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 invention are capable of blocking IL-3 activity in target cells and are useful in the prevention and treatment of diseases or dysfunctions associated with elevated IL3 levels, such as inflammatory diseases, autoimmune diseases, fibrotic diseases, hematological malignancies and other diseases.

Description

Technical Field

[0001] The present disclosure relates to specific antibodies and antibody fragments of 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 suitable for preventing and treating diseases or malfunctions associated with elevated IL3 levels, such as inflammatory diseases, autoimmune diseases, fibrotic diseases, hematological malignancies and other diseases. Background Art

[0002] Interleukins belong to a large family of proteins called cytokines. Cytokines are polypeptides that influence certain cellular functions after binding to specific cell receptors and are divided into subclasses, namely interleukins, interferons, colony stimulating factors (CSFs), lymphokines, growth factors and monokines. It is well known that cytokines play an important role in cell proliferation and, for example, in inflammatory diseases.

[0003] Cell proliferation is a complex process in which growth factors bind to specific receptors on the cell surface, resulting in endocytosis, where the complex of cytokine and receptor is internalized, causing a cellular response. This cellular response includes specific gene transcription activities, such as DNA synthesis and cell replication. Most cytokines have several different biological effects when tested at relatively high concentrations. Due to these effects of cytokines, research on possible therapeutic uses of these proteins is of great interest.

[0004] Interleukins are mediators of the immune system that are produced primarily in low concentrations in leukocytes. They influence the growth, differentiation, and activity of cells of the immune system and are therefore immune modulators. They also act by binding to receptors on the surface of target cells, thereby altering the transcription rate of certain genes. They play an important role in triggering a variety of cellular responses.

[0005] For example, interleukins are involved in the immune cell activation cascade and the subsequent inflammatory changes. Irregular and / or abnormal inflammation is a major component and factor in many human diseases, one of which is the immune disease rheumatoid arthritis (RA). However, other immune 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 precursor cells and acts as a growth factor for mast cells and basophils. IL3, together with IL5 and GM-CSF, belongs to the family of hematopoietic cytokines with four short α-helical bundles. GM-CSF and IL3 stimulate the formation of neutrophil and eosinophil colonies and macrophages. It further stimulates the formation of mast cell, megakaryocyte and pure and mixed erythroid colonies (D. Metcalf, "The hematopoietic colony-stimulating factors", 1984, Elsevier, Amsterdam).

[0007] Mature IL3 consists of 133 amino acids (excluding the 19 amino acid signal sequence) and is known to stimulate colony formation of human hematopoietic progenitor cells and to stimulate DNA synthesis in human acute myeloid leukemia (AML) blasts. IL-3 binds to a unique receptor, also known as the CD123 antigen. This receptor belongs to the type I cytokine receptor family and is a heterodimer with a unique α-chain paired with a common β-subunit (βC or CDW 131). IL-3 binds to a unique α-receptor subunit. However, signal transduction is mediated by the common β-receptor subunit (βC) of the JAK2-STAT5 pathway.

[0008] Human IL-3 has two potential N-glycosylation sites at positions 15 and 70 of mature IL3. It is well known that IL3 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 activities measured by IL3-induced tumor cell proliferation. For mouse IL-3, it has also been demonstrated 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 indication in the literature that glycosylation affects the ability of monoclonal antibodies to block the biological activity of IL3. The inhibitory activity of the monoclonal antibodies was characterized using E. coli-derived IL3 (J Biol. Chem (1991) 266: 10624-31, J Immunol (1991) 146: 893-8) or glycosylated IL3 (J Biol Chem (1991) 266: 21310-7). Binding of the antibodies to IL3 was tested using glycosylated and non-glycosylated IL3 and was found to be independent of glycosylation. IL3 is primarily produced by activated CD4+ T cells and particularly contributes to the growth, differentiation and survival of CD34+ hematopoietic progenitor cells. In vitro, IL3 has been observed to promote the differentiation of basophils and mast cells from myeloid cells. It has further been observed to induce IL-6 release from mouse basophils and upregulate MHC-II expression and IL-1 secretion in monocytes / macrophages. In addition, IL-3 supports the differentiation of monocytes into dendritic cells and osteoclasts.

[0010] Since IL3 was first detected in the human genome, determining its role in healthy humans and its possible role in disease has been a focus of research. The ability of cytokines to initiate or regulate hematopoiesis is of great interest, particularly in relation to dysfunction or disease of the immune system. Such diseases appear to be associated with disturbances in the hematopoietic system and it is thought that such diseases could be treated by providing viable progenitor cells to the hematopoietic system. Triggering differentiation of such progenitor cells is considered a means of treating the corresponding disease.

[0011] Until a few years ago, little was known about the role of IL3 in autoimmune diseases, especially rheumatoid arthritis (RA). RA is the most common joint disease. The initial disease stage usually develops gradually, but it can also present as a sudden flare-up. Although the pain mainly occurs in the joints of the fingers or toes, other joints may also be affected. The affected joints show swelling and often a high fever. Most often, the disease occurs in several flare-ups, which usually last for weeks to months. Between flare-ups, symptoms usually improve.

[0012] The cause of RA is still unclear. An autoimmune cause is strongly suspected, and viral and bacterial causes are also under discussion. Several authors have reported genetic influences (Arthritis Rheum (2009) 60: 661-8, Arthritis Rheum (2004); 50 3085-92). It is believed that misguided immune cells invade the affected joints and lead to the production of proinflammatory cytokines. According to one theory, the balance between cytokines is disturbed in RA. IL1, IL6 and TNFα are reported to be present in excess in RA and are considered to be responsible for harmful inflammatory processes and osteoclast activation in cartilage tissue.

[0013] The treatment of rheumatoid arthritis is still considered difficult and burdensome for patients, as medications with a high risk of adverse side effects must be used. One approach to treating this disease is symptomatic treatment, mainly with nonsteroidal anti-inflammatory drugs (NSAIDs). These drugs act as anti-inflammatory and analgesic agents and usually only relieve pain. These drugs further interfere with a step in the inflammatory cascade, where prostaglandins are produced by cyclooxygenase. However, NSAIDs do not affect the underlying inflammatory process and therefore cannot slow down joint destruction, which is the most harmful effect of RA.

[0014] To prevent joint destruction and disease activity, another current approach to treating RA is the use of disease-modifying antirheumatic drugs (DMARDs). These drugs actually change the course of the disease. An example of a DMARD is methotrexate, the most commonly used antirheumatic drug, whose action is based on the reversible inhibition of the enzyme dihydrofolate reductase. Another commonly used substance for treating RA is leflunomide, which works by interfering with pyrimidine metabolism. Both drugs are long-acting and therefore must be taken for a longer period of time (usually 12-16 weeks) to show the desired effect. To shorten the time it takes for DMARDs to improve the disease, most patients are given steroids.

[0015] Another approach to treating RA is "biological products" that block cytokines such as TNF, IL6, IL1 or co-stimulatory molecules like B7 or deplete leukocyte subsets (e.g., B cells). Biological products (e.g., the TNF antibody infliximab) are mainly used in severe disease processes and after DMARDs have failed to adequately control disease activity. Biological products affect multiple signaling systems in the immune system and have various serious side effects, including bacterial and viral infections, and a higher risk of tumor development. All known treatments have serious disadvantages and side effects, so one goal is to develop new drugs for the treatment of RA that are more effective than other cytokines, more selectively expressed in patients with autoimmune diseases, especially RA, and have fewer side effects than currently used treatment options.

[0016] 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 stages of rheumatoid arthritis. Although the above patent application mentions that no IL-3 mRNA was detected in the synovium of RA patients, and no effect of IL3 on cultured fibroblasts was observed, genetic analysis found that there was an association between a single nucleotide polymorphism in the IL3 promoter gene and RA. Based on this discovery and further studies that confirmed that IL3 levels were significantly increased in RA patients, WO 2010 / 063488 proposed that the inhibitors are mainly antibodies or antibody fragments, antibody variants or antibody polymers in preventive RA treatment, early treatment of the disease or maintenance treatment. IL3 has similar effects on other diseases, such as systemic lupus erythematosus (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), lung inflammation (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 is still a need for effective antibodies specific for IL3 with high affinity and avidity. Since the in vivo efficacy of antibodies used to treat diseases or functional disorders in patients is crucial, there is also an urgent need for antibodies that are effective in the in vivo environment. Therefore, it is highly desirable to provide antibodies that can effectively and specifically inhibit IL3 activity in vivo, thereby making them useful drugs for treating diseases in patients diagnosed with elevated IL3 levels.

[0018] IL3 antibodies that meet some of these criteria are described in WO2017 / 081218, for example, antibody P8C11 (DSMACC3281). However, antibody P8C11 is a mouse antibody. To date, humanization of the P8C11 antibody has not been successful. The present invention successfully humanized P8C11. The humanization process described herein is very cumbersome and involves a six-step process to produce a humanized derivative of antibody P8C11, referred to as GRT002-H16L2. Unexpectedly, it was found that 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 is also stable at higher temperatures.

[0019] Therefore, GRT002-H16L2 and its fragments, variants or conjugates are very suitable for clinical development for the treatment of patients in need of blocking anti-IL3 antibodies. The antibody can also be used to detect human IL-3 expressed in human cells. Summary of the invention

[0020] 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. Unexpectedly, the inventors not only successfully humanized the antibody P8C11, but also the humanized antibody exhibits higher stability at higher temperatures and a wider pH range, particularly at acidic pH.

[0021] The present disclosure relates to human IL3-specific humanized antibodies and antibody fragments that block IL3 activity in TF1 cells and human basophils.

[0022] The present disclosure also relates to human IL3-specific humanized antibodies and antibody fragments that bind to amino acids 41-67 of human IL3 (SEQ ID NO: 1).

[0023] The present disclosure also relates to human IL3-specific humanized antibodies and antibody fragments that do not bind human IL-5 and human GM-CSF.

[0024] The preferred antibodies and antibody fragments of the present disclosure are of human IgG class. The more preferred antibodies or antibody fragments of the present disclosure are of IgG1 class.

[0025] Preferred antibodies and antibody fragments of the present disclosure are monoclonal antibodies or antibody fragments.

[0026] The present disclosure also relates to human IL3-specific antibodies and antibody fragments having excellent biophysical properties, including high temperature stability and wide pH range stability compared to the parent antibody P8C11.

[0027] Preferred humanized antibodies and antibody fragments of the present disclosure include the HCDR1 region of SEQ ID NO: 3, the HCDR2 region of SEQ ID NO: 4, the HCDR3 region of SEQ ID NO: 5, the LCDR1 region of SEQ ID NO: 10, the LCDR2 region of SEQ ID NO: 11, and the LCDR3 region of SEQ ID NO: 12.

[0028] Preferred antibodies and antibody fragments of the present disclosure include the VH of SEQ ID NO: 36 and the VL of SEQ ID NO: 16.

[0029] The present disclosure also relates to vectors comprising such nucleic acids or nucleic acid compositions.

[0030] The present disclosure also relates to host cells comprising such nucleic acids, nucleic acid compositions or vectors.

[0031] The antibodies and antibody fragments of the present disclosure are suitable 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.

[0032] The present disclosure also relates to compositions comprising the antibodies and antibody fragments of the present disclosure and a pharmaceutically acceptable drug carrier. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Analysis of the binding properties of antibody GRT002-H16L2 compared to the parental antibody P8C11 is shown.

[0034] Figure 2 The epitope mapping results of antibody GRT002-H16L2 are shown.

[0035] Figure 3 This showed that antibodies P8C11 and GRT002-H16L2 blocked IL-3 activity in TF1 cells to the same extent.

[0036] Figure 4 This showed that antibodies P8C11 and GRT002-H16L2 blocked IL-3 activity in human basophils to the same extent.

[0037] Figure 5 Shown are the results of melting temperature determinations of antibody P8C11 by differential scanning calorimetry.

[0038] Figure 6 Shown are the results of measuring the melting temperature of antibody GRT002-H16L2 by differential scanning calorimetry.

[0039] Figure 7 It was shown that repeated freezing and thawing had no effect on the functional activity of GRT002-H16L2, as measured by the expression level of CD131 in basophils. FTC = freeze-thaw cycle.

[0040] Figure 8 The effect of GRT002-H16L2 and chimeric P8C11 exposed to higher temperatures is shown, as measured by the expression level of CD131 in basophils. Antibody GRT002-H16L2 has a higher temperature stability than chimeric antibody P8C11.

[0041] Fig. 9 The effect of culturing GRT002-H16L2 and chimeric P8C11 at pH 2 is shown, as measured by the expression level of CD131 in basophils. Antibody GRT002-H16L2 has a higher pH stability than antibody P8C11.

[0042] Fig.10 showed that incubation of antibody GRT002-H16L2 in human plasma for at least 14 days had no effect on functional activity, as measured by the expression level of CD131 in basophils.

[0043] definition

[0044] The present disclosure relates to antibodies that specifically bind to IL3.

[0045] The term "IL3" or "IL-3" refers to a member of the interleukin protein family having the following amino acid sequence (Uni-Prot: P08700):

[0046]

[0047] The signal sequence of IL3 is underlined in the above sequence. Potential N-glycosylation sites are shown in bold and underlined.

[0048] The term "antibody" as used herein refers to a protein comprising at least two heavy chains (H) and two light chains (L), which are interconnected by disulfide bonds and interact with antigens. Each heavy chain consists of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region consists of three domains (CH1, CH2, and CH3). Each light chain consists of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region consists of one domain CL. The VH and VL regions can be further subdivided into highly variable regions, called complementary determining regions (CDRs), alternating with more conservative regions, called framework regions (FRs). Each VH and VL consists of three CDRs and four FRs arranged in the following order from the amino terminus to the carboxyl terminus: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with the antigen. 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, camelid antibodies, and chimeric antibodies. The antibody 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 light and heavy chains are divided into regions of structural and functional homology.

[0049] As used herein, the term "antibody fragment" refers to one or more portions of an antibody that retain the ability to specifically interact with an antigen (e.g., by binding, steric hindrance, stabilizing spatial distribution). Examples of binding fragments include, but are not limited to, Fab fragments, monovalent fragments consisting of VL, VH, CL and CH1 domains; F(ab)2 fragments, bivalent fragments comprising two Fab fragments linked by a disulfide bond at the hinge region; Fd fragments consisting of VH and CH1 domains; Fv fragments consisting of the VL and VH domains of a single arm of an antibody; dAb fragments (Ward et al., (1989) Nature 341: 544-546), consisting of the VH domain; and isolated complementary determining regions (CDRs). In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked using recombinant methods by synthetic linkers that allow them to be made into single-chain protein chains that pair the VL and VH regions to form monovalent molecules (called 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 included in the term "antibody fragment". These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for function in the same manner as intact antibodies. Antibody fragments can also be introduced into single domain antibodies, large antibodies, minibodies, internal antibodies, double antibodies, three antibodies, four antibodies, v-NARs and double-scFv (see, e.g., Hollinger and Hudson, (2005) Nature Biotechnology 23: 1126-1136). 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 monomers). Antibody fragments can be introduced into single-chain molecules comprising a pair of tandem Fv fragments (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).

[0050] The structure and position 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 (see, e.g., Sequences of Proteins of Immunological Interest, US 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 US Department of Health and Human Services; Chothia et al., (1987) J. Mol. Biol. 196:901-917; Chothia et 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)).

[0051] As used herein, "human antibodies" or "human antibody fragments" are antibodies and antibody fragments having variable regions, wherein both the framework region and the CDR region are derived from sequences of human origin. Human antibodies can also be isolated from synthetic libraries or transgenic mice (e.g., xenogeneic mice), provided that the corresponding system is produced in antibodies having variable regions, wherein both the framework region and the CDR region are derived from sequences of human origin. In addition, if the antibody comprises a constant region, the constant region is also derived from such sequences. Human origin includes, for example, human germline sequences, or human germline sequences or mutant versions of antibodies containing consensus framework sequences derived from human framework sequence analysis, for example, as described in Knappik et al., (2000) J Mol Biol 296:57-86).

[0052] "Humanized antibodies" or "humanized antibody fragments" are defined herein as antibody molecules having constant and variable antibody regions or portions thereof derived from human sequences, or only CDRs derived from another species. For example, a humanized antibody can be CDR-grafted, wherein the CDRs of the variable domains are non-human, while one or more frameworks of the variable domains are human, and the constant domains (if present) are human.

[0053] 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 of one species and variable antibody regions derived from another species. Preferably, the constant antibody regions are derived from or correspond to sequences of humans, while the variable antibody regions (e.g., VH, VL, CDR or FR regions) are derived from sequences of non-human animals (e.g., mouse, rat, rabbit or hamster).

[0054] The term "isolated antibody" refers to an antibody or antibody fragment that is substantially free of other antibodies or antibody fragments having different antigenic specificities. In addition, the isolated antibody or antibody fragment may be substantially free of other cellular material and / or chemicals. Thus, in certain aspects, the antibodies provided are isolated antibodies that are separated from antibodies having different specificities. The isolated antibody may be a monoclonal antibody. The isolated antibody may be a recombinant monoclonal antibody. However, an isolated antibody that specifically binds to an epitope, isotype or variant of a target may have cross-reactivity to other related antigens from other species (e.g., species derivatives).

[0055] The term "recombinant antibody" as used herein includes all antibodies prepared, expressed, produced or isolated by a method not present in nature. For example, antibodies isolated from host cells transformed to express the antibody, antibodies selected and isolated from a recombinant combinatorial human antibody library, and antibodies prepared, expressed, produced or isolated by other methods involving splicing all or part of human immunoglobulin genes, sequences to other DNA sequences or antibodies, or from transgenic or transchromosomal animals (e.g., mice) for human immunoglobulin genes or hybridomas prepared therefrom. Preferably, such recombinant antibodies have variable regions, wherein the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when using transgenic animals of human Ig sequences, in vivo somatic mutagenesis), and thus the amino acid sequences of the VH and VL regions of the recombinant antibodies are such sequences, although derived from and related to human germline VH and VL sequences, but may not be naturally present in the human antibody germline library in vivo. The recombinant antibody may be a monoclonal antibody. In one embodiment, the antibodies and antibody fragments disclosed herein are isolated from the HuCAL library (Rothe et al, J. Mol. Biol. (2008) 376, 1182-1200).

[0056] As used herein, an antibody "specifically binds to," "specifically binds to," "specifically recognizes," or "specifically recognizes" an antigen, such as human IL3, if such antibody is able to distinguish such antigen from one or more reference antigens, since binding specificity is not an absolute, but a relative property. For example, a standard ELISA assay or a standard flow cytometry assay can be performed. Scoring can be performed by standard chromogenic methods (e.g., secondary antibodies with horseradish peroxide and tetramethylbenzidine with hydrogen peroxide) or by binding of secondary antibodies labeled with PE or another dye or marker. Reactions in certain wells are scored by optical density (OD), e.g., at 450 nm, or by mean fluorescence intensity (MFI) in flow cytometry. A typical background (= negative reaction) may be 0.1 OD; a typical positive reaction may be 1 OD. Background and positive reaction MFI are highly dependent on instrument settings. Positive / negative differences may exceed 10-fold. Generally speaking, the determination of binding specificity is not by using a single reference antigen, but by using a group of about three to five unrelated antigens, such as milk powder, BSA, transferrin, etc. For flow cytometry, various antigen-negative cells can be used. However, antibodies that specifically bind to antigens may have cross-reactivity to corresponding orthologous antigens from other species (e.g., species derivatives). In some embodiments, this cross-reactivity to orthologous antigens is more preferred.

[0057] As used herein, an antibody is "cross-reactive" or "cross-reactive" if it binds to orthologous antigens from other species. For example, if an antibody binds to human IL3 and marmoset IL3, it is cross-reactive.

[0058] As used herein, the term "affinity" refers to the strength of interaction between a polypeptide and its target at a single site. Within each site, the binding region of a polypeptide interacts with its target at many sites through weak non-covalent forces; the more interactions, the stronger the affinity.

[0059] The term "epitope" includes any protein region that is specifically recognized by an antibody or antibody fragment thereof or that otherwise interacts with a molecule. Typically, an epitope is a chemically active surface grouping of a molecule, such as amino acids or carbohydrate or sugar side chains, and typically may have specific three-dimensional structural characteristics as well as specific charge characteristics. Those skilled in the art will appreciate that virtually anything to which an antibody can specifically bind may be an epitope.

[0060] The term "domain" or "protein domain" refers to a region of a protein polypeptide chain that forms a functional unit and / or independently forms a three-dimensional structure.

[0061] The "compositions" of the present disclosure can be used for therapeutic or preventive applications. Therefore, the present disclosure includes pharmaceutical compositions containing antibodies or antibody fragments disclosed herein and pharmaceutical carriers or excipients. In a related aspect, the present disclosure provides a method for treating inflammatory diseases, autoimmune diseases, fibrotic diseases, hematological malignancies, and potentially other diseases. The method includes the step of administering an effective amount of a pharmaceutical composition containing an antibody or antibody fragment described herein to a subject in need thereof.

[0062] The present disclosure provides methods of treatment, comprising administering to a subject in need of such treatment a therapeutically effective amount of an antibody or antibody fragment disclosed herein. As used herein, "therapeutically effective amount" or "effective amount" refers to the amount of IL3 antibody required to elicit a desired biological response. According to the present disclosure, a therapeutically effective amount is the amount of IL3 antibody required to treat and / or prevent a disease.

[0063] "Administered" or "administration" includes, but is not limited to, delivering the drug by an injectable form, e.g., intravenous, intramuscular, intradermal or subcutaneous route, or by a mucosal route, e.g., as a nasal spray or inhalation aerosol, or as an ingestible solution, capsule or tablet. Preferably, the administration is by an injectable form.

[0064] As used herein, "treatment", "treat" or "treating" and the like refer to clinical interventions that attempt to alter the natural course of a disease in a treated subject, and can be used for prevention or implemented during the clinical pathological process. Desirable effects of treatment include, but are not limited to, preventing the occurrence or recurrence of the disease, alleviating symptoms, reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. In some embodiments, antibodies or antibody fragments according to the present disclosure are used to delay the development of a disease or slow the progression of a disease.

[0065] "Preventing" or "prevention" refers to reducing the risk of acquiring or developing a disease (i.e., so that a subject who may be exposed to a disease-causing agent or who is susceptible to the disease before the onset of the disease does not develop at least one clinical symptom of the disease). "Prevention" also refers to methods intended to prevent the onset of a disease or its symptoms or to delay the onset of a disease or its symptoms.

[0066] "Subject" or "species" or as used in this context refers to any mammal, including rodents, such as mice or rats, and primates, such as cynomolgus monkeys (Macaca fascicularis), Marmoset monkeys (Callithrix jacchus), rhesus monkeys (Macaca mulata), or humans (Homo sapiens). Preferably, the subject is a primate, most preferably a human.

[0067] The term "effector function" refers to biological activities attributable to the Fc region of an antibody, which vary with 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.

[0068] "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) enable these cytotoxic effector cells to specifically bind to target cells bearing antigens and subsequently kill the target cells with cytotoxins. Primary cells that mediate ADCC NK cells express only FcyRIII, while monocytes / macrophages express FcyRI, FcyRII, and FcyRIIII.

[0069] "Complement dependent cytotoxicity" or "CDC" refers to the lysis of target cells in the presence of complement. Activation of the classical complement pathway is initiated by binding of the first component of the complement system (CIq) to an antibody (of the appropriate subclass) of the present disclosure that binds to its cognate antigen.

[0070] "Antibody-dependent cellular phagocytosis" or "ADCP" refers to a mechanism by which antibody-coated target cells are eliminated by internalization by phagocytic cells such as macrophages or dendritic cells.

[0071] Throughout the specification, unless the context requires otherwise, "comprises," "has," and "includes" and their respective variations such as "comprising," "containing," "having," "having," "including," and "comprising," will be understood to imply the inclusion of stated elements or integers or groups of elements or integers but not the exclusion of any other elements or integers or groups of elements or integers.

[0072] As used herein, the term "engineering" or "modification" includes 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, the 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.

[0073] As used herein, "variant" refers to a polypeptide that differs from a reference polypeptide by one or more modifications (e.g., amino acid substitutions, insertions, or deletions). The variant polypeptide generally retains most of the properties of the reference polypeptide, e.g., binding to a target antigen, but introduces new additional features or properties, e.g., the variant polypeptide has a higher affinity for the target antigen than the reference polypeptide, or the variant polypeptide is a humanized version of the reference polypeptide.

[0074] The term "amino acid mutation" as used herein refers to amino acid replacement, deletion, insertion and modification. Any combination of replacement, deletion, insertion and modification can be made, as long as the final construct has the desired characteristics, for example, reduced binding to Fc receptors. Amino acid sequence deletions and insertions include N- and / or C-terminal deletions and insertions of amino acid residues. Specific amino acid mutations are amino acid replacements. Amino acid replacements include replacement with non-natural amino acids or natural amino acid derivatives of twenty standard amino acids. Amino acid mutations can be produced using genetic or chemical methods well known in the art. Genetic methods can include site-directed mutagenesis, PCR, gene synthesis, etc. It is envisioned that methods for changing amino acid residue side chain groups by methods other than genetic engineering, such as chemical modification, can also be useful. Various names can be used herein to represent the same amino acid mutation. For example, the replacement of glycine at position 327 in the Fc region to alanine can be expressed as 327, G327, G327A or Gly327Ala.

[0075] As used herein, the term "EC50" refers to the concentration of an antibody or antibody fragment that induces a half-value response between baseline and maximum in an assay. Thus, it represents the concentration of antibody or ligand at which 50% of the maximum effect is observed.

[0076] The terms "inhibition" or "inhibit", "reduction" or "reduce", "neutralization" or "neutralize" refer to the reduction or cessation of any phenotypic characteristic (such as binding or biological activity or function), or the reduction or cessation of the incidence, degree or likelihood of the characteristic. "Inhibition", "reduction" or "neutralization" need not be complete as long as it can be detected using an appropriate detection method. In some embodiments, "reduction" or "inhibition" or "neutralization" refers to the ability to cause a reduction of 20% or more. In another embodiment, "reduction" or "inhibition" or "neutralization" refers to the ability to cause a reduction of 50% or more. In yet another embodiment, "reduction" or "inhibition" or "neutralization" refers to the ability to cause an overall reduction of 75%, 85%, 90%, 95% or more.

[0077] 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 the biological activity or function or any other phenotypic characteristic of the target antigen.

[0078] A "wild-type" protein is a version or variant of a protein found in nature. The amino acid sequence of a wild-type protein, e.g., the Fc region of a human IgG1 antibody, is the amino acid sequence that occurs in nature for that protein. Due to allotypic differences, there may be more than one amino acid sequence for a wild-type protein. For example, there are several allotypes of the naturally occurring human IGg1 heavy chain constant region (see, e.g., Jeffries et al. (2009) mAbs 1:1).

[0079] "Fc region" is used to define the C-terminal region of an immunoglobulin heavy chain. The Fc region of an immunoglobulin generally includes two constant domains, a CH2 domain and a CH3 domain. Although the boundaries of the IgG heavy chain Fc region may vary slightly, the Fc region of a human IgG heavy chain is generally defined as extending from Cys226 or from 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 indicated herein, the numbering of amino acid residues in the Fc region is in accordance with the EU numbering system, also referred to as the EU index, as described in Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD, 1991. DETAILED DESCRIPTION

[0080] 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 erythroblast cell line created by T. Kitamura in 1987 from the bone marrow of a 35-year-old Japanese male with severe pancytopenia. The growth of TF1 cells is completely dependent on the presence of IL-3 or GM-CSF. Therefore, a test based on TF1 cell proliferation can be used to determine whether a blockade of IL-3 activity, which in turn results in a reduction or even complete inhibition of TF1 cell growth, occurs.

[0081] In order to use the antibodies in human therapy, the ability to block IL-3 activity should also be assayed in a model that is not as far away from the in vivo environment as the TF1 cell line model. Therefore, the inventors tested the anti-IL3 antibodies of the present disclosure using primary human cells obtained from patients or healthy subjects to be treated with the anti-IL3 antibodies as controls. The use of primary cells and IL3 produced by primary human cells is superior to cell line models.

[0082] The glycosylation status depends on the cell type in which human IL-3 is produced. If human IL-3 is recombinantly produced by E. coli cells, the human IL-3 produced will not be glycosylated. If human IL-3 is produced by insect cells, the human IL-3 produced will be weakly glycosylated. If IL-3 is produced by human cells such as HEK cells, the human IL-3 produced will be more strongly glycosylated.

[0083] The primary cells used to determine the efficiency of the anti-IL3 antibodies of the present disclosure are preferably primary human blood cells obtained from patients with rheumatoid arthritis (RA). Primary human blood cells can also be obtained from healthy subjects and can be used as controls. For testing, the blood sample can be treated with heparin, citrate or EDTA as anticoagulants 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".

[0084] Primary human blood cells contain basophils, 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 cell markers expressed by basophils (such as CD11b, CD123, CCR3 and CD203c). The ability to block the activity of human IL3 can be tested, for example, by analyzing the IL3-induced upregulation of CD203c in basophils (Int J ImmunopatholPharmacol (2007) 20: 267-78). IL-3 also induces upregulation of CD11b and downregulation of CD131 in basophils. In addition, human IL3 induces upregulation of HLA-DR and downregulation of CD131 in plasmacytoid dendritic cells (pDCs), which are also contained in blood samples containing primary human blood cells. pDCs can be detected as known in the art, such as by flow cytometry using a combination of fluorescently labeled antibodies against cell markers expressed by dendritic cells, such as HLA-DR, CD123, and CD4. In addition, IL3-induced CD131 downregulation can be quantified for CD14+CD16+ monocytes, CD14++ monocytes, and eosinophils. Thus, the ability of anti-IL-3 antibodies to effectively 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 IL3 bioactivity in human samples because basophils respond strongly to IL-3 and have little reactivity to closely related cytokines such as IL-5 and GM-CSF.

[0085] Peptides

[0086] 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 a HCDR1 region of SEQ ID NO: 3, a HCDR2 region of SEQ ID NO: 4, a HCDR3 region of SEQ ID NO: 5, a LCDR1 region of SEQ ID NO: 10, a LCDR2 region of SEQ ID NO: 11, and a LCDR3 region of SEQ ID NO: 12.

[0087] 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 a HCDR1 region of SEQ ID NO: 3, a HCDR2 region of SEQ ID NO: 4, a HCDR3 region of SEQ ID NO: 5, a LCDR1 region of SEQ ID NO: 10, a LCDR2 region of SEQ ID NO: 11, and a LCDR3 region of SEQ ID NO: 12, as defined by Kabat.

[0088] 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 a HCDR1 region of SEQ ID NO: 6, a HCDR2 region of SEQ ID NO: 7, a HCDR3 region of SEQ ID NO: 8, a LCDR1 region of SEQ ID NO: 13, a LCDR2 region of SEQ ID NO: 14, and a LCDR3 region of SEQ ID NO: 12, as defined by IMGT.

[0089] 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 the VH of SEQ ID NO: 36 and the VL of SEQ ID NO: 16.

[0090] In one embodiment of the present disclosure, the humanized antibody or antibody fragment is a monoclonal antibody or antibody fragment.

[0091] In one embodiment of the present disclosure, the humanized antibody or antibody fragment is a recombinant antibody or antibody fragment.

[0092] In one embodiment of the present disclosure, the humanized antibody or antibody fragment is of IgG isotype.

[0093] In one embodiment of the present disclosure, the humanized antibody or antibody fragment is of the IgG1 class.

[0094] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for human IL3, wherein the antibody comprises a heavy chain of SEQ ID NO: 44 and a light chain of SEQ ID NO: 45.

[0095] In one embodiment, the present disclosure relates to a humanized antibody or antibody fragment specific for human IL3, wherein the antibody has a heavy chain of SEQ ID NO: 44 and a light chain of SEQ ID NO: 45.

[0096] In one embodiment of the present disclosure, the humanized antibody or antibody fragment is specific to the polypeptide encoded by the amino acid sequence of SEQ ID NO: 1.

[0097] In one embodiment of the present disclosure, the humanized antibody or antibody fragment is specific to a polypeptide comprising the amino acid sequence of SEQ ID NO: 1.

[0098] In another embodiment of the present 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 present disclosure, the humanized antibody or antibody fragment binds to amino acids 22-48 of mature human IL3. In another embodiment of the present disclosure, the humanized antibody or antibody fragment binds to a peptide consisting of amino acids of SEQ ID NO: 37 of human IL3. In another embodiment of the present disclosure, the humanized antibody or antibody fragment binds to a peptide comprising amino acids of SEQ ID NO: 37. In another embodiment of the present disclosure, the humanized antibody or antibody fragment binds to a peptide consisting of amino acids of SEQ ID NO: 37. In another embodiment of the present disclosure, the humanized antibody or antibody fragment binds to a peptide comprising amino acids of SEQ ID NO: 38. In another embodiment of the present disclosure, the humanized antibody or antibody fragment binds to a peptide consisting of amino acids of SEQ ID NO: 38. In another embodiment of the present disclosure, the humanized antibody or antibody fragment binds to a peptide comprising amino acids of SEQ ID NO: 39. In another embodiment of the present 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 VH of SEQ ID NO: 36 and VL of SEQ ID NO: 16.

[0099] In another embodiment of the present disclosure, the humanized antibody or antibody fragment specific for human IL3 does not have cross-reactivity with IL3 from other species. In certain embodiments of the present disclosure, the humanized antibody or antibody fragment specific for human IL3 does not have cross-reactivity with mouse IL3 or rat IL3. In certain embodiments of the present disclosure, the humanized antibody or antibody fragment specific for human IL3 does not have cross-reactivity with rat IL3. In certain embodiments of the present disclosure, the humanized antibody or antibody fragment specific for human IL3 does not have cross-reactivity with mouse IL3. In a preferred embodiment, the antibody or antibody fragment comprises the VH of SEQ ID NO: 36 and the VL of SEQ ID NO: 16.

[0100] In order to determine the usefulness of the monoclonal antibodies obtained in diagnostic analysis, it is important to exclude cross-reactivity with closely related cytokines that are also present in the patient's blood, plasma, serum or other body fluids. IL5 (UniProt: P05113) and GM-CSF (UniProt: P04141) are two closely related cytokines. Therefore, in another embodiment of the present disclosure, the humanized antibody or antibody fragment specific for human IL3 has no cross-reactivity with human IL5 or human GM-CSF. In certain embodiments of the present disclosure, the humanized antibody or antibody fragment specific for human IL3 has no cross-reactivity with human IL5. In certain embodiments of the present disclosure, the humanized antibody or antibody fragment specific for human IL3 has no cross-reactivity with human GM-CSF. In a preferred embodiment, the antibody or antibody fragment comprises the VH of SEQ ID NO: 36 and the VL of SEQ ID NO: 16.

[0101] In another embodiment of the present disclosure, the humanized antibody or antibody fragment specific for human IL3 blocks IL3 activity in TF1 cells and human basophils. In certain embodiments of the present disclosure, the humanized antibody or antibody fragment specific for human IL3 blocks IL3 activity in TF1 cells. In certain embodiments of the present disclosure, the humanized antibody or antibody fragment specific for human IL3 blocks IL3 activity in human basophils. In certain embodiments of the present disclosure, the 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 certain embodiments of the present disclosure, the 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.

[0102] In another embodiment of the present disclosure, the humanized antibody or antibody fragment specific for human IL3 has improved temperature stability. In certain embodiments of the present disclosure, the humanized antibody or antibody fragment specific for human IL3 has improved temperature stability at 75°C. In certain embodiments of the present disclosure, the humanized antibody or antibody fragment specific for human IL3 has improved temperature stability at 75°C compared to an antibody or antibody fragment comprising VH of SEQ ID NO: 2 and VL of SEQ ID NO: 9. In a preferred embodiment, the antibody or antibody fragment comprises VH of SEQ ID NO: 36 and VL of SEQ ID NO: 16.

[0103] In another embodiment of the present disclosure, the humanized antibody or antibody fragment specific for human IL3 has improved pH stability. In certain embodiments of the present disclosure, the humanized antibody or antibody fragment specific for human IL3 has improved stability at pH 2. In certain embodiments of the present disclosure, the humanized antibody or antibody fragment specific for human IL3 has improved stability at pH 2 compared to an antibody or antibody fragment comprising VH of SEQ ID NO: 2 and VL of SEQ ID NO: 9. In a preferred embodiment, the antibody or antibody fragment comprises VH of SEQ ID NO: 36 and VL of SEQ ID NO: 16.

[0104] Nucleic Acids

[0105] 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 the HCDR1 region of SEQ ID NO: 3, the HCDR2 region of SEQ ID NO: 4, the HCDR3 region of SEQ ID NO: 5, the LCDR1 region of SEQ ID NO: 10, the LCDR2 region of SEQ ID NO: 11, and the LCDR3 region of SEQ ID NO: 12.

[0106] 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 sequence comprises the HCDR1 region of SEQ ID NO: 3, the HCDR2 region of SEQ ID NO: 4, the HCDR3 region of SEQ ID NO: 5, the LCDR1 region of SEQ ID NO: 10, the LCDR2 region of SEQ ID NO: 11, and the LCDR3 region of SEQ ID NO: 12.

[0107] 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 the HCDR1 region of SEQ ID NO: 3, the HCDR2 region of SEQ ID NO: 4, the HCDR3 region of SEQ ID NO: 5, the LCDR1 region of SEQ ID NO: 10, the LCDR2 region of SEQ ID NO: 11 and the LCDR3 region of SEQ ID NO: 12, as defined by Kabat.

[0108] 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 sequence contains the HCDR1 region of SEQ ID NO: 3, the HCDR2 region of SEQ ID NO: 4, the HCDR3 region of SEQ ID NO: 5, the LCDR1 region of SEQ ID NO: 10, the LCDR2 region of SEQ ID NO: 11 and the LCDR3 region of SEQ ID NO: 12, as defined by Kabat.

[0109] 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 VH of SEQ ID NO: 36 and VL of SEQ ID NO: 16.

[0110] 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 VH of SEQ ID NO: 36 and VL of SEQ ID NO: 16.

[0111] In one embodiment, the nucleic acid composition and / or the nucleic acid sequence and / or multiple nucleic acid sequences are isolated.

[0112] Carrier

[0113] In one embodiment, the present disclosure provides a vector composition comprising one or more vectors comprising a nucleic acid composition comprising a nucleic acid sequence or multiple nucleic acid sequences encoding a human IL3-specific humanized antibody or antibody fragment according to the present disclosure.

[0114] In one embodiment, the present disclosure provides a vector composition comprising one or more vectors comprising a nucleic acid composition comprising a nucleic acid sequence or multiple nucleic acid sequences encoding any one of the human IL3-specific humanized antibodies or antibody fragments disclosed herein.

[0115] In one embodiment, the carrier composition and / or carrier and / or carriers are isolated.

[0116] Host cells

[0117] In one embodiment, the present disclosure provides a host cell comprising a vector composition comprising one or more vectors comprising a nucleic acid composition comprising a nucleic acid sequence or multiple nucleic acid sequences encoding a human IL3-specific humanized antibody or antibody fragment according to the present disclosure.

[0118] In one embodiment, the present disclosure relates to a host cell comprising a vector composition comprising one or more vectors comprising a nucleic acid composition comprising a nucleic acid sequence or multiple nucleic acid sequences encoding an IL3-specific humanized antibody or antibody fragment, wherein the antibody or antibody fragment comprises the VH of SEQ ID NO: 36 and the VL of SEQ ID NO: 16.

[0119] In one embodiment, the host cell according to the present disclosure can express the human IL3-specific humanized antibody or antibody fragment encoded by the vector composition or the nucleic acid composition.

[0120] 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 a HKB11 cell.

[0121] It will be appreciated by those skilled in the art that the nucleic acid sequence or multiple nucleic acid sequences encoding the heavy chain and / or light chain of the antibodies or antibody fragments of the present disclosure can be cloned into different vectors or into the same vector.

[0122] The vector can be introduced into a suitable host cell such as a prokaryotic (e.g., bacterial) or eukaryotic (e.g., yeast or mammalian) cell by methods well known in the art (see, for example, "Current Protocol in Molecular Biology", Ausubel et al. (eds.), Greene Publishing Assoc and John Wiley Interscience, New York, 1989 and 1992). Many 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 an optional 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 by a vector containing such an expression structure. The coding sequence may or may not contain a signal peptide or a leader sequence. After expression in the host cell, the antibody or antibody fragment of the present disclosure is obtained. As known to those skilled in the art, these steps can be achieved in different ways. In general, these steps generally include transforming or transfecting a suitable host cell with a nucleic acid composition or a vector composition or an infectious particle encoding an antibody or antibody fragment. Moreover, these steps generally include culturing the host cell under conditions suitable for the proliferation (reproduction, growth) of the host cell, and a culturing step under conditions suitable for producing (expressing, synthesizing) the encoded antibody or antibody fragment. Cultivating host cells under conditions suitable for proliferation or expression is generally completed in the presence of a culture medium containing components suitable for cell growth or induced expression. In a specific embodiment, the method for producing the antibody or antibody fragment of the present invention also includes the step of separating and purifying the produced antibody or antibody fragment from the host cell or culture medium. If the expression system secretes the protein into the growth medium, the protein can be directly purified from the culture medium. If the protein is not secreted, it is separated from the cell lysate or recovered from the cell membrane portion. Selecting suitable growth conditions and recovery methods are all within the technical scope of the art. Then, the antibody or antibody fragment of the present invention can be purified by a variety of techniques known to those skilled in the art.

[0123] In one embodiment, the present disclosure relates to a method for producing a humanized antibody or antibody fragment specific for human IL3, wherein the antibody or antibody fragment comprises the VH of SEQ ID NO: 36 and the 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, wherein the method comprises: culturing a host cell under conditions suitable for expressing the antibody or antibody fragment, the host cell comprising a vector composition, the vector composition comprising one or more vectors, the vector comprising a nucleic acid composition, the nucleic acid composition comprising a nucleic acid sequence or multiple nucleic acid sequences encoding the antibody or antibody fragment according to the present disclosure, and isolating the antibody or antibody fragment from the host cell or host cell culture medium. The antibody or antibody fragment isolated as described herein can be purified using purification 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 for purifying a specific antibody or antibody fragment will depend in part on factors such as net charge, hydrophobicity, hydrophilicity, and will be apparent to those skilled in the art. For affinity chromatography purification, an 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.

[0124] Effector function

[0125] The Fc region of an immunoglobulin generally confers favorable pharmacokinetic properties to the antibody, such as an extended half-life in serum, and confers the ability to induce effector functions by binding to Fc receptors expressed on cells. On the other hand, binding to Fc receptors may also lead to the unwanted activation of certain cell surface receptors, resulting in undesirable cytokine release and severe side effects after systemic administration.

[0126] Therefore, for certain therapeutic environments, it is desirable to reduce or eliminate the normal binding of the wild-type Fc region of an antibody (such as a wild-type IgG Fc region) to one or more or all Fc receptors and / or to complement components (such as C1q) to reduce or eliminate the ability of the antibody to induce effector functions. For example, it may be desirable to reduce or eliminate the binding of the antibody Fc region to one or more or all Fcy receptors such as FcyRI, FcyRIla, FcyRI Ib, FcyR IIa. 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, antigen uptake mediated by immune complexes 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.

[0127] Reduction or elimination of binding of the Fc region to Fc receptors and / or C1q is usually achieved by mutating a wild-type Fc region, such as an IgG1 Fc region, more specifically a human IgG1-Fc region, thereby generating a variant of the wild-type Fc region or an engineered Fc region, such as a variant human IgG1 Fc region. Substitutions that result in reduced binding may be applicable. In order to reduce or eliminate the binding properties of the Fc region to the Fc receptor, non-conservative amino acid substitutions are preferred, i.e., substitutions of one amino acid with another amino acid having different structural and / or chemical properties.

[0128] 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 Fc receptors and / or C1q when compared to a wild-type Fc region. In one such embodiment, an 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, an isolated antibody or antibody fragment according to the present disclosure does not substantially induce effector function.

[0129] 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, the separated antibodies or antibody fragments according to the present disclosure do not substantially induce ADCC and / or CDC and / or ADCP. In one embodiment, the separated antibodies or antibody fragments according to the present disclosure do not induce ADCC or ADCP in vitro.

[0130] In one embodiment, the variant Fc region of the humanized antibody or antibody fragment according to the present disclosure comprises one or more amino acid substitutions, which reduce or eliminate the 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 the humanized antibody or antibody fragment according to the present disclosure comprises one or more amino acid substitutions, which reduce or eliminate the ability of the antibody to induce effector functions when compared to the wild-type Fc region. In a specific embodiment, when compared to the wild-type Fc region, one or more amino acid substitutions can reduce the binding affinity of the variant Fc region to one or more Fc receptors and / or to C1q by at least 2 times, at least 5 times, at least 10 times, at least 20 times or even at least 50 times. In alternative embodiments, when compared to the wild-type Fc region, one or more amino acid substitutions can reduce the ability of the isolated antibody or antibody fragment according to the present disclosure to induce effector functions by at least 2 times, at least 5 times, at least 10 times, at least 20 times or even at least 50 times.

[0131] In one embodiment, the variant Fc region of the 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 the antibody according to the present disclosure substantially eliminates the ability of the antibody to induce effector function. In one embodiment, the antibody or antibody fragment according to the present disclosure does not substantially induce effector function. In one embodiment, the effector function is ADCC and / or ADCP and / or CDC. In one embodiment, the antibody or antibody fragment according to the present disclosure does not substantially induce effector function means that the level of induced effector function is not significantly higher than the background measured in the absence of the antibody.

[0132] 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 FcγRIIIa, FcγRI, FcγRIla and / or FcγRIlb.

[0133] 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 an Fc receptor and / or C1q and / or reduce the ability of the antibody to induce effector function when compared to a wild-type Fc region.

[0134] According to the humanized antibody or antibody fragment of the present disclosure, it may or may not be fused with one or more other amino acid residues, polypeptides or parts. Such fusion proteins can be prepared in any suitable manner, including genetic or chemical methods. The connected part may include: a secretory sequence or a leader sequence, a sequence that assists in detection, expression, separation or purification, or a sequence that improves protein stability during, for example, recombinant production. Non-limiting examples of potential moieties include beta-galactosidase, glutathione-S-transferase, luciferase, T7 polymerase fragment, secretory signal peptide, antibody or antibody fragment, toxin, reporter enzyme, a part capable of binding metal ions such as a polyhistidine tag, a tag suitable for detection and / or purification, a homologous or heterologous binding domain, a part that improves protein solubility, or a part that includes an enzyme cleavage site.

[0135] Therefore, the humanized antibodies or antibody fragments according to the present disclosure may optionally include one or more moieties for binding to other targets or target proteins of interest. It should be apparent that such other moieties may or may not provide further function to the antibody and may or may not alter the performance of the isolated antibodies or antibody fragments according to the present disclosure.

[0136] Treatment

[0137] The humanized antibodies or antibody fragments according to the present disclosure can be used in therapeutic methods. The antibodies or antibody fragments according to the present disclosure can be used to treat inflammatory diseases, autoimmune diseases, fibrotic diseases, hematological malignancies and potentially other diseases.

[0138] In one embodiment, the disease is associated with the undesirable presence of human IL3. In another embodiment, the disease is associated with the undesirable presence of cells expressing IL3, in particular T cells expressing human IL3.

[0139] IL3 has significant growth stimulation and differentiation effects on various hematopoietic precursor cells, and is also a growth factor for mast cells. The signal transduction caused by IL3 has a key impact on the immune system. Any disease or medical condition in which IL3 plays a direct or indirect role in development or progression is a candidate for treatment by administering antibodies according to the present disclosure. Such diseases or dysfunctions associated with increased IL3 levels or increased expression of IL3 caused by cells that can produce IL3 (such as T cells, basophils, B cells, NK cells, certain types of cancer / tumors, neurons, etc.) are preferably related to the immune system, mainly 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 allogeneic rejection.

[0140] In one embodiment, the disease to be treated is an autoimmune or inflammatory disease. Non-limiting examples of autoimmune or inflammatory diseases include rheumatoid arthritis (RA), psoriasis, psoriatic arthritis, systemic lupus erythematosus (SLE), lupus nephritis, type I diabetes, Grave's 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) autoinflammatory diseases, cold inflammation associated periodic syndrome (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, autoimmune thyroiditis, scleroderma, systemic sclerosis, osteoarthritis, atopic dermatitis, vitiligo, graft-versus-host disease, Sjogren's syndrome (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 disease, 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 caused by acute or chronic inflammation.

[0141] In one embodiment, the disease to be treated is a proliferative disease. In a specific embodiment, 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.

[0142] Further conditions preferably treated by administration of the pharmaceutical composition of the invention are the inhibition of human basophil activity in humans suffering from allergic reactions and the use for stratification of patients with elevated IL3 levels in serum or plasma or elevated IL3 expression by cells capable of producing IL-3 (e.g., T cells).

[0143] In particular, in view of RA, it has been found that for a large group of patients, elevated levels of hIL3 produced by T cells are associated with worsening of the patient's condition and progression of the disease. IL3 is primarily detected in active RA, while patients in an inactive phase of RA generally do not show elevated IL3 expression or elevated levels. Therefore, the pharmaceutical composition of the present invention is particularly suitable for treating patients with active episodes of autoimmune diseases such as RA, as well as for preventive treatment to avoid the occurrence of active episodes of the disease.

[0144] Since existing therapies are only effective in about 50% of treated patients, providing a pharmaceutical composition according to the present invention is an important step in a mild new approach to treating autoimmune diseases in patients. Based on the lack of an overt phenotype in IL3-deficient mice (Nature (1998) 392: 90-3) and the lack of obvious side effects in mice treated with IL3 antibodies (Arthritis Rheum (2009) 60: 1352-61), IL-3 targeted therapy should show less severe side effects than currently used drugs, especially in terms of infection or tumors. In some cases, it is desirable to combine treatment with other drugs such as methotrexate or leflunomide with the antibodies and pharmaceutical compositions of the present invention. Compared with existing biologics, personalized treatment strategies based on IL3 expression or IL3 levels in plasma, serum or other body fluids present advantages because it is currently impossible to reliably predict which patients will respond to a specific therapy (including biologics). In addition, personalized approaches improve the safety of treatment by reducing the risk of side effects of ineffective treatments and reduce the cost of treating RA.

[0145] In addition, once elevated IL3 expression or elevated IL3 levels are detected in the blood, plasma or serum, treatment with the antibodies of the invention is preferably initiated. Thus, early treatment can be applied to patients whose RA activity is associated with elevated hIL-3 levels, and long-term joint damage can be avoided or kept to a minimum. In addition, if a patient has previously failed treatment with a DMARD or a biologic, treatment with an anti-IL3 antibody will be initiated preferentially. Furthermore, it is expected that the use of the antibodies of the invention can reduce cellular infiltration of synovial tissue, which may be a further negative factor in the pathology of the disease.

[0146] In one embodiment, the present disclosure provides a method of treating a disease.

[0147] In one embodiment, the present disclosure provides a method of treating a disease, the method comprising administering the antibody or antibody fragment of the present disclosure to a patient.

[0148] In one embodiment, the present disclosure provides a method of treating a disease, comprising administering the antibody or antibody fragment of the present disclosure to a subject in need thereof.

[0149] In one embodiment, the present disclosure provides a method of preventing a disease.

[0150] In one embodiment, the present disclosure provides a method for preventing a disease, the method comprising administering the antibody or antibody fragment of the present disclosure to a subject.

[0151] 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 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.

[0152] In one embodiment, the present disclosure provides the use of an antibody or antibody fragment according to the present disclosure for the preparation of a medicament. In one embodiment, the present disclosure provides a humanized antibody or antibody fragment according to the present disclosure, used as a medicament. In one embodiment, the present disclosure provides a humanized antibody or antibody fragment according to the present disclosure, used in medicine. In one embodiment, the present disclosure provides a humanized antibody or antibody fragment according to the present disclosure, used as a medicament for treating a subject in need thereof.

[0153] 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 suffering from a disease, the method comprising administering a therapeutically effective amount of the antibody or antibody fragment according to the present disclosure to the subject.

[0154] 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 specifically a human. For use in therapeutic methods, the humanized antibodies or antibody fragments according to the present disclosure will be formulated, dosed, and administered in a manner consistent with good medical practice.

[0155] Pharmaceutical composition

[0156] 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.

[0157] The pharmaceutical composition may also contain at least one other pharmaceutically active compound. The pharmaceutical composition according to the present disclosure may be used for diagnosing, preventing and / or treating diseases associated with the undesirable presence of IL3 (particularly human IL3). The pharmaceutical composition according to the present disclosure may be used for diagnosing, preventing and / or treating diseases associated with the undesirable presence of IL3-positive cells, particularly IL3-positive human cells. Specifically, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure, which is suitable for preventive, therapeutic and / or diagnostic use in mammals, more particularly humans.

[0158] In general, antibodies or antibody fragments according to the present disclosure can be formulated into pharmaceutical compositions comprising at least one antibody or antigen fragment according to the present disclosure and at least one pharmaceutically acceptable carrier or excipient, and optionally one or more other pharmaceutically active compounds. Such preparations can be suitable for oral, parenteral, topical administration or inhalation administration. Therefore, pharmaceutical compositions comprising at least one antibody or antibody fragment according to the present disclosure can be administered parenterally, such as intravenously, intramuscularly or subcutaneously. Alternatively, antibodies of the present invention can be administered via non-parenteral routes, such as oral or topical administration. In a preferred embodiment, pharmaceutical compositions comprising antibodies or antibody fragments according to the present disclosure are administered intravenously or subcutaneously.

[0159] Specifically, the antibodies or antibody fragments according to the present disclosure can be used in combination with one or more pharmaceutically active compounds that are used or can be used to prevent and / or treat diseases involving target antigens of interest, whereby a synergistic effect may or may not be obtained. Examples of such compounds as well as routes of administration, methods, and pharmaceutical preparations or compositions will be apparent to clinicians.

[0160] In one embodiment, the present disclosure provides a pharmaceutical composition comprising an antibody or antibody fragment according to the present disclosure, for preventing and / or treating a disease associated with the undesirable 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 preventing and / or treating a disease associated with the undesirable 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 preventing and / or treating an autoimmune disease and / or inflammatory disease and / or cancer.

[0161] In one embodiment, the present disclosure provides a method of treating an autoimmune disease and / or 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.

[0162] The present disclosure also provides a method for producing an antibody or antibody fragment according to the present disclosure in a form suitable for in vivo administration, the method comprising (a) obtaining an 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, thereby formulating a preparation of the antibody or antibody fragment for in vivo administration. The pharmaceutical composition according to the present disclosure comprises 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.

[0163] Diagnostic Uses

[0164] In one embodiment, the present disclosure provides the use of humanized antibodies or antibody fragments specific for human IL3 according to the present disclosure for disease diagnosis. In one embodiment, the present disclosure provides the use of antibodies or antibody fragments according to the present disclosure for detecting IL3, particularly human IL3. In one embodiment, the present disclosure provides a method for detecting IL3 in a subject or sample, the method comprising the step of contacting the subject or sample with a humanized antibody or antibody fragment specific for human IL3 according to the present disclosure. In one embodiment, the present disclosure provides a method for diagnosing a disease in a subject, the method comprising the step of 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 the expression level of IL3 in patient cells. IL3 expression levels can be used as therapeutic biomarkers, for example, for patient stratification.

[0165] Example

[0166] Example 1 : Parental Antibody P8C11 (DSM ACC3281)

[0167] The parent mouse antibody P8C11 was originally generated by immunizing Balb / c mice with human eukaryotic glycosylated IL-3; see WO2017 / 081218. The antibody was deposited at the DSMZ on October 7, 2015 (DSM ACC3281).

[0168] P8C11 is a mouse 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. Compared to the germline sequence, the variable heavy chain of antibody P8C11 has three mutations: M for L, A for S, and I for V. These positions are indicated in SEQ ID No. 2 below.

[0169] Table 1:

[0170]

[0171] The antibody P8C11 was originally generated by immunizing Balb / c mice with human eukaryotic glycosylated IL-3; see WO2017 / 081218. The antibody was deposited with the DSMZ on October 7, 2015 (DSM ACC3281). P8C11 inhibits IL3-dependent growth and many other beneficial activities of TF1 cells, suggesting that P8C11 may be a candidate for clinical development (WO2017 / 081218).

[0172] Example 2 :Humanization of antibody P8C11

[0173] Example 2.1 :Grafting CDRs onto new framework sequences

[0174] In the first step, the CDRs of P8C11 were transplanted into 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*3*01 variant, which included the germline mutation of SEQ ID No. 2 and a back mutation of an alanine residue to the original glycine residue because the amino acid is close to HCDR1. The sequences of the variable light chain and variable heavy chain are shown in the table below.

[0175] Table 2:

[0176]

[0177] All three variable heavy chains were paired with all three variable light chains, resulting in nine different antibodies. The inhibitory activity of these nine antibodies was tested and compared with that of a chimeric version of P8C11 (an antibody carrying the P8C11 variable mouse heavy and light chains fused to human IgG1κ). The inhibitory activity was measured by analyzing the ability of the antibody variants to block IL-3-induced CD131 downregulation on primary human basophils. IL-3 (0.2 ng / mL) was preincubated with different 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 another vial, PBS alone (without IL-3) was added to whole human EDTA blood at 37°C for 1 hour without inducing CD131 downregulation. The blood was then stained with direct antibodies against CD123, CD203c, and CD131 for 30 min on ice to identify basophils and measure the surface level of CD131 on basophils. The inhibitory activity of chimeric P8C11 was normalized to 100% and that of PBS in the absence of any antibody was normalized to 0%. If the concentration of the humanized variant needed to be 10-fold higher than that of the chimeric P8C11 to achieve the same level of inhibition of CD31 downregulation, the inhibitory activity was determined to be 10%.

[0178] The inhibitory activities are shown in the table below.

[0179] Table 3:

[0180]

[0181] None of the nine humanized antibodies had inhibitory activity close to that of chimeric P8C11. Of the three variable heavy chains, HC3 was the best variant to advance. The three variable light chains were equally effective. LC2 was chosen for further humanization.

[0182] Example 2.2 : Variable chain recognition leading to loss of activity

[0183] Next, to investigate whether the variable heavy chain or variable light chain receptor framework was responsible for the loss of inhibitory activity, the mouse variable heavy chain was paired with the variable light chain VL2 (muHC × LC2), and the mouse variable light chain was paired with the variable heavy chain HC3 (HC3 × muLC). The inhibitory activity was again compared with the chimeric P8C11. The results are shown in the table below.

[0184] Table 4:

[0185]

[0186] This result suggests that the loss of inhibitory activity is due to humanization of the variable heavy chain, but not of the variable light chain.

[0187] Example 2.3 : Identification of key frames in HC3

[0188] To investigate which part of the framework is responsible for the loss of inhibitory activity, various hybrid frameworks were constructed, consisting of the corresponding parts of the original mouse framework or the HC3 humanized framework.

[0189] Three constructs were generated as shown in the table below.

[0190] Table 5

[0191]

[0192] These variable heavy chains were paired with the mouse variable light chains of P8C11 and their inhibitory activity was measured. The results are shown in the table below.

[0193] Table 6:

[0194]

[0195] This result suggests that framework 3 (FR3) of HC3 is responsible for the loss of inhibitory activity. Framework 4 (FR4) of HC3 is unlikely to be the main cause, as only one amino acid differs from the original mouse framework sequence, and FR4 generally does not contribute much to VH function.

[0196] Example 2.4 : Identification of key residues within HC3 framework 3

[0197] 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 ten 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, which corresponds to the germline mutation of the original mouse antibody P8C11). In one construct, HC5, the first ten 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 consisted of hybrids IGHV1-18*01 and IGHV1-69*02, which are similar to the original mouse framework of P8C11 at both the N-terminus and the C-terminus, respectively. Moreover, the isoleucine residue in framework 3, which corresponds to the germline mutation of the original mouse antibody P8C11, was retained. In addition, the "mouse" valine at position 6 of framework 3 and the "mouse" serine at position 10 of framework 3 were retained.

[0198] The amino acid sequences of the three constructs are shown in the table below.

[0199] Table 7:

[0200]

[0201] These variable heavy chains were paired with humanized variable light chain VL2 and their inhibitory activity was measured. The results are shown in the table below.

[0202] Table 8:

[0203]

[0204] Comparison of heavy chain HC4 and HC5 showed that the first 10 amino acids in framework region 3 are important for maintaining antibody activity. If these 10 amino acids have the original mouse sequence, the antibody is fully active. In addition, H6 has a similar amino acid sequence at this position, and the activity is fully retained.

[0205] Example 2.5 : Optimizing HC6 to improve human origin

[0206] To further improve humanization, four different heavy chains were designed based on HC6. HC7 and HC8 are both based on HC6 but have less residual mouse framework sequences. HC9 and HC10 are also based on HC6 but are closer to germline IGHV1-3*01.

[0207] The amino acid sequences of the four constructs are shown in the table below.

[0208] Table 9:

[0209]

[0210] Again, these variable heavy chains were paired with humanized variable light chain VL2 and the inhibitory activity was measured. The results are shown in the table below.

[0211] Table 10:

[0212]

[0213] Comparison of heavy chains HC7 and HC8 showed that residues A and G of mouse origin in framework 1 were important for activity, whereas residues M of mouse origin in framework 1 and I in framework 3 were not essential for activity. Comparison of heavy chains HC9 and HC10 showed that amino acid M was slightly more active than amino acid I at position 4 in framework 3.

[0214] Example 2.6 : Optimizing HC7 to improve human origin

[0215] Six additional different heavy chains were designed based on HC7 to further improve humanization.

[0216] The amino acid sequences of the three constructs are shown in the table below.

[0217] Table 11:

[0218]

[0219] Again, these variable heavy chains were paired with humanized variable light chain VL2 and the inhibitory activity was measured. The results are shown in the table below.

[0220] Table 12:

[0221]

[0222] Comparison of heavy chains HC11 and HC12 showed that a V at position 6 of framework 3 was important for maintaining activity. An A at position 6 of FR3 resulted in a significant loss of inhibitory activity.

[0223] Comparison of heavy chains HC11 and HC13 showed that at position 10 of framework 3, amino acids T and S had similar inhibitory activity. T at position 10 of framework 3 was preferred to improve humanization.

[0224] Comparison of heavy chains HC11, HC14 and HC15 showed that the G at position 24 of framework 1 is important to maintain inhibitory activity and should not be replaced by A.

[0225] Comparison of heavy chains HC11, HC14 and HC15 also showed that at position 21 of framework 1, amino acid A is not essential for maintaining activity and can be replaced by S to improve humanization.

[0226] 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 shows full inhibitory activity and is most likely of human origin.

[0227] Therefore, as the final candidate, an antibody comprising heavy chain HC16 and variable light chain LC2 was selected. The antibody was named "GRT002-H16L2". The antibody has the following heavy chain sequence:

[0228]

[0229] And the following light chain sequence:

[0230]

[0231] Example 2.7 :Summary of the humanization process

[0232] Humanization of P8C11 was by no means obvious. Ultimately, a six-step iterative process was required to identify humanized derivatives of P8C11 that retained the desired activity (i.e., inhibition of glycosylated IL3). During this process, three different variable light chains and sixteen different variable heavy chains were generated and tested in various combinations. Various key amino acids were identified that were required for full binding and inhibition of IL-3. Many of these residues were directly adjacent to the CDR regions.

[0233] GRT002-H16L2 was identified as the final humanized variant of P8C11. The framework of the variable light chain of GRT002-H16L2 is 100% identical to the human germline. Two amino acids in the framework of the variable heavy chain of GRT002-H16L2 differ from the human germline. These two amino acids are critical for full binding and inhibition of IL-3. GRT002-H16L2 is an IgG1 antibody of the G1m3 (heavy chain) and KM3 (light chain) allotypes that lack a C-terminal lysine.

[0234] Example 3: Generation and purification of GRT002-H16L2

[0235] The 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 using 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.

[0236] The antibody preparation had a monomer content of >97%. The molecular weight determined by SEC using 8 marker proteins was 152.12 kDa.

[0237] Example 4: Determination of GRT002-H16L2 binding properties

[0238] The binding performance of antibody GRT002-H16L2 compared to the parental antibody P8C11 was determined by surface plasmon resonance (SPR) using a Biacore X100 instrument (GE Healthcare). The chimeric antibody P8C11 was used as a reference. The antibodies were covalently bound to a CM5 sensor chip via a dextran matrix. Degassed PBS + 0.05% Tween-20 was used as 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 seconds at a flow rate of 5 µL / min (sample cells only). After stabilization for 300 seconds, IL3 was added for 360 seconds at a flow rate of 15 µL / min (sample cells and reference cells). The dissociation time was 600 seconds. 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, the "steady-state affinity" model was used, and for kinetic fitting, the "1:1 binding" model was used.

[0239] The results are as follows Figure 1 Based on Figure 1 From the curves shown in , the binding properties can be determined and indicate that the binding of antibody GRT002-H16L2 to IL-3 is equal to or even slightly better than that of the parent antibody P8C11.

[0240] Example 5: Epitope mapping of GRT002-H16L2

[0241] The epitope of GRT002-H16L2 was mapped by measuring the binding of the antibody to a linear peptide derived from the IL3 amino acid sequence.

[0242] Peptides were coated overnight at 10 µg / mL (60 µL / well) in PBS in Nunclon 96 flat-bottom clear polystyrene well plates (Thermo Fisher). Cells were then washed three times with wash buffer (0.05% Tween 20 in PBS) and blocked with 1% BSA / PBS for 2 hours at room temperature. After another wash step with wash buffer, antibody GRT002-H16L2 (60 µL 10 µg / mL in 1% BSA / PBS) was added and incubated for 1 hour at room temperature. 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 the final washing step, 100 µL of ABTS (2,2'-aza-bis(3-ethylbenzothiazoline-6-sulfonic acid) was added to each well. Absorption was measured at 405 nm in an infinite 200Pro reader (Tecan).

[0243] For the parent antibody P8C11, amino acids 22-48 of human IL3 (counted in the absence of the signal sequence) were reported as the epitope (see WO2017 / 081218). The same epitope was also confirmed for GRT002-H16L2. To further optimize the epitope, the following amino acids were tested.

[0244] Table 13:

[0245]

[0246] The results are as follows Figure 2 As shown. The epitope of GRT002-H16L2 is identical to that of P8C11. The deletion of amino acids 47 and 48 of the peptides tested resulted in a significant reduction in the binding of GRT002-H16L2. GRT002-H16L2 still binds to peptides in which amino acids 22-25 or 22-27 are deleted, but to a lesser extent. In summary, GRT002-H16L2 binds to a linear epitope contained in the amino acid sequence EIITHLKQPPLLDFNNLNGEDQDIL (SEQ ID No. 37).

[0247] Example 6: IL-3 blockade in TF1 cells

[0248] In the first experiment analyzing the ability of GRT002-H16L2 to block IL-3 activity, TF1 cells were used. TF1 cells (ATCC, order #crl-2003) are human erythroblasts and the cell line was established from the bone marrow of a 35-year-old Japanese male with severe pancytopenia. The growth of TF1 cells is completely dependent on the presence of IL-3 or GMCSF. Therefore, a TF1 cell-based cell proliferation assay can be used to determine the blockade of IL-3 activity, which in turn results in a reduction or even complete inhibition of TF1 cell growth.

[0249] Experimentally, the MTT cell proliferation assay was performed to determine cell viability based on the activity of mitochondrial dehydrogenases. The substrate of the dehydrogenase, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), is yellow in solution and is cleaved by the enzyme activity at the tetrazole ring, resulting in the formation of purple MTT formazane crystals. These crystals can be dissolved in isopropanol, the purple solution is measured in a spectrophotometer, and the result is correlated with the amount of viable TF1 cells.

[0250] Culture of TF1 cells:

[0251] TF1 cells were grown in suspension in medium (RPMI-1640 with 10% FCS (HIA), P / S, and glutamine (1:100)) and supplemented with 5 ng / mL IL-3 or 5 ng / mL GM-CSF, split 1:4 every three days. For storage, cells were transferred from cell culture flasks 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 medium (RPMI-1640 with 10% FCS (HIA) + P / S + glutamine + 5 ng / mL IL-3) and 5% DMSO and 1.5 mL aliquots were dispensed into vials. Cells were pre-frozen in freezing containers in a -80°C freezer and transferred to a liquid nitrogen storage tank after 1-2 days.

[0252] Blocking experiment:

[0253] TF-1 cells, which were split every three days according to the above protocol, were split 1:2 the day before the experiment in medium containing 5 ng / mL human IL-3. For this experiment, cells were centrifuged at 1600 rpm for 5 minutes at room temperature. The medium was removed, and the cells were washed twice in RPMI medium, then resuspended in 1 mL RPMI-1640 + 10% FCS (HIA) + P / S + glutamine (1:100), counted, and supplemented with buffer to a final concentration of 1×105 cells / mL. In a 96-well plate, 10,000 cells in 100 μL of culture medium (RPMI + 10% FCS + P / S + glutamate) were provided to each well. 100 μL of IL-3 was pre-incubated with the antibody to be tested for 60 minutes at 37°C. For the pre-incubation, different final concentrations of antibodies and IL-3 were used. To obtain such final concentrations, the concentrations of the antibody and IL-3 solutions needed to be twice the final concentration. After 5 days of incubation at 37°C, 5% CO2 was added, 100 μL of culture medium was removed from each well, 10 μL of MTT solution (LCG standard-ATCC) was added to each well, and the plate was incubated for another 4 hours in a 37°C and 5% CO2 incubator. After this further incubation, 100 μl of MTT solvent was added and the contents of each well were carefully mixed. After overnight incubation, the optical density was measured at 570 and 690 nm, and the number of viable cells was calculated from this.

[0254] The chimeric antibody P8C11 and the antibody GRT002-H16L2 were tested. Figure 3 Both antibodies blocked IL-3 activity to the same extent in TF1 cells.

[0255] Example 7: Blockade of IL-3 binding to human basophils

[0256] To quantify the blocking effect of CD123 binding on basophils by GRT002-H16L2 and P8C11 chimera (P8C11-Chim), the IL-3 Biotinylated Fluorophore Flow Cytometry Kit (R&D Systems) was used. Antibodies were used at various dilutions in 1% BSA / PBS. 10 µL of biotinylated IL-3 (1 µg / mL) was added to 10 µL of antibody solution. Samples were pre-incubated on ice for 30 minutes in the dark. Thereafter, 60 µL of EDTA blood was added, followed by incubation on ice for 60 minutes in the dark. Then, 20 µL of Avidin-FITC, 3.33 µL of CD123 PE-Cy 5.5 (1:30), and 1 µL of CD203c PE (1:100) were added, followed by incubation on ice for another 30 minutes in the dark. After two washing steps, erythrocytes were lysed by adding 1 mL of FACS lysing solution for 10 minutes at room temperature in the dark. Thereafter, the samples were washed with PBS and centrifuged at 1600 rpm for 6 min at 4° C. The samples were then analyzed for 60 sec in a BD FACS Canto II instrument (Becton Dickinson).

[0257] The results of P8C11 and GRT002-H16L2 antibodies are shown in Figure 4Both antibodies blocked IL-3 binding to human basophils to the same extent.

[0258] Example 8: Biophysical properties of GRT002-H16L2

[0259] For clinical development, it is important that the antibody has biophysical properties that allow it to be produced, stored, formulated and administered in sufficient quantities without any undesirable effects such as off-target stickiness. Therefore, the antibody GRT002-H16L2 was tested in a series of analytical tests and compared to the parent antibody P8C11.

[0260] Example 8.1 : Melting temperature

[0261] 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 in the temperature range of 25-130°C; the temperature was increased at a rate of 1°C / min. Antibodies were in a solution of PBS + 100 mM L-arginine. Antibody concentrations were 7.75 mg / mL and 4.55 mg / mL for GRT002-H16L2 and P8C1-chimera, respectively.

[0262] The recording patterns of P8C11 and GRT002-H16L2 antibodies were Figure 5 and Figure 6 shown.

[0263] •Tm1 = CH2 melting: 70.83°C for P8C11 chimera and 70.81°C for GRT002-H16L2

[0264] •Tm2 = Fab and CH3 melting: 78.60°C for P8C11 chimera and 82.53°C for GRT002-H16L2

[0265] It can be seen that the relevant melting point Tm2 including the Fab region for chimeric P8C11 is much lower than that of GRT002-H16L2, indicating that GRT002-H16L2 has higher temperature stability.

[0266] Example 8.2 : Freezing and thawing

[0267] To test the behavior of the antibody to repeated freezing and thawing, antibody GRT002-H16L2 (7.75 mg / mL, 100 mM L-arginine in PBS) was subjected to repeated freeze-thaw cycles. After ten freeze-thaw cycles, samples were subjected to SEC. No effects on the integrity of the antibody were observed in the SEC chromatograms (data not shown).

[0268] The effect of freeze-thaw on the antibody GRT002-H16L2 was also investigated in more detail by analyzing the functional activity of the antibody by measuring the expression level of CD131 in 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 different 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 at 37°C for 1 hour to induce CD131 downregulation. The blood was then stained on ice for 30 minutes with direct antibodies against CD123, CD203c, and CD131 to identify basophils and measure the surface level of CD131 on basophils.

[0269] The results are as follows Figure 7 This confirms that repeated freezing and thawing has no effect on the functional activity of GRT002-H16L2.

[0270] Example 8.3 : Temperature stability

[0271] To test the temperature stability of the antibodies, GRT002-H16L2 and chimeric P8C11 were studied 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, followed by analytical SEC using Superdex 200 with photometric detection of the proteins at 280 nm.

[0272] The SEC profiles of both antibodies incubated at 55°C for 3 hours were indistinguishable from the reference (i.e., the respective antibodies kept at 4°C). After 3 hours of incubation at 65°C, the antibody P8C11 chimera showed a first shoulder at the retention volume before the main peak, indicating the formation of aggregates. In contrast, the SEC profile of GRT002-H16L2 remained indistinguishable from the reference. After 3 hours of incubation at 75°C, the integrity of both antibodies was significantly reduced, but more pronounced for the chimeric P8C11.

[0273] The effect of exposure to elevated temperature on both antibodies was also investigated by means of analyzing the functional activity of the antibodies by measuring the expression levels of CD131 in basophils.CD131 expression was analyzed as described in Example 8.2.

[0274] Compared with the reference, the results of samples incubated at 75°C for 3 hours are as follows Figure 8 As shown (exposure to 55°C or 65°C for 3 hours did not result in any loss of functional activity for either antibody). This shows that antibody GRT002-H16L2 showed significantly higher functional activity after exposure to 75°C for 3 hours compared to antibody P8C11 chimera. At a certain antibody concentration, the functional activity of GRT002-H16L2 was more than twice that of antibody P8C11 chimera. After exposure to 75°C, the concentration of P8C11 chim needed to be more than 16 times higher than that of GRT002-H16L2 to obtain the same level of IL-3 inhibition.

[0275] Antibody GRT002-H16L2 has higher temperature stability than antibody P8C11-chimera.

[0276] Example 8.4 : pH stability

[0277] To test the behavior of the antibodies, GRT002-H16L2 and chimeric P8C11 were incubated for two hours in buffers of different pH values ​​with different antibody concentrations.

[0278] To this end, 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 in water at a ratio of 1:5 and the pH was adjusted to the desired value with HCl / NaOH. The samples were then incubated at room temperature for 2 hours and the pH was then readjusted to 7.2 with HCl / NaOH.

[0279] The effect of exposure of the two antibodies to different pH values ​​was investigated by analyzing the functional activity of the antibodies by measuring the expression levels of CD131 in basophils. CD131 expression was analyzed as described in Example 10.2.

[0280] Fig. 9Results are shown for samples incubated at pH 2 for 2 hours at room temperature compared to a reference (exposure for 2 hours at pH 3, 4 or 9 did not result in loss of functional activity for both antibodies). It can be seen that antibody GRT002-H16L2 completely retains its functional activity after exposure for 2 hours at pH 2, whereas the functional activity of the antibody P8C11 chimera is strongly reduced. At a certain antibody concentration, the functional activity of GRT002-H16L2 is two-fold, or even three-fold, or four-fold higher than that of the antibody P8C11 chimera. After exposure to pH 2, the P8C1 chimera requires a concentration more than 16 times higher than that of GRT002-H16L2 to obtain the same level of IL-3 inhibition.

[0281] The pH stability of antibody GRT002-H16L2 was higher than that of antibody P8C11 chimera.

[0282] Example 8.5 : Plasma stability

[0283] To test the long-term stability of antibody GRT002-H16L2, the antibody was incubated in PBS and human plasma at different concentrations for 48 hours or 14 days.

[0284] In detail, 23.7 μL of antibody (7.75 mg / mL in PBS + 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. Thereafter, the functional activity of the antibody was determined by measuring the expression level of CD131 in basophils. CD131 expression was analyzed as described in Example 10.2.

[0285] The results are as follows Fig.10 This confirms that the antibody GRT002-H16L2 retains its full functional activity after incubation in human plasma for at least 14 days.

Claims

1. A humanized antibody or antibody fragment specific for human IL3, wherein: The antibody or antibody fragment comprises a HCDR1 region of SEQ ID NO: 3, a HCDR2 region of SEQ ID NO: 4, a HCDR3 region of SEQ ID NO: 5, a LCDR1 region of SEQ ID NO: 10, a LCDR2 region of SEQ ID NO: 11 and a LCDR3 region of SEQ ID NO:

12.

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

16.

3. An isolated antibody or antibody fragment according to any one of the preceding claims, wherein The antibody is of human IgG1 class.

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

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

6. An isolated antibody or antibody fragment according to any one of the preceding claims, wherein Compared to an antibody or antibody fragment comprising the VH of SEQ ID NO: 2 and the VL of SEQ ID NO: 9, the antibody has improved stability at 75°C.

7. An isolated antibody or antibody fragment according to any one of the preceding claims, wherein The antibody has improved stability at pH 2 compared to an antibody or antibody fragment comprising the VH of SEQ ID NO: 2 and the VL of SEQ ID NO:

9.

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

9. The isolated antibody or antibody fragment according to claim 8, wherein The medical use is the treatment of inflammatory diseases, autoimmune diseases or fibrotic diseases or malignant tumors.

10. A nucleic acid composition comprising a nucleic acid sequence or multiple nucleic acid sequences encoding the isolated antibody or antibody fragment according to any one of claims 1-9.

11. A vector comprising the nucleic acid composition according to claim 10.

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

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

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