Anti-S100A4 humanized antibody, use and method

Abstract

This invention provides a humanized anti-S100A4 antibody with an improved safety profile. [Solution] An isolated humanized anti-S100A4 antibody molecule and a method for producing the antibody are provided. Also provided are nucleic acids containing the antibody, a vector, isolated host cells, and a pharmaceutical composition, as well as a therapeutic method comprising administering the antibody.

Description

Technical Field

[0001] The present invention relates to isolated anti-S100A4 humanized antibody molecules and their medical use, and more particularly to isolated anti-S100A4 humanized antibody molecules capable of inhibiting the biological activity of S100A4, for example in promoting chronic inflammation, fibrosis, tumor progression and / or inducing tumor metastasis, and their use in the treatment of fibrotic diseases, inflammatory conditions, and cancer, particularly metastatic cancer.

Background Art

[0002] Fibrosis is defined as the excessive deposition of extracellular matrix proteins. The initial stages of fibrotic diseases are most often characterized by an inflammatory response that attracts, differentiates, and activates fibroblasts, which produce collagen and other extracellular matrix proteins. When these processes become chronic, the physiological tissue function is gradually impaired by the inflammatory and / or fibrotic response, which may lead to organ dysfunction or organ failure. The mechanisms involved in chronic inflammation and fibrosis are the underlying causes of a wide range of diseases with various clinical manifestations, including atherosclerotic diseases, cancer, and neurodegenerative diseases, in addition to pure fibrotic and inflammatory conditions. In cancer, the microenvironment of the diseased tissue provides an essential support mechanism for the growth and metastasis of malignant cells. Activation of the inflammatory and fibrotic pathways plays an important role in the development of the pre-metastatic niche, which provides the conditions necessary for primary tumor cells to metastasize to distant organs.

[0003] S100A4 has been identified as a key protein involved in processes that amplify and maintain the inappropriate activation of inflammatory and fibrotic pathways. S100A4 belongs to the S100 family of low-molecular-weight Ca2+ binding proteins with diverse extracellular and intracellular functions (Donato, 2003). Under physiological conditions, S100A4 is mainly present intracellularly, but is released into the extracellular environment in response to cellular stress or damage (Fei et al., 2017). Extracellular S100A4 forms higher-order oligomers, which bind to pattern recognition receptors (PRRs) and activate multiple inflammatory and fibrotic responses (Ambartsumian et al., 2019; Fei et al., 2017). Through its interaction with PRRs, S100A4 triggers the release of inflammatory mediators from immune cells and stimulates the release of extracellular matrix proteins from fibroblasts, participating in epithelial-mesenchymal transition (Kalluri & Zeisberg, 2006; Tomcik et al., 2015; Neidhart et al., 2019). Overexpression of S100A4 is characteristic of chronic inflammation and fibrosis. Numerous studies have shown elevated S100A4 levels compared to healthy controls in various human diseases, including systemic sclerosis, interstitial pulmonary fibrosis, rheumatoid arthritis, psoriasis, and dermatomyositis (Tomcik et al., 2015; Zibert et al., 2008; Klingelhofer et al., 2007; Cerezo et al., 2011; Akiyama et al., 2020).

[0004] Several studies using cell-based assays or in vivo disease models suggest that S100A4 is involved in the development of inflammation and fibrosis (Ambartsumian et al., 2019). Knockdown of S100A4 in fibroblasts inhibits TGFβ-induced fibroblast activation (Tomcik et al., 2015). Knockout of S100A4 inhibits fibrosis, inflammation, and cancer metastasis in several animal models, including bleomycin-induced dermatofibrosis or pulmonary fibrosis, tight skin 1 dermatofibrosis, and several cancer models (Ambartsumian et al., 2019; Tomcik et al., 2015).

[0005] Numerous studies have linked S100A4 activity to tumor progression and metastasis. This evidence has been accumulated through in vitro studies of cancer cell lines, transgenic and knockout mouse models, and evaluations of the prognostic significance of metastasis in cancer patients (Boye et al., 2010; Helfman et al., 2005; Mishra et al., 2011).

[0006] S100A4 activity is associated with stimulating cancer cell motility and aggression, normal and abnormal proliferation, apoptosis, and differentiation. It is involved in signaling pathways that lead to cell membrane and extracellular matrix remodeling, regulation of cytoskeletal dynamics, acquisition of aggression, and induction of angiogenesis (Sherbet, 2009).

[0007] S100A4 is expressed in certain tumor cells, but more commonly, it is activated and secreted by certain cancer-associated stromal cells, accumulating in the tumor microenvironment. Furthermore, the metastatic microenvironment has been shown to contain more S100A4-positive stromal cells than the primary tumor microenvironment (Cabezon et al., 2007; Grum-Schwensen et al., 2005; 2010; Maelandsmo et al., 2009; Schmidt-Hansen, et al., 2004a).

[0008] Furthermore, S100A4 has been shown to maintain the stem cell characteristics and tumorigenicity of progenitor cancer cells in head and neck cancer and glioblastoma (Lo et al., 2011; Chow et al., 2017). Therefore, the development of drugs that can inhibit the biological activity of S100A4 could present a promising therapeutic option for modulating multiple inflammatory and fibrotic pathways activated in various human diseases. Thus, there is an unmet need for therapeutic anti-S100A4 antibodies, particularly humanized anti-S100A4 antibodies, as they specifically target the extracellular pathogenic portion of S100A4.

[0009] Humanized antibodies are antibodies derived from non-human species whose protein sequences have been modified to increase their similarity to antibody variants naturally produced in humans. The "humanization" process is typically applied to monoclonal antibodies developed for administration to humans (for example, antibodies developed as anticancer drugs). Humanization may be necessary when the process of developing a particular antibody involves production in a non-human immune system (such as the mouse immune system). The protein sequences of antibodies produced in a non-human immune system may differ partially from naturally occurring alloantibodies in humans, and therefore may exhibit immunogenicity when administered to human patients, potentially leading to a loss of therapeutic benefit and causing side effects in patients. [Overview of the Initiative]

[0010] The present invention provides a humanized anti-s100A4 antibody having an improved safety profile.

[0011] The inventors found that mouse IgG1 and humanized IgG1 anti-S100A4 antibodies unexpectedly induced an increase in the pro-inflammatory cytokine TNFα, while simultaneously blocking the increase in IL-6 and IL-10 induced by S100A4 stimulation. The increase in TNFα was induced by FcγRIIA receptor clustering and activation. Surprisingly, this effect depended on both the anti-S100A4 antibody and the S100A4 protein; neither the anti-S100A4 antibody in the absence of S100A4, nor S100A4 combined with an isotype control antibody (either human IgG1 or human IgG4), resulted in FcγRIIA receptor clustering.

[0012] The inventors have found that subclass switching of humanized anti-S100A4 antibody from the IgG1 scaffold to the IgG4 scaffold suppresses this previously unreported S100A4-dependent FcγRIIA receptor clustering and activation, thereby preventing the release of undesirable pro-inflammatory cytokines and improving the antibody's safety profile. FcγRIIA receptor clustering and activation by humanized IgG4 antibody are also significantly reduced compared to those induced by mouse IgG1 anti-S100A4 antibody.

[0013] In one embodiment, an isolated antibody, a) i. Heavy chain complementarity determination region 1 (CDR-H1) containing or consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4, ii. Heavy chain complementarity determination region 2 (CDR-H2) containing or consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5, and iii. Heavy chain complementarity determination region 3 (CDR-H3) containing or consisting of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 6, A heavy chain variable (VH) region including, b) i. CDR-L1 containing or consisting of the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 10, ii. CDR-L2 containing or consisting of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 11, and iii. CDR-L3 containing or consisting of the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 12, A light chain variable (VL) region including, The VH region includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, and 17, and one variant of SEQ ID NOs: 13-17, wherein any one amino acid that is not part of the CDR sequence defined by SEQ ID NOs: 1-6 is modified to another amino acid (provided that no more than five amino acids are modified in this way, for example, 5, 4, 3, 2, or 1 amino acid is modified in this way in each amino acid sequence), and / or the VL region includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 20, 21, and 22, and one variant of SEQ ID NOs: 18-22, wherein any one amino acid that is not part of the CDR sequence defined by SEQ ID NOs: 7-12 is modified to another amino acid (provided that no more than five amino acids are modified in this way, e.g., 5, 4, 3, 2, or 1 amino acid are modified in this way in each amino acid sequence), Isolated antibodies are provided.

[0014] In one embodiment, an isolated antibody, i. An amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, and 17, and a heavy chain variable (VH) region containing or consisting of one variant of SEQ ID NOs: 13-17, in which any one amino acid is modified to another amino acid (provided that five or fewer amino acids are modified in this way, for example, 5, 4, 3, 2, or 1 amino acid is modified in this way in each amino acid sequence), ii. A light chain variable (VL) region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 18, SEQ ID NOs. 19, SEQ ID NOs. 20, SEQ ID NOs. 21, and SEQ ID NOs. 22, and one variant of SEQ ID NOs. 18-22, wherein any one amino acid is modified to another amino acid (provided that five or fewer amino acids are modified in this way, for example, 5, 4, 3, 2, or 1 amino acid is modified in this way in each amino acid sequence), Isolated antibodies containing [the specified substance] are provided.

[0015] In one embodiment, an isolated nucleic acid molecule encoding the antibody described in the “Isolated Anti-S100A4 Antibody Molecule” section of this specification is provided.

[0016] In one embodiment, an expression vector is provided that includes a nucleic acid molecule described herein that encodes an anti-S100A4 antibody molecule.

[0017] In one embodiment, an isolated host cell comprising an isolated nucleic acid molecule or expression vector as described herein is provided.

[0018] In one embodiment, a method for producing an anti-S100A4 antibody molecule is provided, comprising culturing a host cell described herein under conditions in which the antibody is expressed.

[0019] In one embodiment, a pharmaceutical composition is provided comprising an antibody, nucleic acid molecule, expression vector, and / or host cell as described herein, and a pharmaceutically acceptable diluent, carrier, and / or excipient.

[0020] In one embodiment, a method for treating an individual having an S100A4-mediated pathological condition is also provided, which includes administering an antibody or host cell described herein to an individual in need thereof.

[0021] In one embodiment, a method for diagnosing or prognosing S100A4-related pathological conditions in an individual, (a) Contacting a biological sample from an individual with an anti-S100A4 antibody described herein that can bind to the S100A4 polypeptide present in the sample; (b) Measuring the presence and / or amount of a complex formed between the antibody molecule and the S100A4 polypeptide; A method comprising the above is provided.

Brief Description of the Drawings

[0022] [Figure 1A] Shows the effect of monoclonal humanized anti-S100A4 antibody on fibrosis measurement values in bleomycin-administered mice. Shows the effect of anti-S100A4 antibody on dermal thickness. P values are represented as follows: when compared with NaCl, 0.05 > p > 0.01 is *, 0.01 > p > 0.001 is **; when compared with mice injected with NaCl for another 3 weeks after injecting bleomycin for 3 weeks, 0.05 > p > 0.01 is #; 0.01 > p > 0.001 is ##. The results are further described in Example 3. [Figure 1B] Shows the effect of monoclonal humanized anti-S100A4 antibody on fibrosis measurement values in bleomycin-administered mice. Shows the effect of anti-S100A4 antibody on the number of myofibroblasts. P values are represented as follows: when compared with NaCl, 0.05 > p > 0.01 is *, 0.01 > p > 0.001 is **; when compared with mice injected with NaCl for another 3 weeks after injecting bleomycin for 3 weeks, 0.05 > p > 0.01 is #; 0.01 > p > 0.001 is ##. The results are further described in Example 3. [Figure 1C] Shows the effect of monoclonal humanized anti-S100A4 antibody on fibrosis measurement values in bleomycin-administered mice. Shows the effect of anti-S100A4 antibody on hydroxyproline content. P values are represented as follows: when compared with NaCl, 0.05 > p > 0.01 is *, 0.01 > p > 0.001 is **; when compared with mice injected with NaCl for another 3 weeks after injecting bleomycin for 3 weeks, 0.05 > p > 0.01 is #; 0.01 > p > 0.001 is ##. The results are further described in Example 3. [Figure 1D]This study demonstrates the effect of a monoclonal humanized anti-S100A4 antibody on fibrosis measurements in bleomycin-treated mice. Representative images of HE-stained skin sections are shown. The results are further explained in Example 3. [Figure 1E] This study demonstrates the effect of a monoclonal humanized anti-S100A4 antibody on fibrosis measurements in bleomycin-treated mice. Representative images of HE-stained skin sections are shown. The results are further explained in Example 3. [Figure 2A] The data shows the amount of IL-6 secreted by monocytes as analyzed by Luminex assay. The data shown are the mean values ​​from five donors. Monocytes purified from PBMCs were cultured for 6 hours in medium, vehicle, LPS, and S100A4 in the absence or presence of mouse IgG1. The data show the IL-6 levels in the supernatant quantified by Luminex assay. The data are shown as the mean value + SEM obtained from five independent donors. "+" indicates that at least one donor had IL-6 levels above the detection limit (19,200 pg / mL). "-" indicates that at least one donor had IL-6 levels below the detection limit (8.8 pg / mL). The results are further explained in Example 4. [Figure 2B] This shows the amount of IL-6 secreted by monocytes as analyzed by Luminex assay. The data shown are the mean values ​​from five donors. Monocytes purified from PBMCs were cultured for 6 hours in medium, vehicle, LPS, and S100A4 in the absence or presence of human IgG4. The data show the IL-6 levels in the supernatant quantified by Luminex assay. The data are shown as the mean value + SEM obtained from five independent donors. "+" indicates that at least one donor had IL-6 levels above the detection limit (19,200 pg / mL). "-" indicates that at least one donor had IL-6 levels below the detection limit (8.8 pg / mL). The results are further explained in Example 4. [Figure 2C]The data shows the amount of IL-6 secreted by monocytes as analyzed by Luminex assay. The data shown are the mean values ​​from five donors. Monocytes purified from PBMCs were cultured for 6 hours in medium, vehicle, LPS, and S100A4 in the absence or presence of AX-202. The data show the IL-6 levels in the supernatant quantified by Luminex assay. The data are shown as the mean value + SEM obtained from five independent donors. "+" indicates that at least one donor had IL-6 levels above the detection limit (19,200 pg / mL). "-" indicates that at least one donor had IL-6 levels below the detection limit (8.8 pg / mL). The results are further explained in Example 4. [Figure 2D] The levels of IL-6 secreted by monocytes, analyzed by Luminex assay, are shown. The data shown are the mean values ​​from five donors. Monocytes purified from PBMCs were cultured for 6 hours in medium, vehicle, LPS, and S100A4 in the absence or presence of 6B12. The data show the IL-6 levels in the supernatant quantified by the Luminex assay. The data are shown as the mean value + SEM obtained from five independent donors. "+" indicates that at least one donor had IL-6 levels above the detection limit (19,200 pg / mL). "-" indicates that at least one donor had IL-6 levels below the detection limit (8.8 pg / mL). The results are further explained in Example 4. [Figure 3A] The levels of IL-10 secreted by monocytes, analyzed by Luminex assay, are shown. The data shown are the mean values ​​from five donors. Monocytes purified from PBMCs were cultured for 6 hours in medium, vehicle, LPS, and S100A4 in the absence or presence of mouse IgG1. The data show IL-10 cytokines in the supernatant, quantified by Luminex assay. The data are shown as the mean value + SEM obtained from five independent donors. "-" indicates that at least one donor had IL-10 below the detection limit (8.6 pg / mL). The results are further explained in Example 4. [Figure 3B]The levels of IL-10 secreted by monocytes, analyzed by Luminex assay, are shown. The data shown are the mean values ​​from five donors. Monocytes purified from PBMCs were cultured for 6 hours in medium, vehicle, LPS, and S100A4, either in the absence or presence of human IgG4. The data show IL-10 cytokines in the supernatant, quantified by Luminex assay. The data are shown as the mean value + SEM obtained from five independent donors. "-" indicates that at least one donor had IL-10 below the detection limit (8.6 pg / mL). The results are further explained in Example 4. [Figure 3C] The levels of IL-10 secreted by monocytes, analyzed by Luminex assay, are shown. The data shown are the mean values ​​from five donors. Monocytes purified from PBMCs were cultured for 6 hours in medium, vehicle, LPS, and S100A4 in the absence or presence of AX-202. The data show IL-10 cytokines in the supernatant, quantified by Luminex assay. The data are shown as the mean value + SEM obtained from five independent donors. "-" indicates that at least one donor had IL-10 below the detection limit (8.6 pg / mL). The results are further explained in Example 4. [Figure 3D] The levels of IL-10 secreted by monocytes, analyzed by Luminex assay, are shown. The data shown are the mean values ​​from five donors. Monocytes purified from PBMCs were cultured for 6 hours in medium, vehicle, LPS, and S100A4 in the absence or presence of 6B12. The data show IL-10 cytokines in the supernatant, quantified by Luminex assay. The data are shown as the mean value + SEM obtained from five independent donors. "-" indicates that at least one donor had IL-10 below the detection limit (8.6 pg / mL). The results are further explained in Example 4. [Figure 4A]This shows the amount of TNF-α secreted by monocytes as analyzed by Luminex assay. The data shown are the mean values ​​from five donors. Monocytes purified from PBMCs were cultured for 6 hours in medium, vehicle, LPS, and S100A4 in the absence or presence of mouse IgG1. The data show the TNF-α levels in the supernatant quantified by Luminex assay. The data are shown as the mean value + SEM obtained from five independent donors. "-" indicates that at least one donor had TNF-α levels below the detection limit (15.20 pg / mL). The results are further explained in Example 4. [Figure 4B] This shows the amount of TNF-α secreted by monocytes as analyzed by Luminex assay. The data shown are the mean values ​​from five donors. Monocytes purified from PBMCs were cultured for 6 hours in medium, vehicle, LPS, and S100A4 in the absence or presence of human IgG4. The data show the TNF-α levels in the supernatant quantified by the Luminex assay. The data are shown as the mean value + SEM obtained from five independent donors. "-" indicates that at least one donor had TNF-α levels below the detection limit (15.20 pg / mL). The results are further explained in Example 4. [Figure 4C] This shows the amount of TNF-α secreted by monocytes as analyzed by Luminex assay. The data shown are the mean values ​​from five donors. Monocytes purified from PBMCs were cultured for 6 hours in medium, vehicle, LPS, and S100A4 in the absence or presence of AX-202. The data show the TNF-α levels in the supernatant quantified by Luminex assay. The data are shown as the mean value + SEM obtained from five independent donors. "-" indicates that at least one donor had TNF-α levels below the detection limit (15.20 pg / mL). The results are further explained in Example 4. [Figure 4D]This shows the amount of TNF-α secreted by monocytes as analyzed by Luminex assay. The data shown are the mean values ​​from five donors. Monocytes purified from PBMCs were cultured for 6 hours in medium, vehicle, LPS, and S100A4 in the absence or presence of 6B12. The data show the TNF-α levels in the supernatant quantified by Luminex assay. The data are shown as the mean value + SEM obtained from five independent donors. "-" indicates that at least one donor had TNF-α levels below the detection limit (15.20 pg / mL). The results are further explained in Example 4. [Figure 5] The specificity of three humanized variants of 6B12mAb compared to other different S100 family members, as measured by Western blotting, is shown. The results are further explained in Example 5. [Figure 6] As demonstrated in wild-type (wt) and S100A4 knockout (ko) mouse embryonic fibroblasts (MEFs), the human variant is specific to the S100A4 protein and does not cross-react with random cellular proteins. Cells were counterstained with DAPI, and the actin cytoskeleton was counterstained with phalloidin. Parental monoclonal mouse anti-S100A4 antibody (6B12) was used as a control. Bar = 100 μm. Results are further described in Example 5. [Figure 7A] We demonstrate that AX-202 inhibits S100A4-induced TLR4 activation in a concentration-dependent manner. S100A4 activates the NF-κB reporter gene in a concentration-dependent manner in HEKBlue hTLR4 cells. The results are further explained in Example 6. [Figure 7B] We demonstrate that AX-202 inhibits S100A4-induced TLR4 activation in a concentration-dependent manner. S100A4 activation of the NF-κB reporter gene is TLR4-dependent. The results are further explained in Example 6. [Figure 7C] This demonstrates that AX-202 inhibits S100A4-inducible TLR4 activation in a concentration-dependent manner. The results are further explained in Example 6. [Figure 8A] This paper compares the FcγIIaR binding activity of 2 μg / ml 6B12 mIgG1, AX-202 hIgG1, and AX-202 hIgG4 when combined with a 2.5 μg / ml recombinant human S100A4 dimer. The control IgG1 or IgG4 antibodies did not mediate receptor clustering or activation. The results are further explained in Example 7. Bars indicate standard deviation. Results are presented as mean ± standard deviation, with n=3 independent tests. 0 μg / ml S100A4 (n=2) was used as a control. [Figure 8B] This paper compares the FcγIIaR binding activity of 2 μg / ml 6B12 mIgG1, AX-202 hIgG1, and AX-202 hIgG4 when combined with a 2.5 μg / ml recombinant human S100A4 polymer. The control IgG1 or IgG4 antibodies did not mediate receptor clustering or activation. The results are further explained in Example 7. Bars indicate standard deviation. Results are presented as mean ± standard deviation, with n=3 independent tests. 0 μg / ml S100A4 (n=2) was used as a control. [Modes for carrying out the invention]

[0023] definition As used herein, unless otherwise clearly indicated by the context, the singular forms "a," "an," and "the" include multiple references. Therefore, for example, a reference to "antibody" includes multiple such antibodies.

[0024] The S100A4 protein is also known as 18A2, 42A, CAPL, FSP1, MTS1, P9KA, PEL98, and S100 calcium-binding protein A4.

[0025] The term "isolated" refers, for example, to a compound that may be an antibody or antigen-binding moiety that substantially does not contain other antibodies or antigen-binding moieties having different antigen specificities. Furthermore, the antigen-binding moiety of an isolated antibody may not substantially contain other cellular material and / or chemical substances.

[0026] As defined herein, "operably ligated" means that the elements mentioned are bound as part of the same nucleic acid molecule and positioned in the appropriate location and orientation so that transcription is initiated from the promoter. DNA operably ligated to a promoter is either under the control of the promoter's transcription initiation or functionally combined with the promoter.

[0027] As used herein, the term “variant” refers to either a naturally occurring genetic mutant of a DNA sequence or the RNA or protein product that encodes it, or a variant prepared by recombinant techniques of a DNA sequence or the RNA or protein product that encodes it. The term “variant” may also refer to either a naturally occurring variant of a given peptide, or a variant prepared by recombinant techniques of a given peptide or protein in which one or more amino acid residues are modified by amino acid substitution, addition, or deletion.

[0028] As used herein, “inhibition” means that the presence of the antibody of the present invention, in whole or in part, inhibits the binding of a ligand to its receptor and / or neutralizes the signal that the receptor would induce upon ligand binding. This includes, for example, downstream signaling that affects cellular behavior and processes. It also includes other mechanisms that inhibit the downstream effects of a target molecule, such as blocking the dimerization, oligomerization, and / or polymerization of the target molecule. “Inhibition,” “blockage,” and “neutralization” are used herein as synonymous terms.

[0029] Isolated anti-S100A4 antibody molecule In one embodiment, an isolated antibody, a) i. Heavy chain complementarity determination region 1 (CDR-H1) containing or consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4, ii. Heavy chain complementarity determination region 2 (CDR-H2) containing or consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5, and iii. Heavy chain complementarity determination region 3 (CDR-H3) containing or consisting of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 6, A heavy chain variable (VH) region including, b) i. CDR-L1 containing or consisting of the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 10, ii. CDR-L2 containing or consisting of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 11, and iii. CDR-L3 containing or consisting of the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 12, A light chain variable (VL) region including, The VH region includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, and 17, and one variant of SEQ ID NOs: 13-17, wherein any one amino acid that is not part of the CDR sequence defined by SEQ ID NOs: 1-6 is modified to another amino acid (provided that no more than five amino acids are modified in this way, for example, 5, 4, 3, 2, or 1 amino acid is modified in this way in each amino acid sequence), and / or the VL region includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 20, 21, and 22, and one variant of SEQ ID NOs: 18-22, wherein any one amino acid that is not part of the CDR sequence defined by SEQ ID NOs: 7-12 is modified to another amino acid (provided that no more than five amino acids are modified in this way, e.g., 5, 4, 3, 2, or 1 amino acid are modified in this way in each amino acid sequence), Isolated antibodies are provided.

[0030] In some embodiments, the heavy chain variable (VH) region of the isolated antibody is as follows: i. Heavy chain complementarity determination region 1 (CDR-H1) containing or consisting of the amino acid sequence of SEQ ID NO: 1, ii. A heavy chain complementarity determination region 2 (CDR-H2) containing or consisting of the amino acid sequence of SEQ ID NO: 2, and iii. Containing the amino acid sequence of Sequence ID No. 3, or a heavy chain complementarity determination region 3 (CDR-H3) consisting thereof, The light chain variable (VL) region is as follows: i. Light chain complementarity determination region 1 (CDR-L1) containing or consisting of the amino acid sequence of Sequence ID No. 7, ii. Light chain complementarity determination region 2 (CDR-L2) containing or consisting of the amino acid sequence of Sequence ID No. 8, and iii. A light chain variable (VL) region containing the amino acid sequence of SEQ ID NO: 9, or a light chain complementarity determining region 3 (CDR-L3) consisting thereof.

[0031] In some embodiments, the heavy chain variable (VH) region of the isolated antibody is as follows: i. Heavy chain complementarity determination region 1 (CDR-H1) containing or consisting of the amino acid sequence of SEQ ID NO: 4, ii. Heavy chain complementarity determination region 2 (CDR-H2) containing or consisting of the amino acid sequence of SEQ ID NO: 5, and iii. Containing the amino acid sequence of SEQ ID NO: 6, or a heavy chain complementarity determining region 3 (CDR-H3) consisting thereof, The light chain variable (VL) region is as follows: i. Light chain complementarity determination region 1 (CDR-L1) containing or consisting of the amino acid sequence of SEQ ID NO: 10, ii. Light chain complementarity determination region 2 (CDR-L2) containing or consisting of the amino acid sequence of SEQ ID NO: 11, and iii. A light chain variable (VL) region containing the amino acid sequence of SEQ ID NO: 12, or a light chain complementarity determining region 3 (CDR-L3) consisting thereof.

[0032] In one embodiment, an isolated antibody, i. An amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, and 17, and a heavy chain variable (VH) region containing or consisting of one variant of SEQ ID NOs: 13-17, in which any one amino acid is modified to another amino acid (provided that five or fewer amino acids are modified in this way, for example, 5, 4, 3, 2, or 1 amino acid is modified in this way in each amino acid sequence), ii. A light chain variable (VL) region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs. 18, SEQ ID NOs. 19, SEQ ID NOs. 20, SEQ ID NOs. 21, and SEQ ID NOs. 22, and one variant of SEQ ID NOs. 18-22, wherein any one amino acid is modified to another amino acid (provided that five or fewer amino acids are modified in this way, for example, 5, 4, 3, 2, or 1 amino acid is modified in this way in each amino acid sequence), Isolated antibodies containing [the specified substance] are provided.

[0033] Therefore, in some embodiments, the heavy chain variable (VH) region of the antibody includes or consists of the amino acid sequence defined by SEQ ID NO: 13. In some embodiments, the heavy chain variable (VH) region of the antibody includes or consists of the amino acid sequence defined by SEQ ID NO: 14. In some embodiments, the heavy chain variable (VH) region of the antibody includes or consists of the amino acid sequence defined by SEQ ID NO: 15. In some embodiments, the heavy chain variable (VH) region of the antibody includes or consists of the amino acid sequence defined by SEQ ID NO: 16. In some embodiments, the heavy chain variable (VH) region of the antibody includes or consists of the amino acid sequence defined by SEQ ID NO: 17. In some embodiments, the heavy chain variable (VH) region of the antibody includes or consists of a variant of any one of the amino acid sequences defined by SEQ ID NOs: 13 to 17, wherein any one amino acid is modified to another amino acid (provided that five or fewer amino acids are modified in this way, e.g., 5, 4, 3, 2, or 1 amino acid are modified in this way in each amino acid sequence).

[0034] In some embodiments, the light chain variable (VL) region of the antibody includes or consists of the amino acid sequence defined by SEQ ID NO: 18. In some embodiments, the light chain variable (VL) region of the antibody includes or consists of the amino acid sequence defined by SEQ ID NO: 19. In some embodiments, the light chain variable (VL) region of the antibody includes or consists of the amino acid sequence defined by SEQ ID NO: 20. In some embodiments, the light chain variable (VL) region of the antibody includes or consists of the amino acid sequence defined by SEQ ID NO: 21. In some embodiments, the light chain variable (VL) region of the antibody includes or consists of the amino acid sequence defined by SEQ ID NO: 22. In some embodiments, the light chain variable (VL) region of the antibody includes or consists of a variant of any one of the amino acid sequences defined by SEQ ID NOs: 18 to 22, wherein any one amino acid is modified to another amino acid (provided that five or fewer amino acids are modified in this way, e.g., 5, 4, 3, 2, or 1 amino acid are modified in this way in each amino acid sequence).

[0035] In some embodiments, the antibody is a bispecific antibody.

[0036] In some embodiments, the antibody is a full-length antibody. In some embodiments, the antibody is a Fab fragment. In some embodiments, the antibody is an F(ab') fragment. In some embodiments, the antibody is an F(ab')2 fragment. In some embodiments, the antibody is scFv. In some embodiments, the antibody is a diabody. In some embodiments, the antibody is a triabody.

[0037] In some embodiments, the antibody is a human IgG1 immunoglobulin subclass antibody. In some embodiments, the antibody is a human IgG2 immunoglobulin subclass antibody. In some embodiments, the antibody is a human IgG3 immunoglobulin subclass antibody.

[0038] The inventors found that, compared to vehicle controls, S100A4-induced TNFα levels were not increased by mouse IgG1 or human IgG4 isotype controls, but 6B12 (mouse IgG1 anti-S100A4 antibody) significantly increased S100A4-induced TNFα levels, and this increase was not present with humanized IgG4 anti-S100A4 antibody (see Examples 4 and 7 for further details). In the present invention, it may be useful to use an antibody having an immunoglobulin subclass that induces a weak pro-inflammatory response in the host, or does not induce one at all. As shown, human IgG4 subclasses may be particularly useful when a reduction in the effector or crosslinking function of the antibody is desired. In some embodiments, the antibody is therefore a human IgG4 subclass antibody. In certain embodiments, the antibody is a human IgG4 subclass antibody having the HC sequence of SEQ ID NO: 58 and the LC sequence of SEQ ID NO: 59.

[0039] In some embodiments, the antibody includes a human heavy chain constant (CH) region comprising the sequence described in SEQ ID NO: 56, or a region comprising the same. In some embodiments, the antibody includes a variant of SEQ ID NO: 56, which has at least 80%, e.g., at least 81%, e.g., at least 82%, e.g., at least 83%, e.g., at least 84%, e.g., at least 85%, e.g., at least 86%, e.g., at least 87%, e.g., at least 88%, e.g., at least 89%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% sequence identity to the sequence, or includes a CH region comprising the same.

[0040] In some embodiments, the antibody includes a human light chain constant (CL) region comprising the sequence described in SEQ ID NO: 57, or a variant thereof. In some embodiments, the antibody includes a variant of SEQ ID NO: 57, which has at least 80%, e.g., at least 81%, e.g., at least 82%, e.g., at least 83%, e.g., at least 84%, e.g., at least 85%, e.g., at least 86%, e.g., at least 87%, e.g., at least 88%, e.g., at least 89%, e.g., at least 90%, e.g., at least 91%, e.g., at least 92%, e.g., at least 93%, e.g., at least 94%, e.g., at least 95%, e.g., at least 96%, e.g., at least 97%, e.g., at least 98%, e.g., at least 99% sequence identity to the sequence, or includes a CL region comprising such a variant.

[0041] In some embodiments, the antibody comprises an Fc domain having a mutant human IgG constant region. In some embodiments, the antibody comprises a mutant human IgG4 heavy chain constant region. In some embodiments, the mutant human IgG4 heavy chain constant region comprises an S228P substitution numbered according to EU numbering. The S228P substitution may interfere with IgG4 Fab-arm exchange in vivo and in vitro, resulting in a functionally monovalent bispecific antibody (bsAb) of unknown specificity, and therefore potentially reduced therapeutic efficacy. In some embodiments, the terminal lysine of the human IgG4 heavy chain constant region is removed.

[0042] The first humanized antibody was produced in 1986 by Greg Winter's laboratory in Cambridge, UK. Although this antibody showed moderately reduced affinity, the strategy of transplanting mouse CDRs into a human framework was considered successful. The next humanized antibody was the therapeutic antibody Campath-1, but this showed significantly reduced affinity, and it was at this point that the framework amino acids crucial for CDR stability and VH / VL interface stability began to be searched for for the first time.

[0043] Since the 1990s, the humanization of mouse antibodies has attracted considerable attention as a means of creating therapeutic agents tolerable for human use. While it was recognized from the early stages that framework amino acids play a crucial role in presenting CDRs in a manner favorable to antigen binding, there were no automated or standardized methods for identifying and determining which residues to reverse-mutate to successfully enhance antigen affinity. It is important to consider which positions are critical to the stability of the VL / VH interface and the frequency of each amino acid at a given position in similar antibody frameworks. Therefore, reverse mutations in frameworks may be useful for improving the affinity or stability of humanized antibodies against targets.

[0044] Therefore, in some embodiments, the antibody includes an amino acid substitution of the amino acid at position 40 of any one of the VH regions of SEQ ID NOs: 13-17 to phenylalanine. In some embodiments, the antibody includes an amino acid substitution of the amino acid at position 43 of any one of the VH regions of SEQ ID NOs: 13-17 to serine. In some embodiments, the antibody includes an amino acid substitution of the amino acid at position 44 of any one of the VH regions of SEQ ID NOs: 13-17 to lysine.

[0045] In some embodiments, the antibody includes an amino acid substitution of the amino acid at position 42 of any one of the VL regions of SEQ ID NOs. 18-22 to glycine. In some embodiments, the antibody includes an amino acid substitution of the amino acid at position 43 of any one of the VL regions of SEQ ID NOs. 18-22 to threonine. In some embodiments, the antibody includes an amino acid substitution of the amino acid at position 44 of any one of the VL regions of SEQ ID NOs. 18-22 to leucine.

[0046] In some embodiments, the VH region of the antibody includes or consists of the VH region defined by SEQ ID NO: 24. In some embodiments, the VH region of the antibody includes or consists of the VH region defined by SEQ ID NO: 25. In some embodiments, the VH region of the antibody includes or consists of the VH region defined by SEQ ID NO: 26. In some embodiments, the VH region of the antibody includes or consists of the VH region defined by SEQ ID NO: 27. In some embodiments, the VH region of the antibody includes or consists of the VH region defined by SEQ ID NO: 28. In some embodiments, the VH region of the antibody includes or consists of the VH region defined by SEQ ID NO: 29. In some embodiments, the VH region of the antibody includes or consists of the VH region defined by SEQ ID NO: 30. In some embodiments, the VH region of the antibody includes or consists of the VH region defined by SEQ ID NO: 31. In some embodiments, the VH region of the antibody includes or consists of the VH region defined by SEQ ID NO: 32. In some embodiments, the VH region of the antibody includes or consists of the VH region defined by SEQ ID NO: 33. In some embodiments, the VH region of the antibody includes or consists of the VH region defined by SEQ ID NO: 34. In some embodiments, the VH region of the antibody includes or consists of the VH region defined by SEQ ID NO: 35. In some embodiments, the VH region of the antibody includes or consists of the VH region defined by SEQ ID NO: 36. In some embodiments, the VH region of the antibody includes or consists of the VH region defined by SEQ ID NO: 37. In some embodiments, the VH region of the antibody includes or consists of the VH region defined by SEQ ID NO: 38.

[0047] In some embodiments, the VL region of the antibody includes or consists of the VL region defined by SEQ ID NO: 39. In some embodiments, the VL region of the antibody includes or consists of the VL region defined by SEQ ID NO: 40. In some embodiments, the VL region of the antibody includes or consists of the VL region defined by SEQ ID NO: 41. In some embodiments, the VL region of the antibody includes or consists of the VL region defined by SEQ ID NO: 42. In some embodiments, the VL region of the antibody includes or consists of the VL region defined by SEQ ID NO: 43. In some embodiments, the VL region of the antibody includes or consists of the VL region defined by SEQ ID NO: 44. In some embodiments, the VL region of the antibody includes or consists of the VL region defined by SEQ ID NO: 45. In some embodiments, the VL region of the antibody includes or consists of the VL region defined by SEQ ID NO: 46. In some embodiments, the VL region of the antibody includes or consists of the VL region defined by SEQ ID NO: 47. In some embodiments, the VL region of the antibody includes or consists of the VL region defined by SEQ ID NO: 48. In some embodiments, the VL region of the antibody includes or consists of the VL region defined by SEQ ID NO: 49. In some embodiments, the VL region of the antibody includes or consists of the VL region defined by SEQ ID NO: 50. In some embodiments, the VL region of the antibody includes or consists of the VL region defined by SEQ ID NO: 51. In some embodiments, the VL region of the antibody includes or consists of the VL region defined by SEQ ID NO: 52. In some embodiments, the VL region of the antibody includes or consists of the VL region defined by SEQ ID NO: 53.

[0048] In some embodiments, the VH region of the antibody includes or consists of SEQ ID NO: 24 (VH1_H40Phe), and the VL region of the antibody includes or consists of SEQ ID NO: 47 (VL3_L44Leu). In some embodiments, the VH region of the antibody includes or consists of SEQ ID NO: 24 (VH1_H40Phe), and the VL region of the antibody includes or consists of SEQ ID NO: 20 (VL3). In some embodiments, the VH region of the antibody includes or consists of SEQ ID NO: 26 (VH1_H44Lys), and the VL region of the antibody includes or consists of SEQ ID NO: 20 (VL3). In some embodiments, the VH region of the antibody includes or consists of SEQ ID NO: 13 (VH1), and the VL region of the antibody includes or consists of SEQ ID NO: 47 (VL3_L44Leu). In some embodiments, the VH region of the antibody includes or consists of SEQ ID NO: 13 (VH1), and the VL region of the antibody includes or consists of SEQ ID NO: 20 (VL3).

[0049] In some embodiments, the antibody is PEGylated.

[0050] Antibody function and therapeutic effect In some embodiments, the antibody according to the present invention can bind to the native three-dimensional structure of the S100A4 protein. In some embodiments, the antibody can bind to the dimeric form of the S100A4 protein. In some embodiments, the antibody can bind to the oligomeric form of the S100A4 protein. In some embodiments, the antibody can bind to the polymeric form of the S100A4 protein.

[0051] In some embodiments, the antibody can bind to a polypeptide having at least 80% sequence identity to amino acids 1-101 described in SEQ ID NO: 23 (Human S100A4). In some embodiments, the antibody can bind to a polypeptide having at least 85% sequence identity to amino acids 1-101 described in SEQ ID NO: 23. In some embodiments, the antibody can bind to a polypeptide having at least 90% sequence identity to amino acids 1-101 described in SEQ ID NO: 23. In some embodiments, the antibody can bind to a polypeptide having at least 95% sequence identity to amino acids 1-101 described in SEQ ID NO: 23. In some embodiments, the antibody can bind to the human S100A4 polypeptide of SEQ ID NO: 23.

[0052] In some embodiments, the antibody can neutralize the biological activity of S100A4. In some embodiments, the biological activity of S100A4 is in promoting tumor progression and / or inducing tumor metastasis.

[0053] In some embodiments, treatment with an anti-S100A4 antibody alleviates fibrosis. Therefore, in some embodiments, the antibody can alleviate S100A4-mediated fibrosis. Fibrosis is associated with dermal thickness, dermal hydroxyproline content, and dermal CD3 + Cell counts and / or dermal myofibroblast counts can be assessed by measuring them using methods known in the art. Accordingly, in some embodiments, treatment with anti-S100A4 antibodies reduces dermal thickness, dermal collagen or hydroxyproline content, dermal myoblast count, and / or T cell count.

[0054] In some embodiments, the antibody can inhibit the biological activity of S100A4 in promoting tumor progression and / or inducing tumor metastasis and / or inflammation.

[0055] In some embodiments, the antibody can inhibit S100A4-mediated T cell recruitment. In some embodiments, the antibody can inhibit S100A4-mediated macrophage recruitment and / or infiltration.

[0056] In some embodiments, the antibody can inhibit the biological activity of the S100A4 protein in response to stimulation of cell invasion. In some embodiments, the biological activity of the S100A4 protein in response to stimulation of cell invasion is measured in a 3D Matrigel matrix assay or a T cell invasion assay in which S100A4 stimulates T cell infiltration into a fibroblast monolayer. In some embodiments, the biological activity of S100A4 in inducing tumor metastasis is measured in an in vivo mouse xenograft model.

[0057] In some embodiments, the antibody has little or no effector function. In some embodiments, the antibody induces little or no binding, crosslinking, and / or activation of Fc receptor-dependent effector function in host cells.

[0058] Nucleic acids encoding antibodies and expression vectors In one embodiment, an isolated nucleic acid molecule encoding the antibody described in the “Isolated Anti-S100A4 Antibody Molecule” section of this specification is provided. In some embodiments, the nucleic acid molecule is codon-optimized for the cell in which it is expressed.

[0059] In one embodiment, an expression vector is also provided that includes a nucleic acid molecule described herein encoding an anti-S100A4 antibody molecule. In some embodiments, the nucleic acid molecule of the expression vector is operably ligated to a control sequence for directing its expression. Such a control sequence may include regulatory elements that can control the transcription of the sequence encoding the anti-S100A4 antibody molecule, such as a promoter (which may be activated by a transcription factor), an enhancer, or a silencer. In some embodiments, the translation of the mRNA encoding the anti-S100A4 antibody molecule may be controlled by different regulatory elements, such as a riboswitch. Suitable regulatory sequences and vectors are well known in the art.

[0060] Suitable techniques for producing, manipulating, and expressing nucleic acids in cells, such as mammalian cells, are well known to those skilled in the art.

[0061] Host cells containing antibodies In one embodiment, an isolated host cell is provided comprising an isolated nucleic acid molecule or expression vector as described in the “Nucleic Acids and Expression Vectors” section of this specification.

[0062] In some embodiments, the isolated host cells are human cells. In some embodiments, the isolated host cells are Chinese hamster ovary (CHO) cells.

[0063] The present invention can also be used in conjunction with ex vivo gene therapy, in which an expression vector encoding the antibody disclosed herein is transfected or transduced in vitro into patient cells. After transfection, the cells are returned to the patient to express and secrete the antibody. Suitable donor cells for ex vivo gene therapy include T cells.

[0064] Method for producing antibodies as described herein In one embodiment, a method for producing an anti-S100A4 antibody molecule is provided, comprising culturing a host cell as described in the “host cell containing the antibody” section of this specification under conditions in which the antibody is expressed.

[0065] In some embodiments, the method further comprises purifying the antibody and isolating the anti-S100A4 antibody thus produced.

[0066] Pharmaceutical composition In one embodiment, a pharmaceutical composition is provided comprising the antibody described in the “Isolated Anti-S100A4 Antibody Molecule” section of this specification, the nucleic acid molecule and / or expression vector described in the “Nucleic Acid and Expression Vector Encoding an Antibody” section of this specification, and / or the host cell described in the “Host Cell Containing an Antibody” section of this specification, and a pharmaceutically acceptable diluent, carrier, and / or excipient.

[0067] Treatment method In one embodiment, a method for treating an individual having an S100A4-mediated pathological condition is also provided, comprising administering an antibody as described in the “Isolated Anti-S100A4 Antibody Molecule” section of this specification, a nucleic acid molecule and / or expression vector as described in the “Nucleic Acid and Expression Vector Encoding an Antibody” section of this specification, or a host cell as described in the “Host Cell Containing an Antibody” section of this specification, to an individual in need thereof.

[0068] In some embodiments, the S100A4-mediated pathology is a fibrotic pathology.

[0069] In some embodiments, the fibrotic pathology is systemic sclerosis. In some embodiments, the fibrotic pathology is cutaneous fibrosis. In some embodiments, the fibrotic pathology is interstitial pulmonary fibrosis. In some embodiments, the fibrotic pathology is hepatic fibrosis. In some embodiments, the fibrotic pathology is renal fibrosis.

[0070] Combining treatment with an anti-S100A4 antibody with treatment with other compounds useful for treating systemic sclerosis may be beneficial. Therefore, in some embodiments, the antibody is co-administered with another compound for the treatment of systemic sclerosis. In some embodiments, the antibody is co-administered with an angiotensin-converting enzyme inhibitor. In some embodiments, the antibody is co-administered with an angiotensin receptor blocker. In some embodiments, the antibody is co-administered with azathioprine. In some embodiments, the antibody is co-administered with a calcium channel blocker. In some embodiments, the antibody is co-administered with cyclophosphamide. In some embodiments, the antibody is co-administered with hydroxychloroquine. In some embodiments, the antibody is co-administered with mycophenolate. In some embodiments, the antibody is co-administered with methotrexate. In some embodiments, the antibody is co-administered with glucocorticoids. In some embodiments, the antibody is co-administered with phosphodiesterase-5 inhibitors. In some embodiments, the antibody is co-administered with endothelin receptor antagonists. In some embodiments, the antibody is co-administered with an alpha-blocker. In some embodiments, the antibody is co-administered with a prostanoid. In some embodiments, the antibody is co-administered with rituximab. In some embodiments, the antibody is co-administered with a tyrosine kinase inhibitor such as nintedanib. In some embodiments, the antibody is co-administered with tociluzimab.

[0071] In some embodiments, the S100A4-mediated condition is an inflammatory condition. In some embodiments, the inflammatory condition is psoriasis. In some embodiments, the inflammatory condition is rheumatoid arthritis. In some embodiments, the inflammatory condition is inflammatory myopathy.

[0072] In some embodiments, the S100A4-mediated disease is cancer. In some embodiments, the cancer is metastatic cancer.

[0073] In some embodiments, the cancer is gastric cancer. In some embodiments, the cancer is pancreatic cancer. In some embodiments, the cancer is colorectal cancer. In some embodiments, the cancer is thyroid cancer. In some embodiments, the cancer is breast cancer. In some embodiments, the cancer is squamous cell carcinoma. In some embodiments, the cancer is non-small cell lung cancer. In some embodiments, the cancer is prostate cancer. In some embodiments, the cancer is lung cancer. In some embodiments, the cancer is head and neck cancer. In some embodiments, the cancer is brain cancer (including glioblastoma multiforme). In some embodiments, the cancer is renal cell carcinoma (including clear cell renal carcinoma). In some embodiments, the cancer is melanoma. In some embodiments, the cancer is lymphoma. In some embodiments, the cancer is plasmacytoma. In some embodiments, the cancer is sarcoma. In some embodiments, the cancer is glioma. In some embodiments, the cancer is thymoma. In some embodiments, the cancer is leukemia. In some embodiments, the cancer is colon cancer. In some embodiments, the cancer is esophageal cancer. In some embodiments, the cancer is ovarian cancer. In some embodiments, the cancer is cervical cancer. In some embodiments, the cancer is hepatocellular carcinoma.

[0074] In some embodiments, antibodies, nucleic acid molecules, expression vectors, and / or host cells are administered by parenteral administration. Therefore, in some embodiments, antibodies, nucleic acid molecules, expression vectors, and / or host cells are administered subcutaneously. In some embodiments, antibodies, nucleic acid molecules, expression vectors, and / or host cells are administered intramuscularly. In some embodiments, antibodies, nucleic acid molecules, expression vectors, and / or host cells are administered intravenously.

[0075] In some embodiments, antibodies, nucleic acid molecules, expression vectors, and / or host cells are administered once a week or less. In some embodiments, antibodies are administered weekly, with weekly doses ranging from 15 mg to 1000 mg.

[0076] Methods of diagnosis or prognosis In one embodiment, a method for diagnosing or prognosing S100A4-related pathological conditions in an individual, (a) Contacting a biological sample from an individual with an anti-S100A4 antibody described herein that can bind to the S100A4 polypeptide present in the sample, (b) Measuring the presence and / or amount of the complex formed between the antibody molecule and the S100A4 polypeptide, A method including this is provided.

[0077] In some embodiments, the biological sample is blood. In some embodiments, the biological sample is plasma. In some embodiments, the biological sample is serum. In some embodiments, the biological sample is a tissue sample. In some embodiments, the biological sample is interstitial fluid. In some embodiments, the biological sample is saliva. In some embodiments, the biological sample is cerebrospinal fluid. In some embodiments, the biological sample is synovial fluid. [Examples]

[0078] Example 1 - Humanization of mouse anti-S100A4 antibody The mouse monoclonal IgG1 anti-S100A4 antibody 6B12 (the VH and VL regions are defined in SEQ ID NO: 54 and SEQ ID NO: 55, respectively) was humanized.

[0079] Sequence analysis and alignment of humanized variants The CDRs in the VH and VL regions of 6B12 were identified using the IMGT and Kabat antibody numbering systems. These two numbering systems identified different residues in the mouse antibody as belonging to the CDR, and the IMGT / Kabat CDR sequences were used in combination to optimally preserve the CDR-loop structure.

[0080] The V region of the human germline gene closest to the VH region was identified as Homo sapiens IGHV4-34*09. A database of human IgG sequences was searched using the BLAST search algorithm for comparison with mouse VH domains, and candidate human variable domains were selected from the top 200 BLAST results. These were narrowed down to four candidates each based on framework homology, maintained major framework residues, and combinations of standard loop structures. For the fifth acceptor sequence, the closest human germline IGHV4-34*09 was selected.

[0081] The human germline gene V region closest to the VL region was identified as Homo sapiens IGKV1-27. Similarly, a database of human IgK sequences was searched using the BLAST search algorithm for comparison with mouse VL domains, and candidate human variable domains were selected from the top 200 BLAST results. These were narrowed down to four candidates based on framework homology, the combination of maintained major framework residues, and standard loop structure. For the fifth acceptor sequence, the closest human germline IGKV1-27 was selected.

[0082] Next, mouse VH and VL CDRs were grafted onto these acceptor frameworks to obtain five humanized VH variants VH1-VH5 (sequence codes 13-17) and five humanized VL variants VL1-VL5 (sequence codes 18-22).

[0083] Confirmation of humanization Humanized variants were verified to determine whether they were humanized according to the WHO definition of humanized antibodies. The variable domain of the humanized chain contains amino acid sequences in the V region that are closer to those of humans than those of other species when analyzed as a whole (evaluated using the Immunogenetics Information System (IMGT) DomainGapAlign tool).

[0084] All variants were classified as humanized according to the WHO definition of humanized antibodies.

[0085] Heavy chain and light chain framework reverse mutation To improve the affinity of humanized antibodies for their targets, the VL and VH regions were analyzed to obtain superior candidates for framework reverse mutations.

[0086] This analysis was performed by examining the frequency of amino acids at each position in the mouse antibody, particularly by searching for amino acids with very low frequencies, such as those occurring at less than 1%. Further evaluation was conducted to determine if these amino acids might be in structurally important positions, and these amino acids were considered potential candidates for framework reverse mutations.

[0087] In the VL region, L44(L) was identified as abnormal at this position (frequency less than 1%). This position has been shown to be important at the VL / VH interface. In the parental germline, this position is likely valine. Most often, this position is proline. This was considered a good candidate for a reverse mutation. The preceding amino acid, L43(T), was also considered worth retaining as a framework reverse mutation, but this sequence was shown to be present in one of the five humanized sequences. In the nearest human germline sequence, this was identified as alanine or valine. Similarly, L42(G) was also considered a good position for a framework reverse mutation.

[0088] In the VH region, the amino acid H43(S) is a crucial residue at the VH / VL interface, and since this position is typically lysine, glutamine, or arginine, it was considered an excellent candidate for framework reverse mutation. Similarly, H40(F) and H44(K) also had a frequency of 1%, making them excellent locations for framework reverse mutation. Although these were not strictly defined as important positions, they were considered excellent candidates because they were directly adjacent.

[0089] The results of framework reverse mutations regarding the affinity of humanized antibodies for S100A4 are further described in Example 2.

[0090] Example 2 - Dynamic analysis of the interaction between anti-S100A4 antibody and S100A4 Using surface plasmon resonance (SPR), we performed quantitative kinetic analysis of the interactions between 14 humanized antibodies and 2 control antibodies with human S100A4 dimers.

[0091] Materials and methods The analysis was performed using a Biacore T200 instrument, with quantitative kinetic interaction analysis at an analysis temperature of 25°C and a flow rate of 50 μl / min.

[0092] Analytical buffer: 10 mM HEPES pH 7.4, 300 mM NaCl, 1 mM CaCl2, 100 μM EDTA, 0.05% Tween20

[0093] Assay cycle: 1. Preparation of anti-His trapping surface 2. Reversible capture of antigen (S100A4) 3. Quantitative analysis of antibodies in MCK mode (0.78-200 nM) 4. Complete removal of antibody-antigen complexes from the capture surface.

[0094] CM4 sensor chip #2: fc1:6878RU standard fc2:6515RU Capture antigen (S100A4) fc3:6804RU fc4:6540RU

[0095] In this experiment, 14 humanized antibodies were analyzed, some of which had reverse mutations in specific amino acids in the VL and / or VH framework regions. Each antibody contained one of the following heavy chain variable regions (VH): VH0 (Sequence ID 54) VH1 (Sequence ID 13) VH1_H40Phe (Sequence ID 24) VH1_H43Ser(Sequence ID 25) VH1_H44Lys (Sequence ID 26)

[0096] Each antibody contained one of the following light chain variable regions (VLs): VL0: (Sequence ID 55) VL3 (Sequence ID 20) VL3_L42Gly (Sequence ID 45) VL3_L43Thr (Sequence ID 46) VL3_L44Leu (Sequence ID 47) VL3_L42Gly+L44Leu

[0097] Two types of control antibodies were also analyzed. 6B12: Mouse monoclonal IgG1 anti-S100A4 as described in Example 1 VH0 VL0: The VH and VL regions of 6B12 included in the humanized IgG4 isotype framework.

[0098] Results and Conclusions The results of the analysis are shown in Table 1 below.

[0099] [Table 1] All 14 antibodies tested had similar dissociation constants to the 6B12 antibody and showed high binding affinity to S100A4. However, it was clear that some specific framework reverse mutations in the humanized light or heavy chain had an improving effect on the dissociation constant. For example, VH1 VL3 has a K value of 3.47E-09. dWhile this was shown, it improved to 2.25E-09 in VH1 VL3_L44Leu and to 1.99E-09 in VH1 VL3_L42Gly+L44Leu. Therefore, some of these manually designed framework reverse mutations were successful in increasing antibody affinity against S100A4. However, it is important to note that not all framework reverse mutations increased antibody affinity against S100A4. For example, VH1 VL3_L43Thr only increased K to 4.72E-09. d That's all it showed.

[0100] Example 3 - Efficacy of humanized anti-S100A4 antibody in the treatment of bleomycin-induced dermatofibrosis in vivo Systemic sclerosis (SSc) is a rare systemic fibrotic disease with high morbidity and mortality. SSc is the autoimmune rheumatic disease with the highest case-specific mortality rate, with the majority of cases diagnosed ultimately resulting in death. The disease is characterized by the accumulation of extracellular matrix proteins by pathologically activated fibroblasts. To date, no therapeutic approach selectively inhibiting the abnormal release of extracellular matrix in SSc is available. Bleomycin-induced dermatofibrosis is the most commonly used mouse model of SSc, particularly its resemblance to the early inflammatory phase of SSc. This study aimed to evaluate the efficacy of a humanized S100A4 antibody in bleomycin-induced dermatofibrosis.

[0101] Materials and methods Antibody stock solution The humanized IgG4 monoclonal anti-S100A4 antibody AX-202 was used in this study. This antibody contained the heavy chain sequence defined in SEQ ID NO: 58 and the light chain sequence defined in SEQ ID NO: 59. The antibody was dissolved in PBS and stored at -20°C.

[0102] Test animals The experiment was conducted using C57Bl / 6 mice. Number of animals: 64 Early death: None Total testing period for each animal: 6 weeks

[0103] Treatment protocol A 2 mg / mL antibody stock solution was diluted with sterile PBS and administered by intraperitoneal injection in a volume of 100 μL.

[0104] Bleomycin (2.5 mg / kg, Sigma-Aldrich) is applied to the designated marked area (1 cm) on the upper back. 2 Dermatofibrosis was induced by daily subcutaneous injections of bleomycin for up to 6 weeks. Treatment was initiated 3 weeks after the prior administration of bleomycin, with either twice-weekly intraperitoneal injections (ip) or once-weekly intravenous injections (IV) into the tail vein. Results were analyzed 3 weeks after the first bleomycin injection (6 weeks after the first bleomycin injection).

[0105] Test design and test group The control group consisted of 8 mice, while the group treated with humanized antibodies consisted of 10 mice.

[0106] The following experimental groups were used: Group 1: Control / NaCl Group 2: Bleomycin for 3 weeks and NaCl for 3 weeks Group 3: Bleomycin for 6 weeks + NaCl for the last 3 weeks (intraperitoneal administration every 3 days) Group 4: Bleomycin for 6 weeks + AX-202 16 mg / kg for the last 3 weeks (administered intraperitoneally twice a week) Group 5: Bleomycin for 6 weeks + AX-202 24 mg / kg for the last 3 weeks (intravenous injection into the tail vein weekly) Group 6: Bleomycin for 6 weeks + AX-202 8 mg / kg for the last 3 weeks (administered intraperitoneally twice a week)

[0107] Implementation of the test The mice's behavior, activity level, fur texture, and stool consistency were clinically monitored daily. After sacrifice, the lungs and skin were visually evaluated.

[0108] Quantitative determination of subcutaneous thickening After euthanasia due to cervical dislocation, a 1 cm² skin sample was collected from a designated area on the upper back between the scapulae. The skin lesion area was excised, fixed in 4% formalin for 6 hours, and embedded in paraffin. 5 μm sections were cut and stained with hematoxylin and eosin. Dermal thickness was measured at 100x magnification by measuring the distance between the epidermal-dermal junction and the dermal-subcutaneous fat junction at three locations on the skin lesion area of ​​each mouse.

[0109] Detection of myofibroblasts Myofibroblasts are characterized by the expression of α-smooth muscle actin (αSMA). αSMA-positive fibroblasts were detected in paraffin-embedded slides from the upper back by incubation with a monoclonal anti-αSMA antibody (clone 1A4, Sigma-Aldrich, Steinheim, Germany). Expression was visualized with horseradish peroxidase-labeled secondary antibody and 3,3-diaminobenzidine tetrahydrochloride (DAB) (Sigma-Aldrich). Monoclonal mouse IgG antibody (Calbiochem, San Diego, CA, USA) was used as a control. Analysis was performed by a blinded examiner who evaluated myofibroblasts in four sections per sample.

[0110] Hydroxyproline assay The amount of collagen protein in skin samples was measured by a hydroxyproline assay. Punch biopsies (3 mm in diameter) of the total skin thickness, taken from the upper back, were digested in 6 M HCl at 120°C for 3 hours, and then the pH of the samples was adjusted to 6 with 6 M NaOH. Next, 0.06 M chloramine T was added to each sample, and the samples were incubated at room temperature for 20 minutes. Then, 3.15 M perchloric acid and 20% p-dimethylaminobenzaldehyde were added, and the samples were incubated at 60°C for a further 20 minutes. Absorbance was measured at 557 nm using a Spectra MAX 190 microplate spectrophotometer.

[0111] Statistical values All data are expressed as mean ± SEM, and statistical significance of differences between groups was tested using a one-way ANOVA with graph pad8. A p-value less than 0.05 was considered statistically significant. P-values ​​are expressed as follows: when compared to control mice injected with NaCl for 6 weeks, 0.05>p>0.01 is *, 0.01>p>0.001 is **, and p<0.001 is ***; when compared to mice injected with bleomycin for 3 weeks followed by NaCl injection for another 3 weeks, 0.05>p>0.01 is #, 0.01>p>0.001 is ##, and p<0.001 is ###.

[0112] result Dermatofibrosis mouse model When bleomycin was administered, mice developed marked dermatofibrosis. Mice administered bleomycin for 6 weeks showed more pronounced fibrotic changes compared to mice administered bleomycin for 3 weeks and then injected with NaCl, the solvent for bleomycin, for an additional 3 weeks. Mice injected with NaCl for 6 weeks were used as a control.

[0113] General tolerability Treatment with the anti-S100A4 antibody AX-202 was well-tolerated, with no apparent signs of toxicity observed in clinical examinations, macroscopic autopsies, or histological examinations.

[0114] Results of effectiveness Treatment with AX-202 significantly reduced dermal thickness, myofibroblast count, and hydroxyproline content compared to control mice injected with bleomycin for 6 weeks (see Figure 1). The effect was dose-dependent, with the most pronounced effects observed at intraperitoneal doses of 16 mg / kg and weekly intravenous doses of 24 mg / kg (see Figures 1A-C). However, a statistically significant effect of AX-202 was also observed at intraperitoneal administration of 8 mg / kg every 3 days. At intraperitoneal doses of 16 mg / kg and intravenous doses of 24 mg / kg, AX-202 statistically significantly altered dermal thickness and myofibroblast count, and also induced regression of fibrosis, compared to mice injected with bleomycin alone for 3 weeks.

[0115] conclusion Treatment with AX-202 resulted in potent improvement of skin thickening, myofibroblast count, and hydroxyproline levels in bleomycin-induced dermatofibrosis at well-tolerated doses.

[0116] Example 4 - Effect on S100A4-induced cytokine release in vitro Materials and methods Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donors by density centrifugation using FicollPaque PLUS (GE Healthcare; 11778538), and monocytes were isolated using a monocyte isolation kit (StemCell Technologies; 19359).

[0117] Monocytes were plated into 96-well plates (100,000 cells / well) and cultured for 6 hours in the presence of the following: culture medium, Vehicle 1 (TBS with 0.074%) LPS (1.0ng / ml; Invivogen; tlrl-b5lps), S100A4 (2.0 μg / ml), S100A4 (2.0 μg / ml) + Vehicle 2 (3.2% PBS) Vehicle 1 + mouse IgG1 (Biolegend; 401407), Vehicle 1 + Human IgG4 (Biolegend; 403701), Vehicle 1 + AX-202 or 6B12 (32 μg / ml each), S100A4 (2.0 μg / ml) + mouse IgG1 (8.0, 16, or 32 μg / ml), S100A4 (2.0 μg / ml) + Human IgG4 (8.0, 16, or 32 μg / ml), S100A4 (2.0 μg / ml) + AX-202 (8.0, 16, or 32 μg / ml), or S100A4 (2.0 μg / ml) + 6B12 (8.0, 16, or 32 μg / ml).

[0118] 6B12: Mouse monoclonal IgG1 anti-S100A4 antibody as described in Example 1 AX-202: Humanized monoclonal IgG4 anti-S100A4 antibody as described in Example 3

[0119] After 6 hours of incubation, the cell culture supernatant was collected and stored at -20°C for subsequent cytokine analysis. The levels of cytokines (IL-6, TNF-α, and IL-10) in the supernatant were quantified by the Luminex assay according to the manufacturer's instructions (R&D systems; LXSAHM-03).

[0120] result Compared to vehicle controls, monocyte stimulation with either LPS (1.0 ng / ml; positive control) or S100A4 (2.0 μg / ml) induced increases in IL-6, TNFα, and IL-10 levels as measured in cell culture supernatant (see Figures 2 and 3). Compared to isotype controls, AX-202 or 6B12 reduced the S100A4-induced increases in IL-6 and IL-10 at all concentrations tested (see Figures 2C-D and 3C-D). Compared to vehicle controls, S100A4-induced TNFα levels were not increased by mouse IgG1 or human IgG4 isotype controls (see Figures 4A-B), but 6B12, rather than AX-202, significantly increased S100A4-induced TNFα levels, and the increase induced by 6B12 showed a dose-dependent trend (see Figures 4C-D).

[0121] conclusion As expected, S100A4 induced increases in the levels of IL-6, TNFα, and IL-10 (see Figures 2–4). The IL-6 and IL-10 releases induced by S100A4 were reduced by both AX-202 and 6B12 (see Figures 2C–D and 3C–D).

[0122] Importantly, combining S100A4 with mouse IgG1 control, human IgG4 control, or AX-202 did not result in a significant increase in TNFα levels compared to S100A4 alone (see Figures 4A-B). In contrast, levels of TNFα, a pro-inflammatory cytokine induced by S100A4, increased in a dose-dependent manner with treatment with the 6B12 antibody (see Figures 4C and 4D).

[0123] Surprisingly, this indicates that the humanized anti-S100A4 IgG4 antibody does not increase S100A4-induced pro-inflammatory TNFα levels compared to the mouse anti-S100A4 antibody 6B12.

[0124] Although the aforementioned inventions have been described in some detail as illustrative examples for clearer understanding, these descriptions and examples should not be construed as limiting the scope of the invention.

[0125] The foregoing specification is deemed sufficient to enable a person skilled in the art to implement the present disclosure. However, it will be understood that, despite how detailed the foregoing is in the text, the present disclosure can be implemented in many ways, and the present disclosure should be interpreted in accordance with the attached claims and equivalents.

[0126] Example 5 - The humanized anti-S100A4 antibody does not cross-react with other S100 family members and is specific to S100A4 from multiple species.

[0127] The ability of different humanized variants (VH1_40Phe:VL3, VH1:VL3_Leu44, and VH1_40Phe:VL3_Leu44) to selectively react with S100A4 was tested by Western blotting. All variants detected mouse and human S100A4 protein and did not cross-react with other S100 family members (see Figure 5).

[0128] To confirm the specificity of the same human variant that recognizes the S100A4 protein, and to rule out the possibility that the variant does not cross-react with random cell proteins, fluorescence microscopy experiments were performed using wild-type (wt) and S100A4 knockout (ko) mouse embryonic fibroblasts (MEFs). IF staining revealed that all three humanized variants of 6B12 (Phe40, Leu44, and Phe40 / Leu44) did not cross-react with the knockout fibroblast proteins, but wild-type MEFs showed typical perinuclear cytoplasmic staining for S100A4 (see Figure 6).

[0129] These results are consistent with previous findings that 6B12 recognizes only S100A4 (Klingelhofer et al., 2012). Furthermore, no cross-reactivity was observed between S100A2, which has the most common homology to the epitope sequence, and S100A5, which exhibits the highest overall homology among all members of the S100 family.

[0130] The AX-202 described in Example 3 was shown to bind to S100A4 with similar affinity across four different species (monkeys, rats, humans, and mice).

[0131] Example 6 - Humanized anti-S100A4 antibody reduces activation of the S100A4-induced inflammatory pathway.

[0132] HEK-Blue hTLR4 (InvivoGen, #hkb-htlr4) cells were incubated in HEK-Blue detection medium (InvivoGen, #hb-det2) and stimulated with incremental concentrations of recombinant human S100A4 multimer or 1.25 ng / ml of LPS-EK ultrapure (positive control; InvivoGen, #tlrl-peklps). After 20 hours of incubation, NF-κB-inducible secreted embryonic alkaline phosphatase (SEAP) levels were measured by reading the OD at 620 nm. S100A4 was shown to clearly activate the NF-κB reporter gene in a concentration-dependent manner in HEKBlue hTLR4 cells (see Figure 7A).

[0133] HEK-Blue hTLR4 and HEK-Blue Null2 (control, InvivoGen, #hkb-null2) cells were incubated in HEK-Blue detection medium and stimulated with 1.25 ng / ml LPS-EK ultrapure (InvivoGen, #tlrl-peklps) or either 1.25 or 2.5 μg / ml S100A4. Cells treated with 50 ng / ml TNF-α (InvivoGen, #rcyc-htnfa) were used as a positive control for HEK-Blue Null2 cell activation. S100A4 activation of the NF-κB reporter gene was shown to be TLR4-dependent, as S100A4 addition completely suppressed reporter gene activation in Null2 cells (see Figure 7B).

[0134] HEK-Blue hTLR4 cells were incubated in HEK-Blue detection medium and stimulated with 1.25 μg / ml of S100A4 alone and with gradually increasing concentrations of the S100A4 neutralizing antibody AX-202 (described in Example 3). Reporter gene LPS activation was not affected by AX-202, but AX-202 was shown to inhibit S100A4-induced TLR4 activation in a concentration-dependent manner (see Figure 7C).

[0135] Example 7 - Changes in FcR-mediated effector function by IgG subclass switching of humanized antibodies The inventors were particularly interested in whether subclass switching could affect the activation of FcγRIIa, and which of these could affect the therapeutic effect. FcγRIIa is an activated FcγR, which has low affinity for a single "monomer" IgG molecule but high binding affinity to ICs containing IgG (Arman et al., 2015).

[0136] In leukocytes, FcγRIIA binding initiates potent effector functions that are key to immune and inflammatory responses, including cytokine release and ADCC (antibody-dependent cell-mediated cytotoxicity), which may adversely affect the safety profile of antibody drugs. Furthermore, in human platelets, FcγRIIa is involved in heparin-induced thrombocytopenia, which is a well-documented pro-thrombotic drug side effect (Sun et al., 2013).

[0137] This study aims to investigate whether the subclass shift from mouse IgG1 to human IgG4 of the parental 6B12 antibody AX-202 affects the binding affinity to FcγRIIa. A lower FcγRIIA binding affinity of the antibody-immune complex may indicate a better safety profile.

[0138] Materials and methods FcYRIIA Reporter Assay Three-fold concentrations of the final antigen (S100A4) and three-fold concentrations of the final antibody were prepared in assay buffer, and 25 μl of each was added to the appropriate wells of a 96-well plate and incubated at 37°C for 15–25 minutes. A suitable number of cells were thawed and transferred to assay buffer. 50,000 cells were seeded per well into 25 μl of assay buffer, resulting in a final volume of 75 μl / well. The plate was covered with a lid and incubated at 37°C and 5% CO2 for 18 hours. After incubation, the assay plate was removed from the incubator and equilibrated at ambient temperature (22–25°C) for 15 minutes. 50 μl of Bio-Glo® reagent was added to each assay plate and incubated at room temperature for 15 minutes. Luminescence was measured using a luminescence plate reader (see protocol section 10.01).

[0139] Analysis of results The data points were imported into Prism Graph Pad (version 9.1.0) and analyzed using an independent t-test (two-tailed test). An asterisk indicates a significant association when P < 0.05 based on n=3.

[0140] biological activity All assays were performed using negative and positive control cells to compare receptor activation. All positive and negative controls showed the expected results.

[0141] result Changes in FcR-mediated effector function due to IgG subclass switching Previous results have shown that AX-202, which possesses mouse IgG1 6B12 and human IgG4 scaffolds, can inhibit S100A4-induced cytokine release in monocytes. Stimulation of monocytes with S100A4 induced levels of IL-6, TNFα, and IL-10 in the cell culture supernatant. Both AX-202 and 6B12 blocked the increase in IL-6 and IL-10 induced by S100A4 stimulation, but unexpectedly induced an increase in TNFα. This suggests that an immune complex composed of oligomeric S100A4 and antibody (6B12) triggers TNFα release via FcγR.

[0142] Therefore, the selection of IgG subclasses can significantly impact unwanted cytokine release. To investigate this possibility, we performed an FcγRIIA reporter assay to evaluate how strongly different antibodies induce FcγR-dependent promoter activity.

[0143] IgG class switching from human IgG1 to IgG4 significantly reduces antigen-specific FcγRIIa activation.

[0144] The Promega FcγRIIa-H ADCP reporter bioassay was used to measure the differences in antibody subtypes in the potency of antibody-antigen complexes that activate FcγRIIa. This assay consisted of human FcγRIIa-H (high-affinity H131 variant) and human Jurkat cells stably expressing NFAT-induced luciferase. The test included two different AX-202 antibodies with either class 1 or 4 hIgG. In addition, the parental mouse antibody 6B12 IgG1 was also included. The results are shown in Figure 8. Immune complexes (ICs) were formed by mixing the antibody with either dimeric S100A4 protein (A) or multimeric S100A4 protein (B).

[0145] Receptor clustering was significantly higher with AX-202 hIgG1 compared to AX-202 hIgG4, with a factor of 5.00 and 3.83 difference in receptor activation between S100A4-D and S100A4-M, respectively (see Figure 8). 6B12, possessing a mouse IgG1 scaffold, had a lower ability to induce IC-dependent FcγRIIa than its human counterpart (AX-202 hIgG1). However, 6B12 still showed 3.23-fold (dimer) and 2.87-fold (multimer) higher activation than AX-202 hIgG4. Since neither the IgG1 nor IgG4 control antibody mediated receptor clustering, antibody-antigen IC formation is shown to be important for FcγRIIa clustering and activation.

[0146] conclusion Antibodies have recently been found to mediate inflammation and immunomodulation by inducing cell differentiation, activation, and cytokine release (A van Erp et al., 2019). This can lead to the activation of undesirable pro-inflammatory pathways and potentially counteract the mechanism of action of therapeutic S100A4 antibodies. In this specification, we have found that an S100A4 neutralizing antibody with an IgG4 scaffold has a far superior safety profile and approximately one-third the potency in inducing FcγR activation compared to the same antibody with a human IgG1 scaffold.

[0147] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6] [Table 2-7] [Table 2-8] [Table 2-9] [Table 2-10] [Table 2-11] [Table 2-12] [Table 2-13]

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Claims

1. Isolated antibodies, a) i. Heavy chain complementarity determination region 1 (CDR-H1) containing or consisting of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4, ii. Heavy chain complementarity determination region 2 (CDR-H2) containing or consisting of the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5, and iii. Heavy chain complementarity determination region 3 (CDR-H3) containing or consisting of the amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 6, A heavy chain variable (VH) region including, b) i. CDR-L1 containing or consisting of the amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 10, ii. CDR-L2 containing or consisting of the amino acid sequence of SEQ ID NO: 8 or SEQ ID NO: 11, and iii. CDR-L3 containing or consisting of the amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 12, A light chain variable (VL) region including, The VH region includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, and 17, and one variant of SEQ ID NOs: 13 to 17, wherein any one amino acid that is not part of the CDR sequence defined by SEQ ID NOs: 1 to 6 is modified to another amino acid (provided that five or fewer amino acids are modified in this way, for example, five, four, three, two, or one amino acid are modified in this way in each amino acid sequence). and / or the VL region includes or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 20, 21, and 22, and one variant of SEQ ID NOs: 18 to 22, wherein any one amino acid that is not part of the CDR sequence defined by SEQ ID NOs: 7 to 12 is modified to another amino acid (provided that five or fewer amino acids are modified in this way, for example, five, four, three, two, or one amino acid are modified in this way in each amino acid sequence). The isolated antibody mentioned above.

2. The heavy chain variable (VH) region is (a) Heavy chain complementarity determination region 1 (CDR-H1) containing or consisting of the amino acid sequence of Sequence ID No. 1, (b) Heavy chain complementarity determination region 2 (CDR-H2) containing or consisting of the amino acid sequence of SEQ ID NO: 2, and (c) comprising the amino acid sequence of SEQ ID NO: 3, or comprising a heavy chain complementarity determining region 3 (CDR-H3) consisting thereof, The light chain variable (VL) region, which includes the light chain variable (VL) region, i. Light chain complementarity determination region 1 (CDR-L1) containing or consisting of the amino acid sequence of Sequence ID No. 7, ii. Light chain complementarity determination region 2 (CDR-L2) containing or consisting of the amino acid sequence of SEQ ID NO: 8, and iii. A light chain complementarity determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO: 9, The antibody according to claim 1.

3. The heavy chain variable (VH) region is (a) Heavy chain complementarity determination region 1 (CDR-H1) containing or consisting of the amino acid sequence of Sequence ID No. 4, (b) Heavy chain complementarity determination region 2 (CDR-H2) containing or consisting of the amino acid sequence of SEQ ID NO: 5, and (c) comprising the amino acid sequence of SEQ ID NO: 6, or comprising a heavy chain complementarity determining region 3 (CDR-H3) consisting thereof, The light chain variable (VL) region, which includes the light chain variable (VL) region, i. Light chain complementarity determination region 1 (CDR-L1) containing or consisting of the amino acid sequence of Sequence ID No. 10, ii. Light chain complementarity determination region 2 (CDR-L2) containing or consisting of the amino acid sequence of SEQ ID NO: 11, and iii. A light chain complementarity determining region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO: 12, The antibody according to claim 1.

4. Isolated antibodies, (a) A heavy chain variable (VH) region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 14, 15, 16, and 17, and one variant of any of SEQ ID NOs: 13-17, wherein any one amino acid is modified to another amino acid (provided that five or fewer amino acids are modified in this way, for example, 5, 4, 3, 2, or 1 amino acid is modified in this way in each amino acid sequence), (b) an amino acid sequence selected from the group consisting of SEQ ID NOs: 18, 19, 20, 21, and 22, and a light chain variable (VL) region comprising or consisting of one variant of SEQ ID NOs: 18-22, wherein any one amino acid is modified to another amino acid (provided that five or fewer amino acids are modified in this way, for example, 5, 4, 3, 2, or 1 amino acid is modified in this way in each amino acid sequence), The isolated antibody, including the following.

5. The antibody according to any one of the prior claims, wherein the antibody is a bispecific antibody.

6. The antibody consists of a full-length antibody, a Fab fragment, an F(ab') fragment, and an F(ab') ２ An antibody according to any one of the prior claims, selected from the group consisting of a fragment, scFv, diabody, and triabody.

7. The antibody according to any one of the prior claims, wherein the antibody is an immunoglobulin subclass antibody selected from the group consisting of human IgG1, human IgG2, human IgG3, and human IgG4.

8. The antibody according to any one of the prior claims, wherein the antibody is a human IgG4 immunoglobulin subclass antibody.

9. The antibody according to claim 8, wherein the antibody comprises a human heavy chain constant (CH) region comprising or consisting of the sequence described in SEQ ID NO: 56 or a variant thereof having at least 80% sequence identity thereto, and a human light chain constant (CL) region comprising or consisting of the sequence described in SEQ ID NO: 57 or a variant thereof having at least 80% sequence identity thereto.

10. The antibody according to any one of the prior claims, wherein the antibody comprises an Fc domain having a mutated human IgG constant region.

11. The antibody according to claim 10, wherein the antibody comprises a mutant human IgG4 heavy chain constant region.

12. The antibody according to claim 11, wherein the mutant human IgG4 heavy chain constant region comprises an S228P substitution numbered according to EU numbering.

13. The aforementioned antibody (a) an amino acid substitution of the amino acid at position 40 of any one of the VH regions of SEQ ID NOs: 13-17 to phenylalanine, an amino acid substitution of the amino acid at position 43 of any one of the VH regions of SEQ ID NOs: 13-17 to serine, and / or an amino acid substitution of the amino acid at position 44 of any one of the VH regions of SEQ ID NOs: 13-17 to lysine, and / or (b) an amino acid substitution of the amino acid at position 42 of any one VL region of SEQ ID NOs: 18-22 to glycine, an amino acid substitution of the amino acid at position 43 of any one VL region of SEQ ID NOs: 18-22 to threonine, and / or an amino acid substitution of the amino acid at position 44 of any one VL region of SEQ ID NOs: 18-22 to leucine The antibody according to any one of the prior claims.

14. The antibody according to any one of the prior claims, wherein the VH region includes or consists of VH regions selected from the group consisting of SEQ ID NOs: 24 to 38.

15. The antibody according to any one of the prior claims, wherein the VL region includes or consists of VL regions selected from the group consisting of SEQ ID NOs: 39 to 53.

16. The antibody according to any one of the prior claims, wherein the VH region includes or consists of SEQ ID NO: 24 (VH1_H40Phe), and the VL region includes or consists of SEQ ID NO: 47 (VL3_L44Leu).

17. The antibody according to any one of the prior claims, wherein the VH region includes or consists of SEQ ID NO: 24 (VH1_H40Phe), and the VL region includes or consists of SEQ ID NO: 20 (VL3).

18. The antibody according to any one of the prior claims, wherein the VH region includes or consists of SEQ ID NO: 26 (VH1_H44Lys), and the VL region includes or consists of SEQ ID NO: 20 (VL3).

19. The antibody according to any one of the prior claims, wherein the VH region includes or consists of SEQ ID NO: 13 (VH1), and the VL region includes or consists of SEQ ID NO: 47 (VL3_L44Leu).

20. The antibody according to any one of the prior claims, wherein the VH region includes or consists of SEQ ID NO: 13 (VH1), and the VL region includes or consists of SEQ ID NO: 20 (VL3).

21. The antibody according to any one of the prior claims, wherein the antibody is PEG-modified.

22. The antibody according to any one of the prior claims, wherein the antibody can bind to the S100A4 protein in its natural three-dimensional structure.

23. The antibody according to any one of the prior claims, wherein the antibody can bind to the dimeric, oligomeric, and / or polymeric forms of the S100A4 protein.

24. The antibody according to any one of the prior claims, wherein the antibody can bind to a polypeptide having at least 80% sequence identity with amino acids 1 to 101 described in SEQ ID NO:

23.

25. The antibody according to any one of the prior claims, wherein the antibody can bind to the human S100A4 polypeptide of SEQ ID NO:

23.

26. The antibody according to any one of the prior claims, wherein administration of the antibody to a target reduces or completely prevents clustering and activation of S100A4-dependent FcγIIaR.

27. The antibody according to any one of the prior claims, wherein administration of the antibody to a target does not decrease or increase TNFα.

28. The antibody according to any one of the prior claims, wherein the antibody can reduce S100A4-mediated fibrosis.

29. The antibody according to any one of the prior claims, wherein the antibody can neutralize the biological activity of S100A4.

30. The antibody according to claim 29, wherein the biological activity of S100A4 is in promoting tumor progression and / or inducing tumor metastasis.

31. The antibody according to claim 30, wherein the antibody can inhibit the biological activity of S100A4 in promoting tumor progression and / or inducing tumor metastasis and / or inflammation.

32. The antibody according to any one of the prior claims, wherein the antibody can inhibit T cell mobilization mediated by S100A4.

33. The antibody according to any one of the prior claims, wherein the antibody can inhibit the recruitment and / or infiltration of macrophages mediated by S100A4.

34. The antibody according to any one of the prior claims, wherein the antibody can inhibit the biological activity of the S100A4 protein in response to stimulation of cell invasion.

35. The antibody according to claim 34, wherein the biological activity of the S100A4 protein in response to stimulation of cell invasion is measured in a 3D Matrigel matrix assay or a T cell invasion assay in which S100A4 stimulates T cell infiltration into a fibroblast monolayer, or the biological activity of S100A4 in inducing tumor metastasis is measured in an in vivo mouse xenograft model.

36. An isolated nucleic acid molecule encoding an antibody according to any one of claims 1 to 35.

37. The nucleic acid molecule according to claim 36, wherein the nucleic acid molecule is codon-optimized for the expressing cell.

38. An expression vector comprising a nucleic acid molecule encoding the anti-S100A4 antibody molecule according to claim 36 or 37.

39. The expression vector according to claim 38, wherein the nucleic acid molecule is operably linked to a control sequence for instructing its expression.

40. An isolated host cell comprising an isolated nucleic acid molecule according to claim 36, or an expression vector according to claim 38 or 39.

41. The isolated host cell according to claim 40, wherein the cell is a human cell.

42. The isolated host cells according to claim 40, wherein the cells are Chinese hamster ovary (CHO) cells.

43. A method for producing an anti-S100A4 antibody molecule, comprising culturing a host cell according to any one of claims 40 to 42 under conditions in which the antibody is expressed.

44. The method according to claim 43, further comprising purifying the antibody and isolating the anti-S100A4 antibody thus produced.

45. A pharmaceutical composition comprising an antibody according to any one of claims 1 to 35, a nucleic acid molecule according to claim 36 or 37, an expression vector according to claim 38 or 39, and / or a host cell according to any one of claims 40 to 42, and a pharmaceutically acceptable diluent, carrier, and / or excipient.

46. A method for treating an individual having an S100A4-mediated pathological condition, comprising administering an antibody according to any one of claims 1 to 35, a nucleic acid or expression vector according to any one of claims 36 to 39, or a host cell according to any one of claims 40 to 42 to an individual in need thereof.

47. The treatment method according to claim 46, wherein the S100A4-mediated pathological condition is cancer or an inflammatory pathological condition.

48. The treatment method according to claim 47, wherein the cancer is metastatic cancer, or the inflammatory condition is psoriasis, rheumatoid arthritis, or inflammatory myopathy.

49. The treatment method according to claim 47 or 48, wherein the cancer is gastric cancer, pancreatic cancer, colorectal cancer, thyroid cancer, breast cancer, squamous cell carcinoma, non-small cell lung cancer, prostate cancer, lung cancer, head and neck cancer, brain cancer (including glioblastoma multiforme), renal cell carcinoma (including clear cell renal carcinoma), melanoma, lymphoma, plasmacytoma, sarcoma, glioma, thymoma, leukemia, colon cancer, esophageal cancer, ovarian cancer, cervical cancer, or hepatocellular carcinoma.

50. A method for diagnosing or predicting the prognosis of S100A4-related pathological conditions in an individual, (a) Contacting a biological sample from the individual with an anti-S100A4 antibody according to any one of claims 1 to 35 that can bind to the S100A4 polypeptide present in the sample, (b) Measuring the presence and / or amount of the complex formed between the antibody molecule and the S100A4 polypeptide, The method, including the method described above.

51. The method according to claim 50, wherein the biological sample is blood, plasma, serum, tissue sample, interstitial fluid, cerebrospinal fluid, synovial fluid, or saliva.

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