binding sites (Zapata et al., (1995) Protein Eng. 8:1057-1062; and U.S. Pat. No. 5,641,870).

[0041] The structure and location of immunoglobulin variable domains, e.g., CDRs, can be defined using well-known numbering schemes, such as the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia (e.g., Sequences of Proteins of Immunological Interest, U.S. Department of Health and Human Services (1991), eds. Kabat et al.; Lazikani et al., (1997) J. Mol. Bio. 273:927-948; Kabat et al., (1991) Sequences of Proteins of Immunological Interest, 5th edit., NIH Publication no. 91-3242 U.S. Department of Health and Human Services; Chothia et al., (1987) J. Mol. Biol. 196:901-917; Chothia et al. al., (1989) Nature 342:877-883; and Al-Lazikani et al., (1997) J. Mol. Biol. 273:927-948; Annals of the New York Academy of Sciences, 764, 47-49 (1995); Nucleic Acids Research, 25, 206-211 (1997).

[0042] As used herein, "human antibodies" or "human antibody fragments" refer to antibodies and antibody fragments having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Human antibodies can also be isolated from synthetic libraries or transgenic mice (e.g., Xenomouse), providing a respective yield of antibodies having variable regions in which both the framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region is also derived from such sequences. Human origins include, for example, human germline sequences or mutated versions of human germline sequences or antibodies containing consensus framework sequences derived from human framework sequence analysis, as described, for example, in Knappik et al., (2000) J Mol Biol 296:57-86).

[0043] A "humanized antibody" or "humanized antibody fragment" is defined herein as an antibody molecule having constant antibody regions derived from sequences of human origin, with only the variable antibody regions or portions thereof, or CDRs, derived from another species. For example, a humanized antibody can be CDR-grafted, in which the CDRs of the variable domain are of non-human origin, while one or more frameworks of the variable domain are of human origin, and the constant domains, if any, are of human origin.

[0044] The term "chimeric antibody" or "chimeric antibody fragment" is defined herein as an antibody molecule having constant antibody regions derived from or corresponding to sequences found in one species and variable antibody regions derived from another species. Preferably, the constant antibody regions are derived from or correspond to sequences found in humans, and the variable antibody regions (e.g., VH, VL, CDR, or FR regions) are derived from sequences found in a non-human animal, such as a mouse, rat, rabbit, or hamster.

[0045] The term "isolated antibody" refers to an antibody or antibody fragment that is substantially free of other antibodies or antibody fragments with different antigen specificities. Furthermore, an isolated antibody or antibody fragment may be substantially free of other cellular material and / or chemicals. Thus, in some embodiments, the provided antibody is an isolated antibody that is separated from antibodies with different specificities. An isolated antibody may be a monoclonal antibody. An isolated antibody may be a recombinant monoclonal antibody. However, an isolated antibody that specifically binds to a target epitope, isoform, or variant may have cross-reactivity with other related antigens, such as antigens from other species (e.g., species homologs).

[0046] As used herein, the term "recombinant antibody" includes all antibodies prepared, expressed, generated, or isolated by non-naturally occurring means. For example, antibodies isolated from host cells transformed to express the antibody, antibodies selected and isolated from recombinant combinatorial human antibody libraries, and antibodies prepared, expressed, generated, or isolated by any other means involving splicing of all or part of a human immunoglobulin gene, sequence to other DNA sequences, or antibodies isolated from animals (e.g., mice) that are transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom. Preferably, such recombinant antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies can be subjected to in vitro mutagenesis (or, when using animals transgenic for human Ig sequences, in vivo somatic mutagenesis), such that the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but are sequences that may not naturally occur within the human antibody germline repertoire in vivo. The recombinant antibody can be a monoclonal antibody. In one embodiment, the antibodies and antibody fragments disclosed herein are isolated from a HuCAL library (Rothe et al., J. Mol. Biol. (2008) 376, 1 182-1200).

[0047] As used herein, binding specificity is a relative, not absolute, property; therefore, an antibody "binds specifically to," "specifically binds to," "specific for," or "specifically recognizes" an antigen, such as human IL3, if it can distinguish between such antigen and one or more reference antigens. For example, a standard ELISA assay or a standard flow cytometry assay can be performed. Scoring can be performed by standard color development (e.g., secondary antibodies containing tetramethylbenzidine with horseradish peroxide and hydrogen peroxide) or by binding of a secondary antibody labeled with PE or another dye or marker. Reactions in a particular well are scored, for example, by optical density (OD) at 450 nm or mean fluorescence intensity (MFI) in flow cytometry. A typical background (=negative reaction) can be 0.1 OD, and a typical positive reaction can be 1 OD. The background and positive reaction MFIs are highly dependent on instrument settings. The difference between positive and negative reactions can be more than 10-fold. Typically, binding specificity is determined not using a single reference antigen, but using a set of approximately 3-5 unrelated antigens, such as milk powder, BSA, transferrin, etc. A variety of antigen-negative cells can be used for flow cytometry. However, antibodies that specifically bind to an antigen may have cross-reactivity to their respective orthologous antigens from other species (e.g., species homologs). In certain embodiments, such cross-reactivity to orthologous antigens is even preferable.

[0048] As used herein, an antibody has "cross-reactivity" or is "cross-reactive" if it binds to orthologous antigens from other species. For example, an antibody is cross-reactive if it binds to human IL3 and marmoset IL3.

[0049] As used herein, the term "affinity" refers to the strength of the interaction between a polypeptide and its target at a single site. Within each site, the binding region of the polypeptide interacts with its target at multiple sites through weak non-covalent forces; the more interactions, the stronger the affinity.

[0050] The term "epitope" includes any proteinaceous region that is specifically recognized by an antibody or antibody fragment thereof or that otherwise interacts with a molecule. Generally, an epitope is a chemically active surface grouping of molecules such as amino acids or carbohydrate or sugar side chains, and generally may have specific three-dimensional structural characteristics and specific charge characteristics. As will be understood by those skilled in the art, virtually anything to which an antibody can specifically bind can be an epitope.

[0051] The term "domain" or "protein domain" refers to a region of a polypeptide chain of a protein that forms a functional unit and / or forms an independent three-dimensional structure.

[0052] The "compositions" of the present disclosure can be used for therapeutic or prophylactic purposes. Accordingly, the present disclosure includes pharmaceutical compositions containing the antibodies or antibody fragments disclosed herein and a pharmaceutically acceptable carrier or excipient therefor. In a related aspect, the present disclosure provides methods for treating inflammatory diseases, autoimmune diseases, fibrotic diseases, hematological malignancies, and potentially other diseases. Such methods comprise administering to a subject in need thereof an effective amount of a pharmaceutical composition containing the antibodies or antibody fragments described herein.

[0053] The present disclosure provides a method of treatment comprising administering a therapeutically effective amount of an antibody or antibody fragment disclosed herein to a subject in need of such treatment. As used herein, a "therapeutically effective amount" or "effective amount" refers to the amount of an IL3 antibody required to induce a desired biological response. According to the present disclosure, a therapeutically effective amount is the amount of an IL3 antibody required to treat and / or prevent a disease.

[0054] "Administered" or "administration" includes, but is not limited to, delivery of a drug by an injectable form, such as, for example, intravenous, intramuscular, intradermal, or subcutaneous routes, or by a mucosal route, for example, as a nasal spray or aerosol for inhalation, or as an ingestible solution, capsule, or tablet. Preferably, administration is by an injectable form.

[0055] As used herein, "treatment," "treat," or "treating," etc., refer to a clinical intervention that seeks to alter the natural history of disease in the subject being treated and can be performed either prophylactically or during the course of clinical pathology. Desirable effects of treatment include, but are not limited to, prevention of disease onset or recurrence, alleviation of symptoms, reduction of any direct or indirect pathological consequences of disease, prevention of metastasis, slowing the rate of disease progression, amelioration or palliation of disease state, and remission or improved prognosis. In some embodiments, antibodies or antibody fragments of the present disclosure are used to delay disease onset or slow the progression of disease.

[0056] "Preventing" or "prevention" refers to a reduction in the risk of acquiring or developing a disease (i.e., not developing at least one clinical symptom of a disease in a subject who may be exposed to a pathogen or who is susceptible to the disease prior to the onset of the disease). "Prevention" also refers to methods aimed at preventing the onset of a disease or its symptoms, or delaying the onset of a disease or its symptoms.

[0057] "Subject" or "species," as used in this context, refers to any mammal, including rodents, such as mouse orrats, and primates, such as cynomolgus monkeys (Macaca fascicularis), marmoset monkeys (Callithrix jacchus), rhesus monkeys (Macaca mulatta), or humans (Homo sapiens). Preferably, the subject is a primate, most preferably a human.

[0058] The term "effector function" refers to the biological activity attributable to the Fc region of an antibody, which varies depending on the antibody isotype. Non-limiting examples of antibody effector functions include C1q binding and complement-dependent cytotoxicity (CDC); Fc receptor binding and antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cellular phagocytosis (ADCP); downregulation of cell surface receptors (e.g., B cell receptors); and direct cell activation or direct cell inhibition.

[0059] "Antibody-dependent cell-mediated cytotoxicity" or "ADCC" refers to a form of cytotoxicity in which antibodies bound to Fc receptors (FcRs) present on certain cytotoxic cells (e.g., NK cells, neutrophils, and macrophages) allow these cytotoxic effector cells to specifically bind to antigen-bearing target cells and subsequently kill the target cells with cytotoxins. The primary cells for mediating ADCC, NK cells, express only FcyRIII, whereas monocytes / macrophages express FcyRI, FcyRII, and FcyRIII.

[0060] "Complement-dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to an antibody (of the appropriate subclass) of the present disclosure that is bound to its cognate antigen.

[0061] "Antibody-dependent cellular phagocytosis" or "ADCP" refers to a mechanism for elimination of antibody-coated target cells by internalization by phagocytic cells such as macrophages or dendritic cells.

[0062] Throughout this specification, unless the context requires otherwise, the words "comprise," "have," and "include," and their respective variations such as "comprises," "comprising," "has," "having," "includes," and "including," are understood to imply the inclusion of a stated element or integer or group of elements or integers, but not the exclusion of any other element or integer or group of elements or integers.

[0063] As used herein, the terms "engineered" or "modified" include the manipulation of nucleic acids or polypeptides by synthetic means (e.g., by recombinant techniques, in vitro peptide synthesis, enzymatic or chemical coupling of peptides, or some combination of these techniques). Preferably, antibodies or antibody fragments according to the present disclosure are engineered or modified to improve one or more properties, such as antigen binding, stability, half-life, effector function, immunogenicity, safety, etc.

[0064] As used herein, "variant" refers to a polypeptide that differs from a reference polypeptide by one or more modifications, such as amino acid substitution, insertion, or deletion. A variant polypeptide typically retains most of the properties of the reference polypeptide, such as binding to a target antigen, but introduces new additional features or properties, for example, the variant polypeptide has a higher affinity for the target antigen compared to the reference polypeptide, or the variant polypeptide is a humanized version of the reference polypeptide.

[0065] As used herein, the term "amino acid mutation" is intended to encompass amino acid substitution, deletion, insertion, and modification. Any combination of substitution, deletion, insertion, and modification can be performed as long as the final construct possesses the desired characteristics, such as reduced binding to Fc receptors. Deletions and insertions in the amino acid sequence include deletions and insertions at the N- and / or C-terminus of amino acid residues. A specific amino acid mutation is an amino acid substitution. Amino acid substitutions include replacement with unnatural amino acids or with derivatives of the 20 standard amino acids. Amino acid mutations can be generated using genetic or chemical methods well known in the art. Genetic methods may include site-directed mutagenesis, PCR, gene synthesis, etc. It is contemplated that methods for modifying the side chain groups of amino acid residues by methods other than genetic engineering, such as chemical modification, may also be useful. Various names may be used herein to refer to the same amino acid mutation. For example, a substitution of Gly to Alanine at position 327 in the Fc region can be designated as 327, G327, G327A, or Gly327Ala.

[0066] As used herein, the term "EC50" refers to the concentration of an antibody or antibody fragment that induces a response in an assay halfway between baseline and maximum, and thus represents the antibody or ligand concentration at which 50% of the maximum effect is observed.

[0067] The terms "inhibition" or "inhibiting" or "reduction" or "reducing" or "neutralization" or "neutralizing" refer to a decrease or cessation of any phenotypic characteristic (such as binding or biological activity or function), or a decrease or cessation of the incidence, degree, or likelihood of that characteristic. "Inhibition," "reduction," or "neutralization" need not be complete, as long as it is detectable using an appropriate assay. In some embodiments, "reduce" or "inhibit" or "neutralizing" refers to the ability to cause a 20% or greater reduction. In other embodiments, "reduce" or "inhibit" or "neutralizing" refers to the ability to cause a 50% or greater reduction. In yet other embodiments, "reduce" or "inhibit" or "neutralizing" refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95%, or greater.

[0068] As used herein, the term "antagonist" antibody refers to an antibody or antibody fragment that interacts with an antigen and partially or completely inhibits or neutralizes a biological activity or function or any other phenotypic characteristic of the target antigen.

[0069] A "wild-type" protein is a version or variant of a protein found in nature. The amino acid sequence of a wild-type protein, such as the Fc region of a human IgG1 antibody, is the amino acid sequence of a naturally occurring protein. Due to allotypic differences, a wild-type protein may have two or more amino acid sequences. For example, there are several allotypes of the naturally occurring human IGg1 heavy chain constant region (see, e.g., Jeffries et al. (2009) mAbs1:1).

[0070] The term "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The Fc region of an immunoglobulin generally contains two constant domains, a CH2 domain and a CH3 domain. Although the boundaries of the Fc region of an IgG heavy chain may vary slightly, the human IgG heavy chain Fc region is usually defined as extending from Cys226 or Pro230 to the C-terminus of the heavy chain. However, the C-terminal lysine (Lys447) of the Fc region may or may not be present. Unless otherwise specified herein, the numbering of amino acid residues in the Fc region follows the EU numbering system, also known as the EU index, as described by Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991.

[0071] Embodiments of the invention In the prior art, the efficacy of potential IL3-specific antibodies was tested on leukemia cell lines or TF1 cells. TF1 is a model human erythroid cell line established by T. Kitamura in 1987 from the bone marrow of a 35-year-old Japanese man suffering from severe pancytopenia. The growth of TF1 cells is completely dependent on the presence of IL-3 or GM-CSF. Therefore, a test based on cell proliferation of TF1 cells can be used to determine whether blocking IL-3 activity results in a reduction or even complete inhibition of TF1 cell growth.

[0072] To use an antibody in human treatment, its ability to block IL-3 activity should also be assayed in a model not too far removed from the in vivo situation, such as the TF1 cell line model. Therefore, the inventors tested the anti-IL3 antibody of the present disclosure using primary human cells obtained from patients treated with the anti-IL3 antibody or primary human cells obtained from healthy subjects as a control. The use of primary cells and IL3 produced by primary human cells is superior to cell line models.

[0073] The glycosylation state depends on the type of cells producing human IL-3. When human IL-3 is recombinantly produced by E. coli cells, the resulting human IL-3 is not glycosylated. When human IL-3 is produced by insect cells, the resulting human IL-3 is weakly glycosylated. When IL-3 is produced by human cells such as HEK cells, the resulting human IL-3 is more heavily glycosylated.

[0074] The primary cells used to determine the efficacy of the anti-IL3 antibodies of the present disclosure are preferably primary human blood cells obtained from patients suffering from rheumatoid arthritis (RA). Primary human blood cells can also be obtained from healthy subjects and used as a control. To test the blood sample, it can be treated with heparin, citrate, or EDTA as an anticoagulant, as known in the art. Preferably, EDTA is added to the blood sample as an anticoagulant. This type of blood sample is also called "EDTA blood."

[0075] Primary human blood cells contain basophilic granulocytes, also known as "basophils," which can be detected by known methods, such as flow cytometry, using labeled antibodies, such as a combination of fluorescently labeled antibodies against basophil-expressed cell markers, such as CD11b, CD123, CCR3, and CD203c. The ability to block human IL3 activity can be tested, for example, by assaying the IL3-induced upregulation of CD203c in basophilic granulocytes (Int J Immunopathol Pharmacol (2007) 20:267-78). IL-3 also induces the upregulation of CD11b and the downregulation of CD131 in basophils. Furthermore, human IL3 induces the upregulation of HLA-DR and the downregulation of CD131 in plasmacytoid dendritic cells (pDCs), which are also contained in blood samples containing primary human blood cells. pDCs can be detected by flow cytometry using a combination of fluorescently labeled antibodies against dendritic cell-expressed cell markers, such as HLA-DR, CD123, and CD4, as is known in the art. Additionally, IL3-induced downregulation of CD131 can be quantified in CD14+CD16+ monocytes, CD14++ monocytes, and eosinophils. Therefore, the ability of anti-IL-3 antibodies to efficiently block hIL-3 activity can be assayed by known tests, such as by quantifying upregulation of CD11b and CD203c in basophils, or upregulation of HLA-DR in pDCs, or downregulation of CD131 in various cell types, including basophils, pDCs, monocytes, or eosinophils. Basophils are a preferred cell type for quantifying the biological activity of IL-3 in human samples because they respond strongly to IL-3 and show little reactivity to closely related cytokines, such as IL-5 and GM-CSF.

[0076] Polypeptides In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for human IL3, wherein the antibody or antibody fragment comprises an HCDR1 region of SEQ ID NO: 3, an HCDR2 region of SEQ ID NO: 4, an HCDR3 region of SEQ ID NO: 5, an LCDR1 region of SEQ ID NO: 10, an LCDR2 region of SEQ ID NO: 11, and an LCDR3 region of SEQ ID NO: 12.

[0077] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for human IL3, wherein the antibody or antibody fragment comprises an HCDR1 region of SEQ ID NO: 3, an HCDR2 region of SEQ ID NO: 4, an HCDR3 region of SEQ ID NO: 5, an LCDR1 region of SEQ ID NO: 10, an LCDR2 region of SEQ ID NO: 11, and an LCDR3 region of SEQ ID NO: 12, as defined by Kabat.

[0078] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for human IL3, wherein the antibody or antibody fragment comprises an HCDR1 region of SEQ ID NO: 6, an HCDR2 region of SEQ ID NO: 7, an HCDR3 region of SEQ ID NO: 8, an LCDR1 region of SEQ ID NO: 13, an LCDR2 region of SEQ ID NO: 14, and an LCDR3 region of SEQ ID NO: 12, as defined by IMGT.

[0079] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for human IL3, wherein said humanized antibody or antibody fragment comprises a VH of SEQ ID NO:36 and a VL of SEQ ID NO:16.

[0080] In one embodiment of the present disclosure, the humanized antibody or antibody fragment is a monoclonal antibody or antibody fragment.

[0081] In one embodiment of the present disclosure, the humanized antibody or antibody fragment is a recombinant antibody or antibody fragment.

[0082] In one embodiment of the present disclosure, the humanized antibody or antibody fragment is of the IgG isotype.

[0083] In one embodiment of the present disclosure, the humanized antibody or antibody fragment is of the IgG1 class.

[0084] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for human IL3, wherein said humanized antibody or antibody fragment comprises a heavy chain of SEQ ID NO:44 and a light chain of SEQ ID NO:45.

[0085] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for human IL3, said antibody having a heavy chain of SEQ ID NO:44 and a light chain of SEQ ID NO:45.

[0086] In one embodiment of the present disclosure, the humanized antibody or antibody fragment is specific for a polypeptide encoded by the amino acid sequence of SEQ ID NO:1.

[0087] In one embodiment of the present disclosure, the humanized antibody or antibody fragment is specific for a polypeptide comprising the amino acid sequence of SEQ ID NO:1.

[0088] In another embodiment of the disclosure, the humanized antibody or antibody fragment binds to amino acids 41-67 of human IL3 (SEQ ID NO: 1). In another embodiment of the disclosure, the humanized antibody or antibody fragment binds to amino acids 22-48 of mature human IL3. In another embodiment of the disclosure, the humanized antibody or antibody fragment binds to a peptide stretch of human IL3 consisting of the amino acids of SEQ ID NO: 37. In another embodiment of the disclosure, the humanized antibody or antibody fragment binds to a peptide comprising the amino acids of SEQ ID NO: 37. In another embodiment of the disclosure, the humanized antibody or antibody fragment binds to a peptide consisting of the amino acids of SEQ ID NO: 37. In another embodiment of the disclosure, the humanized antibody or antibody fragment binds to a peptide comprising the amino acids of SEQ ID NO: 38. In another embodiment of the disclosure, the humanized antibody or antibody fragment binds to a peptide comprising the amino acids of SEQ ID NO: 39. In another embodiment of the disclosure, the humanized antibody or antibody fragment binds to a peptide consisting of the amino acids of SEQ ID NO: 39. In a preferred embodiment, the antibody or antibody fragment comprises a VH of SEQ ID NO:36 and a VL of SEQ ID NO:16.

[0089] In another embodiment of the disclosure, the humanized antibody or antibody fragment specific for human IL3 is not cross-reactive with IL3 from other species. In a specific embodiment of the disclosure, the humanized antibody or antibody fragment specific for human IL3 is not cross-reactive with mouse IL3 or rat IL3. In a specific embodiment of the disclosure, the humanized antibody or antibody fragment specific for human IL3 is not cross-reactive with rat IL3. In a specific embodiment of the disclosure, the humanized antibody or antibody fragment specific for human IL3 is not cross-reactive with mouse IL3. In a preferred embodiment, the antibody or antibody fragment comprises a VH of SEQ ID NO: 36 and a VL of SEQ ID NO: 16.

[0090] To determine the usefulness of a resulting monoclonal antibody for diagnostic assays, it is important to be able to exclude cross-reactivity with closely related cytokines that are also present in a patient's blood, plasma, serum, or other bodily fluids. IL5 (UniProt: P05113) and GM-CSF (UniProt: P04141) are two such closely related cytokines. Thus, in another embodiment of the present disclosure, a humanized antibody or antibody fragment specific for human IL3 is not cross-reactive with human IL5 or human GM-CSF. In a specific embodiment of the present disclosure, a humanized antibody or antibody fragment specific for human IL3 is not cross-reactive with human IL5. In a specific embodiment of the present disclosure, a humanized antibody or antibody fragment specific for human IL3 is not cross-reactive with human GM-CSF. In a preferred embodiment, the antibody or antibody fragment comprises a VH of SEQ ID NO: 36 and a VL of SEQ ID NO: 16.

[0091] In another embodiment of the present disclosure, a humanized antibody or antibody fragment specific for human IL3 blocks IL3 activity in TF1 cells and human basophils. In a specific embodiment of the present disclosure, a humanized antibody or antibody fragment specific for human IL3 blocks IL3 activity in TF1 cells. In a specific embodiment of the present disclosure, a humanized antibody or antibody fragment specific for human IL3 blocks IL3 activity in human basophils. In a specific embodiment of the present disclosure, a humanized antibody or antibody fragment specific for human IL3 blocks IL3 activity in TF1 cells at least to the same extent as an antibody or antibody fragment comprising a VH of SEQ ID NO:2 and a VL of SEQ ID NO:9. In a specific embodiment of the present disclosure, a humanized antibody or antibody fragment specific for human IL3 blocks IL3 activity in human basophils at least to the same extent as an antibody or antibody fragment comprising a VH of SEQ ID NO:2 and a VL of SEQ ID NO:9. In a preferred embodiment, the antibody or antibody fragment comprises a VH of SEQ ID NO:36 and a VL of SEQ ID NO:16.

[0092] In another embodiment of the disclosure, the humanized antibody or antibody fragment specific for human IL3 has increased temperature stability. In a specific embodiment of the disclosure, the humanized antibody or antibody fragment specific for human IL3 has increased temperature stability at 75°C. In a specific embodiment of the disclosure, the humanized antibody or antibody fragment specific for human IL3 has increased temperature stability at 75°C compared to an antibody or antibody fragment comprising a VH of SEQ ID NO:2 and a VL of SEQ ID NO:9. In a preferred embodiment, the antibody or antibody fragment comprises a VH of SEQ ID NO:36 and a VL of SEQ ID NO:16.

[0093] In another embodiment of the disclosure, the humanized antibody or antibody fragment specific for human IL3 has increased pH stability. In a specific embodiment of the disclosure, the humanized antibody or antibody fragment specific for human IL3 has increased stability at pH 2. In a specific embodiment of the disclosure, the humanized antibody or antibody fragment specific for human IL3 has increased stability at pH 2 compared to an antibody or antibody fragment comprising a VH of SEQ ID NO: 2 and a VL of SEQ ID NO: 9. In a preferred embodiment, the antibody or antibody fragment comprises a VH of SEQ ID NO: 36 and a VL of SEQ ID NO: 16.

[0094] nucleic acid In one embodiment, the disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or sequences encoding a humanized antibody or antibody fragment specific for human IL3, wherein the antibody or antibody fragment comprises an HCDR1 region of SEQ ID NO: 3, an HCDR2 region of SEQ ID NO: 4, an HCDR3 region of SEQ ID NO: 5, an LCDR1 region of SEQ ID NO: 10, an LCDR2 region of SEQ ID NO: 11, and an LCDR3 region of SEQ ID NO: 12.

[0095] In one embodiment, the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or sequences encoding a humanized antibody or antibody fragment specific for human IL3, wherein the antibody or antibody fragment contains an HCDR1 region of SEQ ID NO: 3, an HCDR2 region of SEQ ID NO: 4, an HCDR3 region of SEQ ID NO: 5, an LCDR1 region of SEQ ID NO: 10, an LCDR2 region of SEQ ID NO: 11, and an LCDR3 region of SEQ ID NO: 12.

[0096] In one embodiment, the present disclosure relates to a nucleic acid composition comprising one or more nucleic acid sequences encoding a humanized antibody or antibody fragment specific for human IL3, wherein the antibody or antibody fragment comprises an HCDR1 region of SEQ ID NO: 3, an HCDR2 region of SEQ ID NO: 4, an HCDR3 region of SEQ ID NO: 5, an LCDR1 region of SEQ ID NO: 10, an LCDR2 region of SEQ ID NO: 11, and an LCDR3 region of SEQ ID NO: 12, as defined by Kabat.

[0097] In one embodiment, the present disclosure relates to a nucleic acid composition comprising one or more nucleic acid sequences encoding a humanized antibody or antibody fragment specific for human IL3, wherein the antibody or antibody fragment contains an HCDR1 region of SEQ ID NO: 3, an HCDR2 region of SEQ ID NO: 4, an HCDR3 region of SEQ ID NO: 5, an LCDR1 region of SEQ ID NO: 10, an LCDR2 region of SEQ ID NO: 11, and an LCDR3 region of SEQ ID NO: 12, as defined by Kabat.

[0098] In one embodiment, the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or multiple nucleic acid sequences encoding a humanized antibody or antibody fragment specific for human IL3, wherein the antibody or antibody fragment comprises a VH of SEQ ID NO: 36 and a VL of SEQ ID NO: 16.

[0099] In one embodiment, the present disclosure relates to a nucleic acid composition comprising a nucleic acid sequence or multiple nucleic acid sequences encoding a humanized antibody or antibody fragment specific for human IL3, wherein the antibody or antibody fragment contains a VH of SEQ ID NO: 36 and a VL of SEQ ID NO: 16.

[0100] In one embodiment, said nucleic acid composition and / or said nucleic acid sequence and / or plurality of nucleic acid sequences are isolated.

[0101] vector In one embodiment, the present disclosure provides a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a humanized antibody or antibody fragment specific for human IL3 according to the present disclosure.

[0102] In one embodiment, the present disclosure provides a vector composition comprising a vector or a plurality of vectors comprising a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding any one of the humanized antibodies or antibody fragments specific for human IL3 disclosed herein.

[0103] In one embodiment, said vector composition and / or vector and / or plurality of vectors is isolated.

[0104] host cell In one embodiment, the present disclosure provides a host cell comprising a vector comprising a nucleic acid composition, or a vector composition comprising a plurality of vectors, comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a humanized antibody or antibody fragment specific for human IL3 according to the present disclosure.

[0105] In one embodiment, the present disclosure refers to a host cell comprising a vector or a vector composition comprising a plurality of vectors that comprises a nucleic acid composition comprising a nucleic acid sequence or a plurality of nucleic acid sequences encoding a humanized antibody or antibody fragment specific for IL3, wherein said antibody or antibody fragment comprises a VH of SEQ ID NO: 36 and a VL of SEQ ID NO: 16.

[0106] In one embodiment, a host cell according to the present disclosure is capable of expressing a humanized antibody or antibody fragment specific for human IL3 encoded by a vector composition or a nucleic acid composition.

[0107] In a further embodiment, the host cell is an isolated host cell. In a further embodiment, the host cell is a mammalian cell. In one embodiment, the mammalian cell is a human cell. In another embodiment, the mammalian cell is a CHO cell. In one embodiment, the cell is a HEK cell. In another embodiment, the cell is a PERC.6 cell. In one embodiment, the cell is an HKB11 cell.

[0108] One skilled in the art will understand that one or more nucleic acid sequences encoding the heavy and / or light chains of an antibody or antibody fragment of the present disclosure can be cloned into different vectors or the same vector.

[0109] The vector can be introduced into an appropriate host cell, such as a prokaryotic (e.g., bacterial) or eukaryotic (e.g., yeast or mammalian) cell, by methods well known in the art (e.g., "Current Protocols in Molecular Biology," Ausubel et al. (eds.), Greene Publishing Assoc and John Wiley Interscience, New York, 1989 and 1992). Numerous cloning vectors are known to those skilled in the art, and the selection of an appropriate cloning vector is a matter of choice. The gene can be placed under the control of a promoter, a ribosome binding site (for bacterial expression), and optionally an operator (collectively referred to herein as "control" elements) so that the nucleic acid sequence encoding the desired protein is transcribed into RNA in a host cell transformed with the vector containing this expression construct. The coding sequence may or may not contain a signal peptide or leader sequence. Upon expression in a host cell, the antibody or antibody fragment of the present disclosure is obtained. These steps can be achieved in different ways, as known to those skilled in the art. In general, such processes typically involve transforming or transfecting suitable host cells with a nucleic acid composition, vector composition, or infectious particle encoding the antibody or antibody fragment. Furthermore, such processes typically involve culturing the host cells under conditions suitable for their proliferation (reproduction, growth) and production (expression, synthesis) of the encoded antibody or antibody fragment. Culturing host cells under conditions suitable for proliferation or expression is typically achieved in the presence of a medium containing components suitable for inducing cell growth or expression. In particular, in embodiments, the disclosed methods for producing antibodies or antibody fragments further include isolating and purifying the produced antibody or antibody fragment from the host cells or medium. If the expression system secretes the protein into the growth medium, the protein can be purified directly from the medium. If the protein is not secreted, it is isolated from a cell lysate or recovered from a cell membrane fraction. The selection of appropriate growth conditions and recovery methods is within the skill of one of ordinary skill in the art.The antibodies or antibody fragments of the present disclosure can then be purified by several techniques known to those of skill in the art.

[0110] In one embodiment, the present disclosure refers to a method for producing a humanized antibody or antibody fragment specific for human IL3, wherein the antibody or antibody fragment comprises a VH of SEQ ID NO: 36 and a VL of SEQ ID NO: 16. In one embodiment, a method for producing a humanized antibody or antibody fragment according to the present disclosure is provided, comprising culturing a host cell containing a vector or vector composition comprising a plurality of vectors, under conditions suitable for expression of the antibody or antibody fragment, and isolating the antibody or antibody fragment from the host cell or host cell culture medium. Antibodies or antibody fragments isolated as described herein can be purified using techniques known in the art, such as high-performance liquid chromatography (HPLC), ion exchange chromatography, gel electrophoresis, affinity chromatography, size exclusion chromatography, etc. The conditions used to purify a particular antibody or antibody fragment will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, etc., and will be apparent to one of skill in the art. For affinity chromatography purification, the antibody, ligand, receptor, or antigen to which the antibody or antibody fragment binds can be used. For example, for affinity chromatography purification of antibodies or antibody fragments according to the present disclosure, a matrix with Protein A or Protein G can be used. The purity of the antibody or antibody fragment can be determined by any of a variety of well-known analytical methods, including gel electrophoresis, high pressure liquid chromatography, and the like.

[0111] Effector function The Fc region of an immunoglobulin generally confers favorable pharmacokinetic properties to antibodies, such as extended serum half-life and the ability to induce effector functions via binding to Fc receptors expressed on cells. However, Fc receptor binding can also result in the undesired activation of certain cell surface receptors, potentially resulting in undesired cytokine release and severe side effects upon systemic administration.

[0112] Thus, in certain therapeutic situations, it may be desirable to reduce or eliminate the normal binding of a wild-type Fc region of an antibody, such as a wild-type IgG Fc region, to one or more or all of the Fc receptors and / or binding to complement components such as C1q, to ​​reduce or eliminate the antibody's ability to induce effector functions. For example, it may be desirable to reduce or eliminate binding of the Fc region of an antibody to one or more or all of the Fcy receptors, such as FcyRI, FcyRI1a, FcyRIIb, and FcyRIIIa. Effector functions may include, but are not limited to, one or more of the following: complement-dependent cytotoxicity (CDC), antibody-dependent cell-mediated cytotoxicity (ADCC), antibody-dependent cellular phagocytosis (ADCP), cytokine secretion, immune complex-mediated antigen uptake by antigen-presenting cells, binding to NK cells, binding to macrophages, binding to monocytes, binding to polymorphonuclear cells, direct signaling to induce apoptosis, cross-linking of target-bound antibodies, dendritic cell maturation, or T cell priming.

[0113] Reducing or eliminating the binding of an Fc region to an Fc receptor and / or C1q is typically achieved by mutating a wild-type Fc region, such as an IgG1 Fc region, more specifically a human IgG1 Fc region, to produce a mutant or engineered Fc region of the wild-type Fc region, such as a mutant human IgG1 Fc region. Substitutions that result in reduced binding can be useful. To reduce or eliminate the binding properties of an Fc region to an Fc receptor, non-conservative amino acid substitutions, i.e., replacing one amino acid with another amino acid having different structural and / or chemical properties, are preferred.

[0114] Thus, in one embodiment, an isolated antibody or antibody fragment specific for human IL3 according to the present disclosure comprises a variant Fc region that has reduced or eliminated binding to an Fc receptor and / or C1q when compared to the wild-type Fc region. In one such embodiment, the isolated antibody or antibody fragment according to the present disclosure comprises a variant Fc region that reduces or eliminates the ability of the antibody to induce effector function. In a further embodiment, the isolated antibody or antibody fragment according to the present disclosure does not substantially induce effector function.

[0115] In certain embodiments, the effector function is one or more selected from the group consisting of CDC, ADCC, and ADCP. In one embodiment, the effector function is ADCC. In one embodiment, the effector function is CDC. In one embodiment, the effector function is ADCP. In one embodiment, an isolated antibody or antibody fragment according to the present disclosure does not substantially induce ADCC and / or CDC and / or ADCP. In one embodiment, an isolated antibody or antibody fragment according to the present disclosure does not induce ADCC or ADCP in vitro.

[0116] In one embodiment, the variant Fc region of a humanized antibody or antibody fragment according to the present disclosure comprises one or more amino acid substitutions that reduce or eliminate binding of the variant Fc region to one or more Fc receptors and / or C1q when compared to the wild-type Fc region. In one embodiment, the variant Fc region of a humanized antibody or antibody fragment according to the present disclosure comprises one or more amino acid substitutions that reduce or eliminate the ability of the antibody to induce effector function when compared to the wild-type Fc region. In certain embodiments, the one or more amino acid substitutions may reduce the binding affinity of the variant Fc region to one or more Fc receptors and / or C1q by at least two-fold, at least five-fold, at least ten-fold, at least twenty-fold, or even at least fifty-fold when compared to the wild-type Fc region. In alternative embodiments, the one or more amino acid substitutions may reduce the ability of an isolated antibody or antibody fragment according to the present disclosure to induce effector function by at least two-fold, at least five-fold, at least ten-fold, at least twenty-fold, or even at least fifty-fold when compared to the wild-type Fc region.

[0117] In one embodiment, the variant Fc region of a humanized antibody or antibody fragment according to the present disclosure does not substantially bind to one or more Fc receptors and / or C1q. In one embodiment, the variant Fc region of an antibody according to the present disclosure substantially eliminates the ability of the antibody to induce effector function. In one embodiment, an antibody or antibody fragment according to the present disclosure does not substantially induce effector function. In one embodiment, the effect function is ADCC and / or ADCP and / or CDC. In one embodiment, an antibody or antibody fragment according to the present disclosure does not substantially induce effector function, meaning that the level of induced effector function is not significantly above background measured in the absence of the antibody.

[0118] In one embodiment, the Fc receptor is a human Fc receptor. In one embodiment, the Fc receptor is a Fey receptor. In one embodiment, the Fc receptor is human FcyRIIIa, FcyRI, FcyRIla and / or FcyRIlb.

[0119] In one embodiment, a humanized antibody or antibody fragment according to the present disclosure comprises a variant human IgG1 Fc region comprising one or more amino acid substitutions compared to a wild-type human IgG1 Fc region, in one embodiment, the one or more amino acid substitutions reduce or eliminate binding of the variant Fc region to Fc receptors and / or C1q and / or reduce the ability of the antibody to induce effector function when compared to the wild-type Fc region.

[0120] Humanized antibodies or antibody fragments according to the present disclosure may or may not be fused to one or more other amino acid residues, polypeptides, or moieties. Such fusion proteins can be prepared by any suitable method, including genetic or chemical approaches. The linked moieties may contain secretory or leader sequences, sequences that aid in detection, expression, separation, or purification, or sequences that provide increased protein stability, for example, during recombinant production. Non-limiting examples of potential moieties include beta-galactosidase, glutathione-S-transferase, luciferase, a T7 polymerase fragment, a secretory signal peptide, an antibody or antibody fragment, a toxin, a reporter enzyme, a moiety capable of binding to a metal ion such as a polyhistidine tag, a tag suitable for detection and / or purification, a homo- or hetero-association domain, a moiety that increases protein solubility, or a moiety containing an enzymatic cleavage site.

[0121] Thus, a humanized antibody or antibody fragment according to the present disclosure may optionally contain one or more moieties for binding to other targets or target proteins of interest. It will be apparent that such additional moieties may or may not provide additional functionality to the antibody and may or may not modify the properties of the isolated antibody or antibody fragment according to the present disclosure.

[0122] Treatment method Humanized antibodies or antibody fragments according to the present disclosure can be used in therapeutic methods: for the treatment of inflammatory diseases, autoimmune diseases, fibrotic diseases, hematological malignancies, and potentially other diseases.

[0123] In one embodiment, the disease is associated with the unwanted presence of human IL3, hi another embodiment, the disease is associated with the unwanted presence of IL3-expressing cells, particularly human IL3-expressing T cells.

[0124] IL3 has significant growth-stimulating and differentiation effects on various hematopoietic progenitor cells and is also a growth factor for mast cells. IL3-induced signaling has a profound effect on the immune system. Any disease or medical condition in which IL3 plays a direct or indirect role in the development or progression is a candidate for treatment by administering an antibody according to the present disclosure. Preferably, such diseases or dysfunctions associated with elevated levels of IL3 or elevated expression of IL3 by cells capable of producing IL3, such as T cells, basophils, B cells, NK cells, certain cancers / tumors, and neurons, are associated with the immune system, primarily autoimmune, inflammatory, or fibrotic diseases, particularly systemic lupus, systemic sclerosis, RA, PsA, or acute or chronic graft-versus-host disease and multiple sclerosis, acute or chronic allograft rejection.

[0125] In one embodiment, the disease to be treated is an autoimmune or inflammatory disease. Non-limiting examples of autoimmune or inflammatory diseases include autoinflammatory diseases such as rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, systemic lupus erythematosus (SLE), lupus nephritis, type I diabetes, Graves' disease, inflammatory bowel disease (IBD), Crohn's disease (CD), ulcerative colitis (UC), irritable bowel syndrome, multiple sclerosis (MS), Guillain-Barré syndrome, familial Mediterranean fever (FMF), cryopyrin-associated periodic syndromes (CAPS), IL-1 receptor antagonist deficiency (DIRA), hyper IgD syndrome (HIDS), autoimmune myocarditis, Kawasaki disease, coronary artery disease, chronic obstructive pulmonary disease (COPD), interstitial lung disease, and autoimmune diseases such as bronchitis, bronchitis, and bronchial asthma. These include immune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atopic dermatitis, vitiligo, graft-versus-host disease, Sjogren's syndrome, autoimmune nephritis, Goodpasture's syndrome, chronic inflammatory demyelinating polyneuropathy, ANCA-associated vasculitis, uveitis, scleroderma, bullous pemphigoid, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's chorea, cystic fibrosis, gout, age-related macular degeneration, allergies, asthma, antiphospholipid syndrome (APS), atherosclerosis, C3 glomerulopathy and IgA nephropathy, ischemia / reperfusion injury, peritonitis, sepsis, and other autoimmune diseases that result in either acute or chronic inflammation.

[0126] In one embodiment, the disease to be treated is a proliferative disease.In certain embodiments, the disease is cancer.Non-limiting examples of cancer include hematological malignancies such as chronic myelomonocytic leukemia (CMML), acute myeloid leukemia (AML), myelodysplastic syndrome, mastocytosis, basophilic leukemia, pDC cancer, and non-hematological malignancies such as bladder cancer, brain cancer, head and neck cancer, pancreatic cancer, lung cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, endometrial cancer, esophageal cancer, colon cancer, colorectal cancer, rectal cancer, gastric cancer, prostate cancer, blood cancer, sarcoma, skin cancer, squamous cell carcinoma, bone cancer, melanoma, renal cell carcinoma and kidney cancer.

[0127] Further conditions that are preferably treated by administering a pharmaceutical composition according to the invention are use in suppressing the activity of human basophils in people suffering from allergic reactions and for stratifying patients with elevated levels of IL3 in serum or plasma or elevated expression of IL3 by cells capable of producing IL-3 (e.g., T cells).

[0128] Particularly considering RA, it has been found that in many patient groups, the increased level of hIL3 produced by T cells correlates with the deterioration and disease progression caused in patients.IL3 is mainly detected in active RA, while patients with inactive stage of RA usually do not show increased expression or level of IL3.Therefore, the pharmaceutical composition of the present invention is particularly useful for treating patients with active episodes of autoimmune diseases such as RA, and for preventive treatment to avoid the occurrence of active episodes of disease.

[0129] Because available treatments are only effective in approximately 50% of treated patients, providing pharmaceutical compositions according to the present invention represents a major step toward new and gentler treatments for autoimmune diseases in patients. Based on the lack of a clear phenotype in IL3-deficient mice (Nature (1998) 392:90-3) and the apparent side effects in mice treated with antibodies against IL3 (Arthritis Rheum (2009) 60:1352-61), IL-3-targeted treatments should exhibit less severe side effects than currently used pharmaceuticals, particularly with regard to infection or neoplasia. In certain cases, it may be desirable to combine treatment with the antibodies and pharmaceutical compositions of the present invention with other medications, such as methotrexate or leflunomide. Individualized treatment strategies based on IL3 expression or IL3 levels in plasma, serum, or other body fluids offer advantages over available biologics, as it is currently impossible to reliably predict which patients will respond to a particular treatment (including biologics). Furthermore, a personalized approach improves the safety of treatment by reducing the risk of side effects of ineffective treatment and reduces the cost of treating RA.

[0130] Furthermore, treatment with the antibody of the present invention will be preferentially initiated as soon as elevated IL3 expression or elevated IL3 levels are detected in blood, plasma, or serum. Therefore, early treatment can be applied to patients whose RA activity is correlated with elevated hIL-3 levels, and long-term joint damage can be avoided or minimized. In addition, if a patient has failed previous treatment with DMARDs or biologics, treatment with an anti-IL3 antibody will be preferentially initiated. Furthermore, it is expected that the use of the antibody of the present invention can reduce cellular infiltration of synovial tissue, which may be an additional negative factor in disease pathology.

[0131] In one embodiment, the present disclosure provides a method for treating a disease.

[0132] In one embodiment, the present disclosure provides a method for treating a disease comprising administering to a patient an antibody or antibody fragment of the present disclosure.

[0133] In one embodiment, the present disclosure provides a method for treating a disease comprising administering to a subject in need thereof an antibody or antibody fragment of the present disclosure.

[0134] In one embodiment, the present disclosure provides a method for preventing a disease.

[0135] In one embodiment, the present disclosure provides a method for preventing a disease comprising administering to a subject an antibody or antibody fragment of the present disclosure.

[0136] In one embodiment, the present disclosure provides a humanized antibody or antibody fragment according to the present disclosure for treating a disease. In one embodiment, the present disclosure provides a humanized antibody or antibody fragment according to the present disclosure for use in treating a disease. In one embodiment, the present disclosure provides a humanized antibody or antibody fragment according to the present disclosure for use in treating a disease in a subject in need thereof.

[0137] In one embodiment, the present disclosure provides the use of an antibody or antibody fragment according to the present disclosure for the manufacture of a medicament. In one embodiment, the present disclosure provides a humanized antibody or antibody fragment according to the present disclosure for use as a medicament. In one embodiment, the present disclosure provides a humanized antibody or antibody fragment according to the present disclosure for use in medicine. In one embodiment, the present disclosure provides a humanized antibody or antibody fragment according to the present disclosure for use as a medicament to treat a subject in need thereof.

[0138] In one embodiment, the present disclosure provides a humanized antibody or antibody fragment specific for human IL3 according to the present disclosure for use in a method of treating a subject having a disease, comprising administering to the subject a therapeutically effective amount of an antibody or antibody fragment according to the present disclosure.

[0139] In one embodiment, the method further comprises administering to the subject a therapeutically effective amount of at least one additional therapeutic agent. The subject in need of treatment is typically a mammal, more particularly a human. For use in therapeutic methods, the humanized antibody or antibody fragment according to the present disclosure will be formulated, dosed, and administered in a manner consistent with good medical practice.

[0140] Pharmaceutical Composition In one embodiment, the present disclosure provides a pharmaceutical composition comprising an isolated antibody or antibody fragment according to the present disclosure and a pharmaceutically acceptable carrier or excipient.

[0141] The pharmaceutical composition may further comprise at least one other pharmaceutically active compound. The pharmaceutical composition according to the present disclosure can be used for the diagnosis, prevention, and / or treatment of diseases associated with the undesired presence of IL3, particularly human IL3. The pharmaceutical composition according to the present disclosure can be used for the diagnosis, prevention, and / or treatment of diseases associated with the undesired presence of IL3-positive cells, particularly IL3-positive human cells. In particular, the present disclosure provides pharmaceutical compositions comprising an antibody or antibody fragment according to the present disclosure that are suitable for prophylactic, therapeutic, and / or diagnostic use in mammals, more particularly humans.

[0142] Generally, antibodies or antibody fragments according to the present disclosure can be formulated as pharmaceutical compositions comprising at least one antibody or antibody fragment according to the present disclosure, at least one pharmaceutically acceptable carrier or excipient, and optionally one or more additional pharmaceutically active compounds. Such formulations can be suitable for oral, parenteral, topical, or inhalation administration. Thus, pharmaceutical compositions comprising at least one antibody or antibody fragment according to the present disclosure can be administered parenterally, for example, intravenously, intramuscularly, or subcutaneously. Alternatively, antibodies of the present invention can be administered via the oral route, for example, orally or topically. In a preferred embodiment, pharmaceutical compositions comprising antibodies or antibody fragments according to the present disclosure are administered intravenously or subcutaneously.

[0143] In particular, antibodies or antibody fragments according to the present disclosure are or can be used for the prevention and / or treatment of diseases involving the target antigen of interest, and may therefore be used in combination with one or more pharmaceutically active compounds, which may or may not result in a synergistic effect. Examples of such compounds, as well as routes, methods and pharmaceutical formulations or compositions for administering them, will be apparent to the clinician.

[0144] In one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure for use in the prevention and / or treatment of a disease associated with the undesired presence of IL3, particularly human IL3. In one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure for use in the prevention and / or treatment of a disease associated with the undesired presence of IL3-positive cells, particularly IL3-positive human cells. In one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure for use as a medicament. In one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure for use in the prevention and / or treatment of an autoimmune disease and / or inflammatory disease and / or cancer.

[0145] In one embodiment, the present disclosure provides a method for treating an autoimmune disease and / or an inflammatory disease and / or cancer in a subject in need thereof using a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure.

[0146] Further provided is a method for producing an antibody or antibody fragment according to the present disclosure in a form suitable for in vivo administration, comprising: (a) obtaining the antibody or antibody fragment by a method according to the present disclosure; and (b) formulating the antibody or antibody fragment with at least one pharmaceutically acceptable carrier or excipient, whereby the antibody or antibody fragment preparation is formulated for in vivo administration. Pharmaceutical compositions according to the present disclosure comprise a therapeutically effective amount of one or more antibodies or antibody fragments according to the present disclosure dissolved in a pharmaceutically acceptable carrier or excipient.

[0147] Diagnostic Use In one embodiment, the present disclosure provides the use of a humanized antibody or antibody fragment specific to human IL3 according to the present disclosure for the diagnosis of disease. In one embodiment, the present disclosure provides the use of an antibody or antibody fragment according to the present disclosure for the detection of IL3, particularly human IL3. In one embodiment, the present disclosure provides a method for detecting IL3 in a subject or sample, comprising contacting the subject or sample with a humanized antibody or antibody fragment specific to human IL3 according to the present disclosure. In one embodiment, the present disclosure provides a method for diagnosing disease in a subject, comprising contacting the subject or sample with a humanized antibody or antibody fragment according to the present disclosure. The antibody can also be used to determine IL3 expression levels in cells derived from a patient. IL3 expression levels can serve as a therapeutic biomarker, for example, for patient stratification. [Example]

[0148] Example 1 Parent antibody P8C11 (DSM ACC3281) The parental murine antibody P8C11 was originally produced by immunizing Balb / c mice with human eukaryotic glycosylated IL-3, see WO 2017 / 081218. The antibody was deposited at DSMZ on October 7, 2015 (DSM ACC3281).

[0149] P8C11 is a murine IgG1 monoclonal antibody. The sequences of the variable heavy chain (IGHV1S137*01), variable light chain (IGKV15-103*01) and CDRs are shown in Table 1 below. The variable heavy chain of antibody P8C11 has three mutations compared to the germline sequence: M instead of L, A instead of S, and I instead of V. These positions are shown in SEQ ID NO:2 below.

[0150] [Table 1]

[0151] Antibody P8C11 was originally produced by immunizing Balb / c mice with human eukaryotic glycosylated IL-3 (see WO 2017 / 081218). The antibody was deposited with DSMZ on October 7, 2015 (DSM ACC3281). P8C11 inhibits the IL3-dependent growth and many other beneficial activities of TF1 cells, indicating that P8C11 may be a candidate for clinical development (WO 2017 / 081218).

[0152] Example 2 Humanization of antibody P8C11

[0153] Example 2.1 CDR grafting onto new framework sequences In a first step, the CDRs of P8C11 were grafted onto the frameworks of the three most closely related human germline genes - IGKV1-12*01, IGKV1-33*01, and IGKV1-27*01 for the variable light chain. For the variable heavy chain, IGHV1-3*01 and IGHV7-4-1*02 were selected, as well as the IGHV1-3*01 variant, which contains the germline mutation of SEQ ID NO: 2, with one alanine residue backmutated to the original glycine residue due to the proximity of this amino acid to HCDR1. The sequences of the variable light and heavy chains are shown in the table below.

[0154] [Table 2]

[0155] All three variable heavy chains were paired with all three variable light chains to yield nine different antibodies. The inhibitory activity of these nine antibodies was tested and compared to that of a chimeric version of P8C11 (an antibody with the variable murine heavy and light chains of P8C11 fused to human IgG1 kappa). Inhibitory activity was measured by analyzing the ability of the antibody variants to block IL-3-induced downregulation of CD131 on primary human basophils. IL-3 (0.2 ng / ml) was preincubated with various concentrations of chimeric P8C11 or humanized variants, or PBS as a control, for 1 hour at 37°C, and then added to whole human EDTA blood for 1 hour at 37°C. In a separate vial, PBS alone (without IL-3) was added to whole human EDTA blood for 1 hour at 37°C to induce no downregulation of CD131. The blood was then stained with direct antibodies against CD123, CD203c, and CD131 on ice for 30 minutes to identify basophils and measure the surface levels of CD131 on basophils. The inhibitory activity of chimeric P8C11 was normalized to 100%, and the inhibitory activity of PBS in the absence of antibody was normalized to 0%. If the concentration of the humanized variant needed to be 10 times higher than that of chimeric P8C11 to achieve the same level of inhibition of CD31 downregulation, the inhibitory activity was determined to be 10%.

[0156] The inhibitory activity is shown in the table below.

[0157] [Table 3]

[0158] None of the nine humanized antibodies possessed inhibitory activity approaching that of chimeric P8C11. Of the three variable heavy chains, HC3 was the best variant to proceed. The three variable light chains were equally effective. LC2 was selected for further humanization.

[0159] Example 2.2 Identification of the variable chain involved in the loss of activity Next, to investigate whether the variable heavy or variable light chain acceptor framework was responsible for the loss of inhibitory activity, the murine variable heavy chain was paired with the variable light chain VL2 (muHCxLC2) and the murine variable light chain was paired with the variable heavy chain HC3 (HC3xmuLC). The inhibitory activity was again compared with that of chimeric P8C11. The results are shown in the table below.

[0160] [Table 4]

[0161] This result indicated that the loss of inhibitory activity was due to humanization of the variable heavy chain and not the variable light chain.

[0162] Example 2.3 Identification of important frameworks within HC3 To investigate which part of the framework is responsible for the loss of inhibitory activity, various hybrid frameworks consisting of either the original murine framework or respective parts of the humanized framework of HC3 were constructed.

[0163] Three constructs were generated as shown in the table below.

[0164] [Table 5]

[0165] These variable heavy chains were paired with the murine variable light chain of P8C11 and the inhibitory activity was measured, the results are shown in the table below.

[0166] [Table 6]

[0167] This result indicates that framework 3 (FR3) of HC3 is involved in the loss of inhibitory activity. Because only one amino acid differs from the original mouse framework sequence and FR4 usually does not contribute significantly to VH function, it is highly unlikely that framework 4 (FR4) of HC3 is involved.

[0168] Example 2.4 Identification of key residues within framework 3 of HC3 To investigate which part of framework 3 in HC3 is responsible for the loss of inhibitory activity, various hybrids of framework 3 were generated. All constructs had identical framework regions 1 and 2, and all three CDRs were identical. In one construct, HC4, the first 10 amino acids of framework 3 were those of the original mouse framework, and the remaining amino acids were those of the human germline sequence IGHV1-3*01 (except for one isoleucine residue corresponding to the germline mutation in the original mouse antibody P8C11). In one construct, HC5, the first 10 amino acids of framework 3 were those of the human germline sequence IGHV1-3*01, and the remaining amino acids were those of the original mouse framework. In the third construct, HC6, framework 3 was composed of hybrids IGHV1-18*01 and IGHV1-69*02, both of which share similarity with the original mouse framework of P8C11 at the N- and C-termini, respectively. Again, the isoleucine residue in framework 3 corresponding to the germline mutation in the original mouse antibody P8C11 was maintained. In addition, the "murine" valine at position 6 of framework 3 and the "murine" serine at position 10 of framework 3 were maintained.

[0169] The amino acid sequences of the three constructs are shown in the table below.

[0170] [Table 7]

[0171] These variable heavy chains were paired with the humanized variable light chain VL2 and the inhibitory activity was measured, the results are shown in the table below.

[0172] [Table 8]

[0173] Comparison of heavy chain HC4 and HC5 shows that the first 10 amino acids of framework region 3 are important for preserving antibody activity. If these 10 amino acids have the original murine sequence, the antibody is fully active. H6, which has a similar amino acid sequence in this position, also fully preserves activity.

[0174] Example 2.5 Optimization of HC6 for enhanced humanization To further increase the degree of humanization, four different heavy chains were designed based on HC6: HC7 and HC8 are both based on HC6 but contain less remaining mouse framework sequence; HC9 and HC10 are also both based on HC6 but are closer to the germline IGHV1-3*01.

[0175] The amino acid sequences of the four constructs are shown in the table below.

[0176] [Table 9]

[0177] Again, these variable heavy chains were paired with the humanized variable light chain VL2 and the inhibitory activity was measured, the results are shown in the table below.

[0178] [Table 10]

[0179] A comparison of heavy chains HC7 and HC8 shows that the remaining murine-derived amino acids A and G in framework 1 are important for activity, while the remaining murine-derived amino acid M in framework 1 and I in framework 3 are not essential for activity. A comparison of heavy chains HC9 and HC10 shows that at position 4 in framework 3, amino acid M confers slightly higher activity than amino acid I.

[0180] Example 2.6 Optimization of HC7 for enhanced humanization To further increase the degree of humanization, six additional different heavy chains were designed based on HC7.

[0181] The amino acid sequences of the three constructs are shown in the table below.

[0182] [Table 11]

[0183] Again, these variable heavy chains were paired with the humanized variable light chain VL2 and the inhibitory activity was measured, the results are shown in the table below.

[0184] [Table 12]

[0185] Comparison of heavy chains HC11 and HC12 shows that a V at position 6 of framework 3 is important for preserving activity. An A at position 6 of FR3 results in a significant loss of inhibitory activity.

[0186] Comparison of heavy chains HC11 and HC13 shows that amino acids T and S at position 10 of framework 3 result in similar inhibitory activity. T at position 10 of framework 3 is preferred for increasing the degree of humanization.

[0187] Comparison of heavy chains HC11, HC14 and HC15 indicates that G at position 24 of framework 1 is important for preserving inhibitory activity and should not be replaced with A.

[0188] Comparison of heavy chains HC11, HC14 and HC15 also shows that at position 21 of framework 1, amino acid A is not essential for preserving activity and can be replaced by S to increase the degree of humanization.

[0189] The heavy chain HC16 contains a G at position 24 of framework 1, a V at position 6 of framework 3 and a T at position 10 of FR3. It exhibits full inhibitory activity and the best possible degree of humanization.

[0190] Therefore, an antibody comprising the heavy chain HC16 and the variable light chain LC2 was selected as the final candidate. This antibody was designated "GRT002-H16L2". The antibody has the following heavy chain sequence: QVQLVQSGAEVKKPGASVKVSCKGSGFTFTDYALHWVRQAPGQRLEWMGLISTYYGDTTYNQRFKGRVTMTVDKSTSTAYMELSSLRSEDTAVYYCVITTVAGTDAMDYWGQGT LVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHT CPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG (SEQ ID NO: 44) and the following light chain sequence: DIQMTQSPSSLSASVGDRVTITCHASQNINVWLSWYQQKPGKAPKLLIYKASNLHTGVPSRFSGSGSGTDFTFTISSLQPEDIATYYCQQGYSYPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 45).

[0191] Example 2.7 Overview of the humanization process The humanization of P8C11 was far from straightforward. Finally, a six-step iterative process was required to identify a humanized derivative of P8C11 that retained the desired activity, i.e., inhibition of glycosylated IL-3. During this process, three different variable light chains and 16 different variable heavy chains were generated, which were tested in various combinations. Various critical amino acids required for complete IL-3 binding and inhibition were identified. Many of these residues are directly adjacent to the CDR regions.

[0192] GRT002-H16L2 was identified as the final humanized variant of P8C11. The variable light chain framework of GRT002-H16L2 is 100% identical to the human germline. Two amino acids in the variable heavy chain framework of GRT002-H16L2 are not identical to the human germline. These two amino acids are essential for full binding and inhibition of IL-3. GRT002-H16L2 is an IgG1 antibody of the G1m3 (heavy chain) and KM3 (light chain) allotypes lacking the C-terminal lysine.

[0193] Example 3 Generation and purification of GRT002-H16L2 Antibody GRT002-H16L2 was expressed and purified by Evitria AG (Schlieren, Switzerland) using standard techniques. Briefly, CHO cells were stably transfected with a vector encoding the GRT002-H16L2 antibody. Cells were grown in serum-free medium. Expression levels were >100 mg / L. The antibody was purified with Protein A (Schlieren, Switzerland). The antibody was dissolved in PBS + 100 mM L-arginine at a concentration of 7.75 mg / ml and stored in aliquots at -20°C until further use.

[0194] The antibody preparation had a monomer content of >97% and a molecular weight of 152.12 kDa as determined by SEC using eight marker proteins.

[0195] Example 4 Determination of the binding properties of GRT002-H16L2 The binding properties of antibody GRT002-H16L2 were determined by surface plasmon resonance (SPR) in comparison with the parent antibody P8C11 using a Biacore X100 instrument (GE Healthcare). Chimeric antibody P8C11 was used as a reference. The antibody was covalently coupled to a CM5 sensor chip via a dextran matrix. Degassed PBS + 0.05% Tween-20 was used as the running buffer. The antibody was used at a concentration of 1.5 μg / ml, and the IL3 concentration was varied. Capture was performed for 45 min at a flow rate of 5 μl / min (sample cells only). After 300 s of stabilization, IL3 was added for 360 s at a flow rate of 15 μl / min (sample and reference cells). The dissociation time was 600 s. The chip was regenerated with 10 mM Gly-HCl pH 1.5 for 30 seconds at a flow rate of 30 μl / min, followed by a wash step with running buffer and 100 mM PBS pH 7.4. Data were analyzed using Biacore evaluation software, version 2.0.1. For affinity fitting, a "steady-state affinity" model was used, and a "1:1 binding" model was used for kinetic fitting.

[0196] The results are shown in Figure 1. Based on the curves shown in Figure 1, the binding characteristics could be determined, which showed that the binding of antibody GRT002-H16L2 to IL-3 was the same as or even slightly better than that of the parent antibody P8C11.

[0197] Example 5 Epitope mapping of GRT002-H16L2 The epitope of GRT002-H16L2 was mapped by measuring the binding of the antibody to a linear peptide derived from the amino acid sequence of IL3.

[0198] The peptide was coated overnight in PBS on a Nunclon 96 flat-bottom clear polystyrene plate (Thermo Fisher) at a concentration of 10 μg / ml (60 μl / well). The cells were then washed three times with wash buffer (0.05% Tween 20 in PBS) and subsequently blocked with 1% BSA / PBS at room temperature for 2 hours. After another wash step with wash buffer, antibody GRT002-H16L2 was added (60 μl of a 10 μg / ml solution in 1% BSA / PBS) and incubated at room temperature for 1 hour. 1% BSA / PBS served as a control. After another wash step, GRT002-H16L2 was incubated with a secondary antibody (HRP-conjugated AffinePure fragment goat anti-human IgG, Jackson Immuno Research) for 1 hour at room temperature. After a final washing step, 100 μl of ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) was added to each well. Absorbance was measured at 405 nm in an Infinity 200 Pro reader (Tecan).

[0199] For the parent antibody P8C11, amino acids 22-48 (counting without the signal sequence) of human IL3 were reported as the epitope (see WO 2017 / 081218). The same epitope was confirmed for GRT002-H16L2. To further refine the epitope, the following amino acids were tested:

[0200] [Table 13]

[0201] The results are shown in Figure 2. The epitope of GRT002-H16L2 is identical to that of P8C11. Deletion of amino acids 47 and 48 of the tested peptide results in a dramatic decrease in binding of GRT002-H16L2. GRT002-H16L2 still binds to peptides lacking amino acids 22-25 or 22-27, but to a slightly lesser extent. In summary, GRT002-H16L2 binds to a linear epitope contained in the amino acid sequence EIITHLKQPPLPLLDFNNLNGEDQDIL (SEQ ID NO: 37).

[0202] Example 6 IL-3 blockade in TF1 cells In the first experiment to analyze the ability of GRT002-H16L2 to block IL-3 activity, TF1 cells were used. TF1 cells (ATCC, Order No. #crl-2003) are human erythroblasts, and this cell line was established from the bone marrow of a 35-year-old Japanese man suffering from severe pancytopenia. The growth of TF1 cells is completely dependent on the presence of IL-3 or GMCSF. Therefore, a test based on cell proliferation of TF1 cells can be used to determine the blockade of IL-3 activity, which results in a reduction or even complete inhibition of TF1 cell growth.

[0203] Experimentally, an MTT cell proliferation assay was performed to determine cell viability based on the activity of mitochondrial dehydrogenases. The dehydrogenase substrate MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), which exhibits a yellow color in solution, is cleaved at the tetrazolium ring by enzymatic activity, resulting in the formation of purple MTT formazan crystals. Such crystals can be dissolved in isopropanol, and the purple solution is measured spectrophotometrically. The results correlate with the amount of viable TF1 cells.

[0204] TF1 cell culture: TF1 cells were grown in suspension in culture medium (RPMI-1640 containing 10% FCS (HIA), P / S, and glutamine (1:100)) supplemented with either 5 ng / ml IL-3 or 5 ng / ml GM-CSF and split 1:4 every 3 days. For storage, cells were transferred from cell culture bottles to 50 ml or 15 ml cell culture flasks (BD Falcon™). After centrifugation at 1400 rpm for 5 minutes at room temperature, the supernatant was completely removed. Cells were resuspended in culture medium (RPMI-1640 containing 10% FCS (HIA) + P / S + glutamine + 5 ng / ml IL-3) with 5% DMSO, and 1.5 ml aliquots were filled into vials. Cells were pre-frozen in a freezing container in a -80°C freezer and transferred to a liquid nitrogen storage tank after 1-2 days.

[0205] Interruption Test: TF-1 cells, split every 3 days according to the above protocol, are split 1:2 into medium containing 5 ng / ml human IL-3 the day before the experiment. For the experiment, the cells are centrifuged at 1600 rpm for 5 minutes at room temperature. After removing the culture medium and washing the cells twice with RPMI medium, the cells are resuspended in 1 ml of RPMI-1640 + 10% FCS (HIA) + P / S + glutamine (1:100), counted, and collected at 1 x 10 5The buffer is supplemented to a final concentration of 10,000 cells / ml in 100 μl of medium (RPMI + 10% FCS + P / S + glutamine) in a 96-well plate. 100 μl of IL-3 is pre-incubated with the antibody to be tested for 60 minutes at 37°C. Different final concentrations of antibody and IL-3 are used for pre-incubation. To achieve these final concentrations, the concentrations of the antibody and IL-3 solutions need to be twice the final concentrations. After 5 days of incubation at 37°C and 5% CO2, 100 μl of medium is removed from each well, 10 μl of MTT solution (LCG Standard - ATCC) is added to each well, and the plate is incubated for an additional 4 hours in an incubator at 37°C and 5% CO2. After this further incubation, 100 μl of MTT solution is added, and the contents of the wells are carefully mixed. After overnight incubation, the optical density is determined at 570 and 690 nm, from which the number of viable cells is calculated.

[0206] Experiments were performed with the chimeric antibody P8C11 and the antibody GRT002-H16L2, and the results are shown in Figure 3. Both antibodies block IL-3 activity in TF1 cells to a similar extent.

[0207] Example 7 Blockade of IL-3 binding to human basophils To quantify the GRT002-H16L2- and P8C11-Chim-mediated blockade of IL-3 binding to CD123 on basophil granulocytes, an "IL-3 Biotinylated Fluorokine Flow Cytometry Kit" (R&D Systems) was used. Antibodies were used at various dilutions in 1% BSA / PBS. To 10 μl of antibody solution, 10 μl of biotinylated IL-3 (1 μg / ml) was added. Samples were preincubated on ice in the dark for 30 minutes. 60 μl of EDTA-blood was then added, followed by incubation on ice in the dark for 60 minutes. Next, 20 μl of avidin-FITC, 3.33 μl of CD123PE-Cy5.5 (1:30), and 1 μl of CD203c PE (1:100) were added, followed by further incubation on ice in the dark for 30 minutes. After two washing steps, red blood cells were lysed by adding 1 ml of FACS lysing solution for 10 minutes at room temperature in the dark. Samples were then washed with PBS and centrifuged at 1,600 rpm and 4°C for 6 minutes. Samples were then analyzed for 60 seconds on a BD FACS Canto II instrument (Becton Dickinson).

[0208] The results for antibodies P8C11 and GRT002-H16L2 are shown in Figure 4. Both antibodies block IL-3 binding to human basophils to a similar extent.

[0209] Example 8 Biophysical characterization of GRT002-H16L2 For clinical development, it is important that antibodies have biophysical properties that allow them to be manufactured, stored, formulated, and administered in sufficient quantities without any undesirable effects such as off-target adhesion. Therefore, antibody GRT002-H16L2 was tested in a series of assays and compared to the parent antibody P8C11.

[0210] Example 8.1 Melting temperature Melting points were determined by DSC (differential scanning calorimetry) using a Microcal VP-DSC instrument (Malvern). All samples were degassed (Microcal Thermovac, Malvern). Samples were measured over a temperature range of 25-130 °C, increasing the temperature at 1 °C / min. Antibodies were in a solution of PBS + 100 mM L-arginine. The antibody concentrations were 7.75 mg / ml for GRT002-H16L2 and 4.55 mg / ml for P8C11-Chim.

[0211] The patterns recorded for antibodies P8C11 and GRT002-H16L2 are shown in Figures 5 and 6, respectively. Tm1 = CH2 melting: 70, 83 °C for P8C11-chim and 70, 81 °C for GRT002-H16L2 Tm2 = Fab and CH3 melting: 78.60 °C for P8C11Chim and 82.53 °C for GRT002-H16L2

[0212] The relative melting temperature Tm2 containing the Fab region was found to be much lower for chimeric P8C11 than for GRT002-H16L2, indicating that GRT002-H16L2 has higher thermal stability.

[0213] Example 8.2 Freezing and thawing To test the behavior of antibodies subjected to repeated freezing and thawing, antibody GRT002-H16L2 (7.75 mg / ml in PBS containing 100 mM L-arginine) was subjected to repeated freeze-thaw cycles. After 10 cycles of freezing and thawing, the sample was subjected to SEC. No effect on antibody integrity was observed in the SEC chromatograms (data not shown).

[0214] The effects of freezing and thawing were also investigated in more detail for antibody GRT002-H16L2 to analyze its functional activity by measuring CD131 expression levels on basophils. The inhibitory activity of the antibody was measured by analyzing the ability of the antibody variants to block IL-3-induced downregulation of CD131 on primary human basophils. IL-3 (0.2 ng / ml) was preincubated with various concentrations of chimeric P8C11 or GRT002-H16L2 or PBS as a control for 1 hour at 37°C, and then added to whole human EDTA blood for 1 hour at 37°C. In a separate vial, PBS alone (without IL-3) was added to whole human EDTA blood for 1 hour at 37°C to avoid CD131 downregulation. The blood was then stained with direct antibodies against CD123, CD203c, and CD131 on ice for 30 minutes to identify basophils and measure the surface levels of CD131 on basophils.

[0215] The results are shown in Figure 7. It can be demonstrated that repeated freezing and thawing does not affect the functional activity of GRT002-H16L2.

[0216] Example 8.3 temperature stability To test the temperature stability of the antibodies, GRT002-H16L2 and chimeric P8C11 were investigated by analytical size-exclusion chromatography (SEC). A 7.75 mg / ml solution of GRT002-H16L2 and a 4.55 mg / ml solution of chimeric P8C11 in PBS + 100 mM L-arginine were incubated at 55, 65, or 75°C for 3 hours, respectively, followed by analytical SEC using a Superdex200 and photometric detection of the protein at 280 nm.

[0217] The SEC patterns of both antibodies incubated at 55°C for 3 hours were indistinguishable from the reference, i.e., the respective antibodies maintained at 4°C. After 3 hours of incubation at 65°C, antibody P8C11-chim showed a first shoulder in the retention volume before the main peak, indicating the formation of aggregates. In contrast, the SEC pattern of GRT002-H16L2 remained indistinguishable from the reference. After 3 hours of incubation at 75°C, both antibodies showed a strong decrease in their integrity, but this was more pronounced for chimeric P8C11.

[0218] The effect of exposure to elevated temperatures was also investigated for both antibodies in order to analyze their functional activity by measuring the level of CD131 expression on basophils. CD131 expression was analyzed as described in Example 8.2.

[0219] The results are shown in Figure 8 for samples incubated at 75°C for 3 hours compared to the reference (3 hours of exposure at 55°C or 65°C did not result in any loss of functional activity for both antibodies). It can be seen that antibody GRT002-H16L2 exhibits significantly higher functional activity after 3 hours of exposure at 75°C compared to antibody P8C11-chim. At a given antibody concentration, the functional activity of GRT002-H16L2 was more than twice that of antibody P8C11-chim. After exposure to 75°C, a more than 16-fold higher concentration of P8C11-chim was required than GRT002-H16L2 to achieve the same level of IL-3 inhibition.

[0220] The antibody GRT002-H16L2 has higher temperature stability than the antibody P8C11-chim.

[0221] Example 8.4 pH stability To test the behavior of the antibodies, GRT002-H16L2 and chimeric P8C11 were incubated with various antibody concentrations in buffers of various pH for 2 hours.

[0222] To do so, a 7.75 mg / ml solution of GRT002-H16L2 and a 4.55 mg / ml solution of chimeric P8C11 in PBS + 100 mM L-arginine were dissolved 1:5 in water, and the pH was adjusted to the desired value with HCl / NaOH. The samples were then incubated at room temperature for 2 hours, after which the pH was readjusted to 7.2 with HCl / NaOH.

[0223] The effect of exposure to different pH values ​​was investigated for both antibodies in order to analyze their functional activity by measuring the level of CD131 expression on basophils. CD131 expression was analyzed as described in Example 10.2.

[0224] Results are shown in Figure 9 for samples incubated at pH 2 for 2 hours at room temperature versus the reference (2 hours of exposure at pH 3, 4, or 9 did not result in loss of functional activity of either antibody). It can be seen that antibody GRT002-H16L2 fully retains its functional activity after 2 hours of exposure at pH 2, whereas the functional activity of antibody P8C11-chim is significantly reduced. At certain antibody concentrations, the functional activity of GRT002-H16L2 was two-fold, or even three-fold, or four-fold higher than that of antibody P8C11-chim. After exposure to pH 2, a more than 16-fold higher concentration of P8C11-chim was required than GRT002-H16L2 to achieve the same level of IL-3 inhibition.

[0225] Antibody GRT002-H16L2 has higher pH stability than antibody P8C11-chim.

[0226] Example 8.5 plasma stability To test the long-term stability of antibody GRT002-H16L2, the antibody was incubated at various concentrations in PBS and human plasma for 48 hours or 14 days.

[0227] Specifically, 23.7 μl of antibody (7.75 mg / ml in PBS containing 100 mM L-arginine) was mixed with 176.3 μl of PBS or human lithium heparin plasma (1 mg / ml) and incubated at 37° C. for 0 hours, 48 ​​hours, or 14 days. The functional activity of the antibody was then determined by measuring the level of CD131 expression on basophils. CD131 expression was analyzed as described in Example 10.2.

[0228] The results are shown in Figure 10. It can be demonstrated that antibody GRT002-H16L2 retains its full functional activity upon incubation in human plasma for at least 14 days.

Claims

1. A humanized antibody or antibody fragment specific to human IL3, said antibody or antibody fragment comprising an HCDR1 region of SEQ ID NO: 3, an HCDR2 region of SEQ ID NO: 4, an HCDR3 region of SEQ ID NO: 5, an LCDR1 region of SEQ ID NO: 10, an LCDR2 region of SEQ ID NO: 11, and an LCDR3 region of SEQ ID NO:

12.

2. 2. The isolated antibody or antibody fragment of claim 1, wherein the antibody or antibody fragment comprises a VH of SEQ ID NO: 36 and a VL of SEQ ID NO:

16.

3. 3. The isolated antibody or antibody fragment of claim 1 or 2, wherein the antibody is of the human IgG1 class.

4. The isolated antibody or antibody fragment of any one of claims 1 to 3, wherein the antibody binds to amino acids 41 to 67 of human IL3 (SEQ ID NO: 1).

5. The isolated antibody or antibody fragment of any one of claims 1 to 4, wherein the antibody or antibody fragment blocks IL3 activity in TF1 cells and human basophils.

6. 6. The isolated antibody or antibody fragment of any one of claims 1 to 5, wherein the antibody has increased stability at 75°C compared to an antibody or antibody fragment comprising a VH of SEQ ID NO:2 and a VL of SEQ ID NO:

9.

7. 7. The isolated antibody or antibody fragment of any one of claims 1 to 6, wherein the antibody has increased stability at pH 2 compared to an antibody or antibody fragment comprising a VH of SEQ ID NO: 2 and a VL of SEQ ID NO:

9.

8. An isolated antibody or antibody fragment according to any one of claims 1 to 7 for use in medicine.

9. 9. The isolated antibody or antibody fragment of claim 8, wherein said use in medicine is the treatment of an inflammatory, autoimmune or fibrotic disease or a malignant tumor.

10. A nucleic acid composition comprising one or more nucleic acid sequences encoding the isolated antibody or antibody fragment of any one of claims 1 to 9.

11. A vector comprising the nucleic acid composition of claim 10.

12. A host cell comprising the vector of claim 11 or the nucleic acid composition of claim 10.

13. A pharmaceutical composition comprising the isolated antibody or antibody fragment of claims 1-9 and a pharmaceutically acceptable carrier or excipient.

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