Anti-S100A4 humanized antibodies, uses and methods

Abstract

Provided herein are isolated humanized anti-S100A4 antibody molecules and methods for producing the antibodies, as well as nucleic acids, vectors, isolated host cells, and pharmaceutical compositions comprising the antibodies, and methods of treatment comprising administering the antibodies.

Description

[Technical field]

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

[0002] Fibrosis is defined as the excessive deposition of extracellular matrix proteins. The early stages of fibrotic diseases are most often characterized by an inflammatory response that functions to attract, differentiate, and activate fibroblasts, which produce collagen and other extracellular matrix proteins. When these processes become chronic, inflammatory and / or fibrotic responses can progressively impair physiological tissue functions, leading to organ dysfunction or failure. In addition to pure fibrotic and inflammatory pathologies, mechanisms involved in chronic inflammation and fibrosis are the underlying causes of a wide range of diseases with different clinical manifestations, including atherosclerosis, cancer, and neurodegenerative diseases. In cancer, the microenvironment of diseased tissues provides essential support mechanisms for the proliferation and metastasis of malignant cells. Activation of inflammatory and fibrotic pathways plays a key role in the development of premetastatic niches that provide the necessary conditions 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 inappropriate activation of inflammatory and fibrotic pathways. S100A4 belongs to the S100 family of low molecular weight Ca-binding proteins with diverse extracellular and intracellular functions (Donato, 2003). Under physiological conditions, S100A4 is primarily present intracellularly, but upon cellular stress or injury, it is released into the extracellular environment (Fei et al., 2017). Extracellular S100A4 forms higher-order oligomers that bind to pattern recognition receptors (PRRs) and activate multiple inflammatory and fibrotic responses (Ambartsumian et al., 2019; Fei et al., 2017). Through interaction with PRRs, S100A4 triggers the release of inflammatory mediators from immune cells, stimulates the release of extracellular matrix proteins from fibroblasts, and participates in epithelial-mesenchymal transition (Kalluri & Zeisberg, 2006; Tomcik et al., 2015; Neidhart et al., 2019). Overexpression of S100A4 is a hallmark of chronic inflammation and fibrosis. Numerous studies have revealed that S100A4 levels are elevated in various human diseases, including systemic sclerosis, interstitial pulmonary fibrosis, rheumatoid arthritis, psoriasis, and dermatomyositis, compared to healthy controls (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 causally involved in the development of inflammation and fibrosis (Ambartsumian et al., 2019). Knockdown of S100A4 in fibroblasts blocks TGFβ-induced fibroblast activation (Tomcik et al., 2015). Knockout of S100A4 inhibits fibrosis, inflammation, and cancer metastasis in several animal models, including bleomycin-induced dermal or pulmonary fibrosis, tight skin 1 dermal fibrosis, and several cancer models (Ambartsumian et al., 2019; Tomcik et al., 2015).

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

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

[0007] S100A4 has been shown to be expressed by certain tumor cells, but more commonly is activated and secreted by certain cancer-associated stromal cells and accumulates in the tumor microenvironment. Moreover, 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] Moreover, S100A4 has been shown to maintain the stemness properties and tumorigenicity of cancer-initiating cells in head and neck cancer and glioblastoma (Lo et al., 2011; Chow et al., 2017). Thus, the development of drugs capable of inhibiting the biological activity of S100A4 may present a promising therapeutic option for regulating multiple inflammatory and fibrotic pathways activated in various human diseases. Thus, there is an unmet need for therapeutic anti-S100A4 antibodies, especially humanized anti-S100A4 antibodies, that specifically target the extracellular pathogenic portion of S100A4.

[0009] A humanized antibody is an antibody derived from a non-human species whose protein sequence has been modified to increase its similarity to the antibody variants naturally produced in humans. The process of "humanization" is usually applied to monoclonal antibodies developed for administration to humans (e.g., antibodies developed as anti-cancer drugs). Humanization may be necessary when the process of developing a particular antibody involves production in a non-human immune system (such as the immune system of a mouse). The protein sequence of antibodies produced in a non-human immune system is partially different from the homologous antibody naturally occurring in humans and may therefore be immunogenic when administered to a human patient, which may result in loss of therapeutic benefit and cause side effects in the patient. Summary of the Invention

[0010] The present invention provides humanized anti-s100A4 antibodies with an improved safety profile.

[0011] We found that mouse IgG1 and humanized IgG1 anti-S100A4 antibodies elicited an unexpected increase in the proinflammatory cytokine TNFα while simultaneously blocking the S100A4-stimulated increase in IL-6 and IL-10. The increase in TNFα is elicited by FcγRIIA receptor clustering and activation. Surprisingly, this effect was dependent on both the anti-S100A4 antibody and the S100A4 protein; neither the anti-S100A4 antibody in the absence of S100A4 nor S100A4 in combination with an isotype control antibody (either human IgG1 or human IgG4) resulted in FcγRIIA receptor clustering.

[0012] The inventors found that subclass switching of a humanized anti-S100A4 antibody from an IgG1 to an IgG4 scaffold suppresses this previously unreported S100A4-dependent FcγRIIA receptor clustering and activation, thereby preventing the release of undesirable proinflammatory cytokines and improving the safety profile of the antibody. FcγRIIA receptor clustering and activation by the humanized IgG4 antibody is also significantly reduced compared to that elicited by the murine IgG1 anti-S100A4 antibody.

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

[0014] In one aspect, there is provided an isolated antibody comprising: i. a heavy chain variable (VH) region comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and a variant of any one of SEQ ID NOs: 13 to 17, in which any one amino acid has been modified to another amino acid (provided that no more than five amino acids have been so modified, e.g., 5, 4, 3, 2, or 1 amino acid has been so modified in each amino acid sequence); ii. a light chain variable (VL) region comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and a variant of any one of SEQ ID NOs: 18 to 22, in which any one amino acid has been modified to another amino acid (provided that no more than five amino acids have been so modified, e.g., 5, 4, 3, 2, or 1 amino acid has been so modified in each amino acid sequence); An isolated antibody is provided, comprising:

[0015] In one aspect, there is provided an isolated nucleic acid molecule encoding an antibody described herein above in the "Isolated anti-S100A4 antibody molecule" section.

[0016] In one aspect, an expression vector is provided that comprises a nucleic acid molecule described herein encoding an anti-S100A4 antibody molecule.

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

[0018] In one aspect, there is provided a method of producing an anti-S100A4 antibody molecule, comprising culturing a host cell as described herein under conditions such that the antibody is expressed.

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

[0020] In one aspect, there is also provided a method of treating an individual having an S100A4-mediated pathology comprising administering to an individual in need thereof an antibody or a host cell as described herein.

[0021] In one aspect, there is provided a method for the diagnosis or prognosis of an S100A4 associated condition in an individual, comprising: (a) contacting a biological sample from an individual with an anti-S100A4 antibody described herein that is capable of binding to an S100A4 polypeptide present in the sample; (b) determining the presence and / or amount of complexes formed between the antibody molecule and the S100A4 polypeptide; A method is provided that includes: [Brief description of the drawings]

[0022] [Figure 1A] Figure 1 shows the effect of monoclonal humanized anti-S100A4 antibody on fibrosis measurements in bleomycin-treated mice. Figure 2 shows the effect of anti-S100A4 antibody on dermal thickness. P values ​​are expressed as follows: *0.05>p>0.01, **0.01>p>0.001 compared to NaCl; #0.05>p>0.01; ##0.01>p>0.001 compared to mice injected with bleomycin for 3 weeks followed by an additional 3 weeks of NaCl. The results are further described in Example 3. [Figure 1B] Figure 1 shows the effect of monoclonal humanized anti-S100A4 antibody on fibrosis measurements in bleomycin-treated mice. Figure 2 shows the effect of anti-S100A4 antibody on myofibroblast counts. P values ​​are expressed as follows: *0.05>p>0.01, **0.01>p>0.001 compared to NaCl; #0.05>p>0.01; ##0.01>p>0.001 compared to mice injected with bleomycin for 3 weeks followed by an additional 3 weeks of NaCl. The results are further described in Example 3. [Figure 1C] Figure 1 shows the effect of monoclonal humanized anti-S100A4 antibody on fibrosis measurements in bleomycin-treated mice. Figure 2 shows the effect of anti-S100A4 antibody on hydroxyproline content. P values ​​are expressed as follows: *0.05>p>0.01, **0.01>p>0.001 compared to NaCl; #0.05>p>0.01; ##0.01>p>0.001 compared to mice injected with bleomycin for 3 weeks followed by an additional 3 weeks of NaCl. The results are further described in Example 3. [Figure 1D]Figure 1 shows the effect of monoclonal humanized anti-S100A4 antibodies on fibrosis measurements in bleomycin-treated mice. Representative images of HE-stained skin sections are shown. Results are further described in Example 3. [Figure 1E] Figure 1 shows the effect of monoclonal humanized anti-S100A4 antibodies on fibrosis measurements in bleomycin-treated mice. Representative images of HE-stained skin sections are shown. Results are further described in Example 3. [Figure 2A] Figure 1 shows the amount of IL-6 secreted by monocytes analyzed by Luminex analysis. Data shown are the average of five donors. Monocytes purified from PBMC were cultured for 6 hours with medium, vehicle, LPS, S100A4 in the absence or presence of mouse IgG1. Data show IL-6 levels in the supernatant quantified by Luminex assay. Data shown as the mean + SEM obtained from five independent donors. "+" indicates at least one donor above the detection limit of IL-6 (19,200 pg / mL). "-" indicates at least one donor below the detection limit of IL-6 (8.8 pg / mL). Results are further described in Example 4. [Figure 2B] Figure 1 shows the amount of IL-6 secreted by monocytes analyzed by Luminex analysis. Data shown are the average of five donors. Monocytes purified from PBMC were cultured for 6 hours with medium, vehicle, LPS, S100A4 in the absence or presence of human IgG4. Data show IL-6 levels in the supernatant quantified by Luminex assay. Data shown as the mean + SEM obtained from five independent donors. "+" indicates at least one donor above the detection limit of IL-6 (19,200 pg / mL). "-" indicates at least one donor below the detection limit of IL-6 (8.8 pg / mL). Results are further described in Example 4. [Figure 2C]Figure 1 shows the amount of IL-6 secreted by monocytes analyzed by Luminex analysis. Data shown are the average of five donors. Monocytes purified from PBMC were cultured for 6 hours with medium, vehicle, LPS, S100A4 in the absence or presence of AX-202. Data show IL-6 levels in the supernatant quantified by Luminex assay. Data shown as the average + SEM obtained from five independent donors. "+" indicates at least one donor above the detection limit of IL-6 (19,200 pg / mL). "-" indicates at least one donor below the detection limit of IL-6 (8.8 pg / mL). Results are further described in Example 4. [Figure 2D] Figure 1 shows the amount of IL-6 secreted by monocytes analyzed by Luminex analysis. Data shown are the average of five donors. Monocytes purified from PBMC were cultured for 6 hours with medium, vehicle, LPS, S100A4 in the absence or presence of 6B12. Data show IL-6 levels in the supernatant quantified by Luminex assay. Data shown as the average + SEM obtained from five independent donors. "+" indicates at least one donor with IL-6 above the detection limit (19,200 pg / mL). "-" indicates at least one donor with IL-6 below the detection limit (8.8 pg / mL). Results are further described in Example 4. [Figure 3A] Figure 1 shows the amount of IL-10 secreted by monocytes analyzed by Luminex analysis. Data shown are the average of five donors. Monocytes purified from PBMC were cultured for 6 hours with medium, vehicle, LPS, S100A4 in the absence or presence of mouse IgG1. Data shows the cytokine IL-10 in the supernatant quantified by Luminex assay. Data shown as the mean + SEM from five independent donors. "-" indicates at least one donor below the detection limit for IL-10 (8.6 pg / mL). Results are further described in Example 4. [Figure 3B]Figure 1 shows the amount of IL-10 secreted by monocytes analyzed by Luminex analysis. Data shown are the average of five donors. Monocytes purified from PBMC were cultured for 6 hours with medium, vehicle, LPS, S100A4 in the absence or presence of human IgG4. Data shows the cytokine IL-10 in the supernatant quantified by Luminex assay. Data shown as the mean + SEM from five independent donors. "-" indicates at least one donor below the detection limit for IL-10 (8.6 pg / mL). Results are further described in Example 4. [Figure 3C] Figure 1 shows the amount of IL-10 secreted by monocytes analyzed by Luminex analysis. Data shown are the average of five donors. Monocytes purified from PBMC were cultured for 6 hours with medium, vehicle, LPS, S100A4 in the absence or presence of AX-202. Data shows the cytokine IL-10 in the supernatant quantified by Luminex assay. Data shown as the mean + SEM from five independent donors. "-" indicates at least one donor below the detection limit for IL-10 (8.6 pg / mL). Results are further described in Example 4. [Figure 3D] Figure 1 shows the amount of IL-10 secreted by monocytes analyzed by Luminex analysis. Data shown are the average of five donors. Monocytes purified from PBMC were cultured for 6 hours with medium, vehicle, LPS, S100A4 in the absence or presence of 6B12. Data shows the cytokine IL-10 in the supernatant quantified by Luminex assay. Data shown as the mean + SEM from five independent donors. "-" indicates at least one donor below the detection limit for IL-10 (8.6 pg / mL). Results are further described in Example 4. [Figure 4A]Figure 1 shows the amount of TNF-α secreted by monocytes analyzed by Luminex analysis. Data shown are the average of five donors. Monocytes purified from PBMC were cultured for 6 hours with medium, vehicle, LPS, S100A4 in the absence or presence of mouse IgG1. Data show the TNF-α levels in the supernatant quantified by Luminex assay. Data shown as the mean + SEM from five independent donors. "-" indicates at least one donor with TNF-α below the detection limit (15.20 pg / mL). Results are further described in Example 4. [Figure 4B] Figure 1 shows the amount of TNF-α secreted by monocytes analyzed by Luminex analysis. Data shown are the average of five donors. Monocytes purified from PBMC were cultured for 6 hours with medium, vehicle, LPS, S100A4 in the absence or presence of human IgG4. Data show the TNF-α levels in the supernatant quantified by Luminex assay. Data shown as the mean + SEM from five independent donors. "-" indicates at least one donor with TNF-α below the detection limit (15.20 pg / mL). Results are further described in Example 4. [Figure 4C] Figure 1 shows the amount of TNF-α secreted by monocytes analyzed by Luminex analysis. Data shown are the average of five donors. Monocytes purified from PBMC were cultured for 6 hours with medium, vehicle, LPS, S100A4 in the absence or presence of AX-202. Data show the TNF-α levels in the supernatant quantified by Luminex assay. Data shown as the mean + SEM from five independent donors. "-" indicates at least one donor with TNF-α below the detection limit (15.20 pg / mL). Results are further described in Example 4. [Figure 4D]Figure 1 shows the amount of TNF-α secreted by monocytes analyzed by Luminex analysis. Data shown are the average of five donors. Monocytes purified from PBMC were cultured for 6 hours with medium, vehicle, LPS, S100A4 in the absence or presence of 6B12. Data show the TNF-α levels in the supernatant quantified by Luminex assay. Data shown as the mean + SEM from five independent donors. "-" indicates at least one donor with TNF-α below the detection limit (15.20 pg / mL). Results are further described in Example 4. [Diagram 5] 1 shows the specificity of three humanized variants of 6B12 mAb against other different S100 family members as measured by Western blotting. The results are further described in Example 5. [Figure 6] As shown in wild-type (wt) and S100A4 knockout (ko) mouse embryonic fibroblasts (MEFs), we show that the human variants are specific for S100A4 protein and do not cross-react with random cellular proteins. Cells were counterstained with DAPI and the actin cytoskeleton was counterstained with phalloidin. The parental monoclonal mouse anti-S100A4 antibody (6B12) was used as a control. Bar = 100 μm. Results are further described in Example 5. [Figure 7A] This shows that AX-202 inhibits S100A4-induced TLR4 activation in a concentration-dependent manner. S100A4 activates the NF-kB reporter gene in HEKBlue hTLR4 cells in a concentration-dependent manner. The results are further described in Example 6. [Figure 7B] We show that AX-202 inhibits S100A4-induced TLR4 activation in a concentration-dependent manner. S100A4 activation of NF-kB reporter gene is dependent on TLR4. The results are further described in Example 6. [Figure 7C] This shows that AX-202 inhibits S100A4-induced TLR4 activation in a concentration-dependent manner. AX-202 inhibits S100A4-induced TLR4 activation in a concentration-dependent manner. The results are further described in Example 6. [Figure 8A] Figure 1 shows a comparison of FcγIIaR binding activity for 6B12 mIgG1, AX-202 hIgG1, and AX-202 hIgG4 at 2 μg / ml in combination with recombinant human S100A4 dimer at 2.5 μg / ml. Control IgG1 or IgG4 antibodies did not mediate receptor clustering and activation. Results are further described in Example 7. Bars indicate standard deviation. Results are presented as mean ± standard deviation, n=3 independent experiments. 0 μg / ml S100A4 n=2 was used as a control. [Figure 8B] Figure 1 shows a comparison of FcγIIaR binding activity for 6B12 mIgG1, AX-202 hIgG1, and AX-202 hIgG4 at 2 μg / ml in combination with recombinant human S100A4 multimer at 2.5 μg / ml. Control IgG1 or IgG4 antibodies did not mediate receptor clustering and activation. Results are further described in Example 7. Bars indicate standard deviation. Results are presented as mean ± standard deviation, n=3 independent experiments. S100A4 at 0 μg / ml, n=2, was used as a control. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] definition As used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise. Thus, for example, a reference to an "antibody" includes a plurality of 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 to a compound which may be, for example, an antibody or antigen-binding portion that is substantially free of other antibodies or antigen-binding portions having different antigen specificities. Furthermore, an antigen-binding portion of an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0026] As defined herein, operably linked refers to the elements referenced being joined as part of the same nucleic acid molecule and appropriately positioned and oriented to allow transcription to be initiated from the promoter. DNA operably linked to a promoter is under the transcription initiation control of the promoter or is in functional association with the promoter.

[0027] As used herein, the term "variant" defines either a naturally occurring genetic mutant of a DNA sequence or its encoded RNA or protein product, or a recombinantly prepared variant of a DNA sequence or its encoded RNA or protein product. The term "variant" may also refer to either a naturally occurring variant of a given peptide, or a recombinantly prepared variant of a given peptide or protein, in which one or more amino acid residues are modified by amino acid substitution, addition, or deletion.

[0028] "Inhibition" as used herein means that the presence of an antibody of the invention, in whole or in part, inhibits the binding of a ligand to its receptor and / or neutralizes the signal that the receptor would elicit upon ligand binding. This includes downstream signaling that affects, for example, cellular behavior and processes. It also includes other mechanisms of inhibiting downstream effects of a target molecule, such as by blocking dimerization, oligomerization, and / or multimerization of the target molecule. "Inhibition," "blocking," and "neutralization" are used as equivalent terms herein.

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

[0030] In some embodiments, the heavy chain variable (VH) region of the isolated antibody comprises the following: i. a heavy chain complementarity determining region 1 (CDR-H1) comprising or consisting of the amino acid sequence of SEQ ID NO:1; ii. a heavy chain complementarity determining region 2 (CDR-H2) comprising or consisting of the amino acid sequence of SEQ ID NO:2; and iii. A heavy chain complementarity determining region 3 (CDR-H3) comprising or consisting of the amino acid sequence of SEQ ID NO:3; The light chain variable (VL) region is i. a light chain complementarity determining region 1 (CDR-L1) comprising or consisting of the amino acid sequence of SEQ ID NO:7; ii. a light chain complementarity determining region 2 (CDR-L2) comprising or consisting of the amino acid sequence of SEQ ID NO:8, and iii. A light chain variable (VL) region comprising a light chain complementarity determining region 3 (CDR-L3) comprising or consisting of the amino acid sequence of SEQ ID NO:9.

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

[0032] In one aspect, there is provided an isolated antibody comprising: i. a heavy chain variable (VH) region comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, SEQ ID NO: 17, and a variant of any one of SEQ ID NOs: 13 to 17, in which any one amino acid has been modified to another amino acid (provided that no more than five amino acids have been so modified, e.g., 5, 4, 3, 2, or 1 amino acid has been so modified in each amino acid sequence); ii. a light chain variable (VL) region comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO: 20, SEQ ID NO: 21, SEQ ID NO: 22, and a variant of any one of SEQ ID NOs: 18 to 22, in which any one amino acid has been modified to another amino acid (provided that no more than five amino acids have been so modified, e.g., 5, 4, 3, 2, or 1 amino acid has been so modified in each amino acid sequence); An isolated antibody is provided, comprising:

[0033] Thus, in some embodiments, the heavy chain variable (VH) region of the antibody comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises or consists of a variant of any one of the amino acid sequences defined by SEQ ID NOs: 13-17, in which any one amino acid has been modified to another amino acid (provided that no more than five amino acids have been so modified, e.g., 5, 4, 3, 2, or 1 amino acid has been so modified in each amino acid sequence).

[0034] In some embodiments, the light chain variable (VL) region of the antibody comprises 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 comprises 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 comprises 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 comprises 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 comprises 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 comprises or consists of a variant of any one of the amino acid sequences defined by SEQ ID NOs: 18-22, in which any one amino acid has been modified to another amino acid (provided that no more than five amino acids have been so modified, e.g., 5, 4, 3, 2, or 1 amino acid has been so modified 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 a F(ab') fragment. In some embodiments, the antibody is a F(ab')2 fragment. In some embodiments, the antibody is an 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, which was absent with humanized IgG4 anti-S100A4 antibodies (see Examples 4 and 7 for further details). In the present case, it may be useful to use antibodies with immunoglobulin subclasses that induce weak or no proinflammatory responses in the host. As shown, human IgG4 subclasses may be particularly useful when reduced effector or cross-linking functions of the antibody are desired. In some embodiments, the antibody is thus a human IgG4 subclass antibody. In a particular embodiment, the antibody is a human IgG4 subclass antibody with the HC sequence of SEQ ID NO:58 and the LC sequence of SEQ ID NO:59.

[0039] In some embodiments the antibody comprises a human heavy chain constant (CH) region comprising or consisting of the sequence set forth in SEQ ID NO: 56. In some embodiments the antibody comprises a CH region comprising or consisting of a variant of SEQ ID NO: 56 having at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99% sequence identity thereto.

[0040] In some embodiments the antibody comprises a human light chain constant (CL) region comprising or consisting of the sequence set forth in SEQ ID NO: 57. In some embodiments the antibody comprises a CL region comprising or consisting of a variant of SEQ ID NO: 57 having at least 80%, such as at least 81%, for example at least 82%, such as at least 83%, for example at least 84%, such as at least 85%, for example at least 86%, such as at least 87%, for example at least 88%, such as at least 89%, for example at least 90%, such as at least 91%, for example at least 92%, such as at least 93%, for example at least 94%, such as at least 95%, for example at least 96%, such as at least 97%, for example at least 98%, such as at least 99% sequence identity thereto.

[0041] In some embodiments, the antibody comprises an Fc domain with a mutated human IgG constant region. In some embodiments, the antibody comprises a mutated human IgG4 heavy chain constant region. In some embodiments, the mutated human IgG4 heavy chain constant region comprises an S228P substitution numbered according to EU numbering. The S228P substitution may prevent IgG4 Fab-arm exchange in vivo and in vitro, resulting in functionally monovalent bispecific antibodies (bsAbs) 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 made in 1986 by the laboratory of Greg Winter in Cambridge, UK. This antibody had a moderate loss of affinity, but the strategy of CDR-grafting mouse CDRs onto a human framework was considered successful. The next antibody to be humanized was the therapeutic antibody Campath-1, which had a significantly reduced affinity, and it was here that researchers first began to explore framework amino acids important for CDR stability and for the stability of the VH / VL interface.

[0043] Since the 1990s, humanization of mouse antibodies has attracted much interest as a means of generating therapeutics that are tolerable for human use. Although it was recognized early on that framework amino acids play an important role in presenting CDRs in a manner that favors antigen binding, there was no automated or routine method to identify and determine which residues to backmutate to successfully increase antigen affinity. It is important to consider which positions are important for the stability of the VL / VH interface and the frequency of each amino acid at a given position in similar antibody frameworks. Thus, framework backmutations can be useful to improve the affinity or stability of humanized antibodies to their targets.

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

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

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

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

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

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

[0050] Function and therapeutic effect of antibodies In some embodiments, the antibody according to the present invention can bind to S100A4 protein in its native conformation. In some embodiments, the antibody can bind to the dimeric form of S100A4 protein. In some embodiments, the antibody can bind to the oligomeric form of S100A4 protein. In some embodiments, the antibody can bind to the multimeric form of S100A4 protein.

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

[0052] In some embodiments, the antibody is capable of neutralizing the biological activity of S100A4, hi 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 reduces fibrosis. Thus, in some embodiments, the antibody can reduce S100A4-mediated fibrosis. Fibrosis is measured by measuring dermal thickness, dermal hydroxyproline content, dermal CD3 + The number of cells and / or the number of myofibroblasts in the dermis can be assessed by methods known in the art. Thus, in some embodiments, treatment with an anti-S100A4 antibody reduces dermal thickness, dermal collagen or hydroxyproline content, dermal myoblast number and / or T cell number.

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

[0055] In some embodiments, the antibody is capable of inhibiting S100A4-mediated T cell recruitment. In some embodiments, the antibody is capable of inhibiting S100A4-mediated macrophage recruitment and / or infiltration.

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

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

[0058] Nucleic acids and expression vectors encoding antibodies In one aspect, an isolated nucleic acid molecule is provided encoding an antibody described herein above in the "Isolated anti-S100A4 antibody molecule" section. In some embodiments, the nucleic acid molecule is codon-optimized for the cell in which it is expressed.

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

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

[0061] Host cells containing antibodies In one aspect, there is provided an isolated host cell comprising an isolated nucleic acid molecule or expression vector described herein above in the "Nucleic Acids and Expression Vectors" section.

[0062] In some embodiments, the isolated host cell is a human cell. In some embodiments, the isolated host cell is a Chinese Hamster Ovary (CHO) cell.

[0063] The present invention can also be used in connection with ex vivo gene therapy, in which patient cells are transfected or transduced in vitro with expression vectors encoding the antibodies disclosed herein. After transfection, the cells are returned to the patient where they express and secrete the antibodies. Suitable donor cells for ex vivo gene therapy include T cells.

[0064] Methods for Producing the Antibodies Described Herein In one aspect, there is provided a method for producing an anti-S100A4 antibody molecule, comprising culturing a host cell as described herein above in the section "Host cells comprising antibodies" 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 so produced.

[0066] Pharmaceutical Compositions In one aspect, there is provided a pharmaceutical composition comprising an antibody as described herein above in the "Isolated anti-S100A4 antibody molecule" section, a nucleic acid molecule and / or an expression vector as described herein above in the "Nucleic acids and expression vectors encoding the antibody" section, and / or a host cell as described herein above in the "Host cells comprising the antibody" section, and a pharma- ceutically acceptable diluent, carrier, and / or excipient.

[0067] Treatment method In one aspect, there is also provided a method for treating an individual having an S100A4-mediated pathology, comprising administering to an individual in need thereof an antibody as described herein above in the "Isolated anti-S100A4 antibody molecule" section, a nucleic acid molecule and / or an expression vector as described herein above in the "Nucleic acids and expression vectors encoding the antibody" section, or a host cell as described herein above in the "Host cells comprising the antibody" section.

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

[0069] In some embodiments, the fibrotic condition is systemic sclerosis. In some embodiments, the fibrotic condition is skin fibrosis. In some embodiments, the fibrotic condition is interstitial pulmonary fibrosis. In some embodiments, the fibrotic condition is liver fibrosis. In some embodiments, the fibrotic condition is renal fibrosis.

[0070] It may be beneficial to combine treatment with an anti-S100A4 antibody with treatment with other compounds useful for the treatment of systemic sclerosis. Thus, 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 a glucocorticoid. In some embodiments, the antibody is co-administered with a phosphodiesterase 5 inhibitor. In some embodiments, the antibody is co-administered with an endothelin receptor antagonist. 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 pathology is an inflammatory pathology. In some embodiments, the inflammatory pathology is psoriasis. In some embodiments, the inflammatory pathology is rheumatoid arthritis. In some embodiments, the inflammatory pathology is an inflammatory myopathy.

[0072] In some embodiments, the S100A4-mediated pathology 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, the antibodies, nucleic acid molecules, expression vectors, and / or host cells are administered parenterally. Thus, in some embodiments, the antibodies, nucleic acid molecules, expression vectors, and / or host cells are administered subcutaneously. In some embodiments, the antibodies, nucleic acid molecules, expression vectors, and / or host cells are administered intramuscularly. In some embodiments, the antibodies, nucleic acid molecules, expression vectors, and / or host cells are administered intravenously.

[0075] In some embodiments, the antibodies, nucleic acid molecules, expression vectors, and / or host cells are administered no more than once a week, hi some embodiments, the antibodies are administered weekly and the weekly dose ranges from 15 mg to 1000 mg.

[0076] Methods of diagnosis or prognosis In one aspect, there is provided a method for the diagnosis or prognosis of an S100A4 associated condition in an individual, comprising: (a) contacting a biological sample from an individual with an anti-S100A4 antibody described herein that is capable of binding to an S100A4 polypeptide present in the sample; (b) determining the presence and / or amount of complexes formed between the antibody molecule and the S100A4 polypeptide; A method is provided that includes:

[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 tissue 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 a murine anti-S100A4 antibody Humanization was performed on the murine monoclonal IgG1 anti-S100A4 antibody 6B12 (VH and VL regions are defined in SEQ ID NO: 54 and SEQ ID NO: 55, respectively).

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

[0080] The closest human germline V region 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 the mouse VH domain, and human variable domain candidates were selected from the top 200 BLAST results. These were narrowed down to four candidates each based on a combination of framework homology, conserved key framework residues, and canonical loop structures. For the fifth acceptor sequence, the closest human germline IGHV4-34*09 was selected.

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

[0082] The CDRs of mouse VH and VL were then grafted into these acceptor frameworks to obtain five humanized VH variants VH1 to VH5 (SEQ ID NOs: 13 to 17) and five humanized VL variants VL1 to VL5 (SEQ ID NOs: 18 to 22).

[0083] Confirmation of humanization The humanized variants were checked to determine whether they were humanized according to the WHO definition of a humanized antibody: the variable domains of the humanized chains have V-region amino acid sequences that, when analyzed as a whole, are closer to human than to other species (assessed using the Immunogenetics Information System® (IMGT®) DomainGapAlign tool).

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

[0085] Heavy and light chain framework backmutations To improve the affinity of the humanized antibody for its target, the VL and VH regions were analyzed to obtain good candidates for framework backmutations.

[0086] This analysis was performed by looking at the frequency of amino acids at each position in the mouse antibody, specifically looking for amino acids with very low frequency, e.g., less than 1%, etc. We further evaluated whether these amino acids might be in structurally important positions, and these amino acids were considered as potential candidates for framework backmutations.

[0087] In the VL region, L44 (L) was identified as unusual at this position (frequency <1%). This position has been shown to be important at the VL / VH interface. In the parental germline, this position is likely to be a valine. Most often, this position is a proline. This was considered a good candidate for backmutation. The previous amino acid, L43 (T), was also considered worth leaving as a framework backmutation, but it was shown to be present in one of the five humanized sequences. In the closest human germline sequences, this was identified as an alanine or valine. Similarly, L42 (G) was also considered a good position for framework backmutation.

[0088] For the VH region, the amino acid at H43(S) was considered a good candidate for framework backmutation because it is a critical residue at the VH / VL interface and this position is typically a lysine, glutamine, or arginine. Similarly, H40(F) and H44(K) were also considered good positions for framework backmutation because they had a frequency of 1%. Although these were not considered to be strictly at critical defined positions, they were considered good candidates because they are directly adjacent.

[0089] The consequences of framework backmutations on the affinity of humanized antibodies for S100A4 are further described in Example 2.

[0090] Example 2 - Kinetic analysis of the interaction of anti-S100A4 antibodies with S100A4 Surface plasmon resonance (SPR) was used to quantitatively analyze the kinetics of the interactions of 14 humanized antibodies and two control antibodies with human S100A4 dimers.

[0091] Materials and Methods The analysis was carried out using a Biacore T200 device to perform quantitative kinetic interaction analysis at an analysis temperature of 25° C. and a flow rate of 50 μl / min.

[0092] Assay buffer: 10 mM HEPES pH 7.4, 300 mM NaCl, 1 mM CaCl2, 100 μM EDTA, 0.05% Tween 20

[0093] Assay cycle: 1. Preparation of Anti-His Capture 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 study, 14 humanized antibodies were analyzed, some of which had specific amino acids backmutated in the VL and / or VH framework regions. Each antibody contained one of the following heavy chain variable regions (VH): VH0 (SEQ ID NO:54) VH1 (SEQ ID NO: 13) VH1_H40Phe (SEQ ID NO: 24) VH1_H43Ser (SEQ ID NO: 25) VH1_H44Lys (SEQ ID NO: 26)

[0096] Each antibody contained one of the following light chain variable regions (VL): VL0: (SEQ ID NO: 55) VL3 (SEQ ID NO: 20) VL3_L42Gly (SEQ ID NO: 45) VL3_L43Thr (SEQ ID NO: 46) VL3_L44Leu (SEQ ID NO: 47) VL3_L42Gly+L44Leu

[0097] Two 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 contained within a 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, demonstrating high binding affinity to S100A4. However, it was clear that some of the specific framework back-mutations in the humanized light or heavy chains had an improving effect on the dissociation constant. For example, VH1 VL3 had a K of 3.47E-09. d, which improved to 2.25E-09 for VH1 VL3_L44Leu and 1.99E-09 for VH1 VL3_L42Gly+L44Leu. Thus, some of these manually designed framework back mutations were successful in increasing the affinity of the antibody for S100A4. However, it is important to note that not all framework back mutations increased the antibody affinity for S100A4. For example, VH1 VL3_L43Thr had a K of only 4.72E-09. d Only it showed.

[0100] Example 3 - Efficacy of humanized anti-S100A4 antibodies in treating bleomycin-induced skin fibrosis in vivo Systemic sclerosis (SSc) is a systemic fibrotic rare disease with high morbidity and mortality. SSc has the highest case-specific mortality rate among autoimmune rheumatic diseases, with the majority of cases diagnosed with the condition ultimately dying as a direct consequence. The disease is characterized by the accumulation of extracellular matrix proteins by pathologically activated fibroblasts. To date, no therapeutic approach is available that selectively inhibits the abnormal release of extracellular matrix in SSc. Bleomycin-induced dermal fibrosis is the most commonly used mouse model of SSc. It is particularly similar to the early inflammatory phase of SSc. Herein, we aimed to evaluate the effect of a humanized S100A4 antibody in bleomycin-induced dermal fibrosis.

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

[0102] Test animals Experiments were performed in C57Bl / 6 mice. Number of animals: 64 Early deaths: None Total study duration for each animal: 6 weeks

[0103] Treatment Protocol Antibody stock solutions of 2 mg / mL were diluted in sterile PBS and injected intraperitoneally in a volume of 100 μL.

[0104] Bleomycin (2.5 mg / kg, Sigma-Aldrich) was administered into a predefined marked area (1 cm) on the upper back. 2 Dermal fibrosis was induced by daily subcutaneous injections of bleomycin (100 mg / kg / day) for up to 6 weeks. Treatment began 3 weeks after pretreatment with bleomycin and consisted of either twice weekly intraperitoneal (ip) injections or weekly intravenous (iv) injections in the tail vein. Results were analyzed 3 weeks after the first bleomycin injection (6 weeks after the first bleomycin injection).

[0105] Study design and study arms N=8 mice in the control group, N=10 mice in the humanized antibody treatment group

[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 (intraperitoneally every 3 days) Group 4: bleomycin for 6 weeks + AX-202 16 mg / kg for the last 3 weeks (intraperitoneally twice weekly) Group 5: bleomycin for 6 weeks + AX-202 24 mg / kg for the last 3 weeks (weekly intravenous injection into the tail vein) Group 6: bleomycin for 6 weeks + AX-202 8 mg / kg for the last 3 weeks (intraperitoneally twice weekly)

[0107] Testing Mice were clinically monitored daily for behavior, activity, fur texture, and stool consistency. After sacrifice, macroscopic evaluation of the lungs and skin was performed.

[0108] Quantification of subcutaneous thickening After sacrifice by cervical dislocation, 1 cm2 skin samples were taken from a designated area on the upper back between the shoulder blades. 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 by measuring the distance between the epidermal-dermal junction and the dermal-subcutaneous fat junction at three locations in the skin lesion of each mouse at 100x magnification.

[0109] Myofibroblast detection 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 a horseradish peroxidase-conjugated secondary antibody and 3,3-diaminobenzidine tetrahydrochloride (DAB) (Sigma-Aldrich). A monoclonal mouse IgG antibody (Calbiochem, San Diego, CA, USA) was used as a control. Analysis was performed by a blinded examiner evaluating myofibroblasts in four sections per sample.

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

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

[0112] result Dermal fibrosis mouse model Upon treatment with bleomycin, the mice developed significant skin fibrosis, and fibrotic changes were more pronounced in mice treated with bleomycin for 6 weeks compared with mice that were treated with bleomycin for 3 weeks followed by injections of NaCl, the solvent for bleomycin, for an additional 3 weeks. Mice injected with NaCl for 6 weeks served as controls.

[0113] General Tolerability Treatment with the anti-S100A4 antibody AX-202 was well tolerated, with no obvious signs of toxicity on clinical examination, gross necropsy, or histology.

[0114] Efficacy Results Treatment with AX-202 significantly reduced dermal thickening, 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 the 16 mg / kg i.p. dose and the 24 mg / kg i.v. dose once a week (see Figure 1A-C). However, a statistically significant effect of AX-202 was also observed when 8 mg / kg was administered i.p. every 3 days. At 16 mg / kg i.p. and 24 mg / kg i.v., AX-202 induced regression of fibrosis as well as statistically significant changes in dermal thickening and myofibroblast count compared to mice injected with bleomycin alone for 3 weeks.

[0115] conclusion Treatment with AX-202 potently improved bleomycin-induced dermal fibrosis in skin thickening, myofibroblast numbers, and hydroxyproline 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 over FicollPaque PLUS (GE Healthcare; 11778538), and monocytes were isolated using a Monocyte Isolation Kit (StemCell Technologies; 19359).

[0117] Monocytes were plated in 96-well plates (100,000 cells / well) and cultured for 6 hours in the presence of: Culture medium, Vehicle 1 (0.074% TBS), 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 (each at 32 μg / ml); S100A4 (2.0 μg / ml) + mouse IgG1 (either 8.0, 16, or 32 μg / ml); S100A4 (2.0 μg / ml) + human IgG4 (either 8.0, 16, or 32 μg / ml); S100A4 (2.0 μg / ml) + AX-202 (either 8.0, 16, or 32 μg / ml), or S100A4 (2.0 μg / ml) + 6B12 (either 8.0, 16, or 32 μg / ml).

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

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

[0120] result Compared to vehicle controls, stimulation of monocytes with either LPS (1.0 ng / ml; positive control) or S100A4 (2.0 μg / ml) induced increases in the levels of IL-6, TNFα, and IL-10 measured in cell culture supernatants (see Figures 2 and 3). Compared to isotype controls, AX-202 or 6B12 at all concentrations tested reduced the S100A4-induced increases in IL-6 and IL-10 (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, but not 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 increased levels of IL-6, TNFα, and IL-10 (see Figures 2-4). S100A4-induced release of IL-6 and IL-10 was reduced by both AX-202 and 6B12 (see Figures 2C-D and 3C-D).

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

[0123] This surprisingly demonstrates that the humanized anti-S100A4 IgG4 antibody does not increase pro-inflammatory TNFα levels induced by S100A4 compared to the murine anti-S100A4 antibody 6B12.

[0124] Although the foregoing invention has been described in some detail by way of illustrations and examples for purposes of clarity of understanding, these illustrations and examples should not be construed as limiting the scope of the invention.

[0125] The foregoing specification is deemed sufficient to enable one skilled in the art to practice the present disclosure. However, no matter how detailed the foregoing appears in text, it will be understood that the present disclosure can be practiced in many ways and that the present disclosure should be construed according to the appended claims and their equivalents.

[0126] Example 5 - Humanized anti-S100A4 antibodies are specific for S100A4 from multiple species without cross-reactivity to other S100 family members.

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

[0128] To confirm the specificity of the same human variants recognizing S100A4 protein and to exclude that the variants do not cross-react with random cellular proteins, we performed fluorescence microscopy experiments 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 protein in knockout fibroblasts, whereas wild-type MEFs showed the typical perinuclear cytoplasmic staining of S100A4 (see Figure 6).

[0129] This result is consistent with previous results showing that 6B12 recognizes only S100A4 (Klingelhofer et al., 2012). Furthermore, no cross-reactivity was observed with S100A2, which shares the most common epitope sequence, and S100A5, which shares the highest overall homology with all members of the S100 family.

[0130] AX-202, described in Example 3, was further shown to bind with similar affinity to S100A4 across four different species (monkey, rat, human, and mouse).

[0131] Example 6 - Humanized anti-S100A4 antibodies reduce activation of S100A4-induced inflammatory pathways

[0132] HEK-Blue hTLR4 (InvivoGen, #hkb-htlr4) cells were incubated in HEK-Blue detection medium (InvivoGen, #hb-det2) and stimulated with increasing concentrations of recombinant human S100A4 multimers or 1.25 ng / ml LPS-EK ultrapure (positive control; InvivoGen, #tlrl-peklps). After 20 hours of incubation, the levels of NF-kB-induced secreted embryonic alkaline phosphatase (SEAP) were measured by reading the OD at 620 nm. S100A4 was shown to clearly activate the NF-kB 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 either 1.25ng / ml LPS-EK ultrapure (InvivoGen, #tlrl-peklps), 1.25 or 2.5μg / ml S100A4. Cells treated with 50ng / ml TNF-α (InvivoGen, #rcyc-htnfa) were used as a positive control for HEK-Blue Null2 cell activation. S100A4 activation of the NF-kB reporter gene was shown to be TLR4 dependent, as addition of S100A4 completely abrogated 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 S100A4 alone and increasing concentrations of the S100A4 neutralizing antibody AX-202 (described in Example 3). While LPS activation of the reporter gene was unaffected by AX-202, AX-202 was shown to inhibit S100A4-induced TLR4 activation in a concentration-dependent manner (see FIG. 7C).

[0135] Example 7 - Alteration of FcR-mediated effector functions by IgG subclass switching of humanized antibodies We were particularly interested in whether subclass switching could affect the activation of FcγRIIa, which may affect therapeutic efficacy. FcγRIIa is an activating FcγR with low affinity for single "monomeric" IgG molecules but high avidity for IgG-containing ICs (Arman et al., 2015).

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

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

[0138] Materials and Methods FcYRIIA reporter assay Three times the final antigen (S100A4) concentration and three times the final antibody concentration 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. The appropriate number of cells was thawed and transferred to assay buffer. 50,000 cells were seeded per well in 25 μl of assay buffer for a final volume of 75 μl / well. Plates were covered with lids and incubated at 37°C and 5% CO2 for 18 hours. After incubation, assay plates were 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] Analyzing the results Data points were imported into Prism Graph Pad (version 9.1.0) and analyzed using unpaired t-tests (two-tailed). Asterisks indicate significant associations at P<0.05 based on n=3.

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

[0141] result Alteration of FcR-mediated effector functions by IgG subclass switching Results obtained so far have shown that mouse IgG1 6B12 and AX-202 with human IgG4 scaffold can inhibit S100A4-induced monocyte cytokine release. Stimulation of monocytes with S100A4 induced IL-6, TNFα, and IL-10 levels in cell culture supernatants. Both AX-202 and 6B12 blocked the increase in IL-6 and IL-10 by S100A4 stimulation, but unexpectedly induced an increase in TNFα. This may suggest that immune complexes composed of oligomeric S100A4 and antibody (6B12) cause TNFα release via FcγR.

[0142] Therefore, the choice of IgG subclass may have a significant impact on unwanted cytokine release.To test this possibility, we performed FcγRIIA reporter assays to evaluate how strongly different antibodies induced FcγR-dependent promoter activity.

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

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

[0145] Receptor clustering was significantly higher for AX-202 hIgG1 compared to AX-202 hIgG4, with fold differences in receptor activation of 5.00 and 3.83 for S100A4-D and S100A4-M, respectively (see FIG. 8). 6B12, which has a mouse IgG1 scaffold, was less able 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. Neither the IgG1 nor the IgG4 control antibodies mediated receptor clustering, indicating that antibody-antigen IC formation is important for FcγRIIa clustering and activation.

[0146] conclusion Antibodies have recently been found to mediate inflammation and immune regulation by inducing cell differentiation, activation, and cytokine release (A van Erp et al., 2019). This can lead to the activation of undesirable proinflammatory pathways, countering the mechanism of action of therapeutic S100A4 antibodies. Herein, we discovered that an S100A4 neutralizing antibody with an IgG4 scaffold has a much better safety profile and is approximately one-third less potent at inducing FcγR activation than 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. An isolated antibody that specifically binds to S100A4, the antibody comprising: a heavy chain variable region (VH) comprising the heavy chain complementarity determining region 1 (CDR-H1), CDR-H2, and CDR-H3 amino acid sequences of the VH amino acid sequence of SEQ ID NO: 13; a light chain variable region (VL) comprising the light chain complementarity determining region 1 (CDR-L1), CDR-L2, and CDR-L3 amino acid sequences of the VL amino acid sequence of SEQ ID NO: 47; Including, An antibody wherein the VH comprises the amino acid sequence of SEQ ID NO: 13 and / or the VL comprises the amino acid sequence of SEQ ID NO:

47.

2. The antibody the CDR-H1 amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4; the CDR-H2 amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5, and CDR-H3 amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 6 The antibody of claim 1, comprising:

3. The antibody the CDR-L1 amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 10; the CDR-L2 amino acid sequence of SEQ ID NO: 8 or YTS, and CDR-L3 amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 12 The antibody of claim 1, comprising:

4. The antibody the CDR-H1 amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 4; the CDR-H2 amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 5; the CDR-H3 amino acid sequence of SEQ ID NO: 3 or SEQ ID NO: 6; the CDR-L1 amino acid sequence of SEQ ID NO: 7 or SEQ ID NO: 10; the CDR-L2 amino acid sequence of SEQ ID NO: 8 or YTS, and CDR-L3 amino acid sequence of SEQ ID NO: 9 or SEQ ID NO: 12 The antibody of claim 1, comprising:

5. The antibody of claim 1, wherein the antibody comprises the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9, respectively.

6. The antibody of claim 1, wherein the antibody comprises the CDR-H1, CDR-H2, CDR-H3, CDR-L1, CDR-L2, and CDR-L3 amino acid sequences of SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:10, YTS, and SEQ ID NO:12, respectively.

7. The antibody of claim 1 , wherein the antibody comprises a heavy chain constant region, or an Fc region thereof.

8. The antibody of claim 7, wherein the heavy chain constant region is selected from the group consisting of human IgG1, human IgG2, human IgG3, and human IgG4.

9. The antibody of claim 7, wherein the heavy chain constant region is human IgG4.

10. The antibody of claim 9, wherein the amino acid sequence of the human IgG4 heavy chain constant region comprises a P at position 228 numbered according to the EU numbering system.

11. The antibody of claim 9, wherein the amino acid sequence of the human IgG4 heavy chain constant region consists of or comprises the amino acid sequence of SEQ ID NO: 56 or the amino acid sequence of a variant having at least 95% sequence identity to SEQ ID NO:

56.

12. The antibody of claim 1 , wherein the antibody comprises a light chain constant region.

13. The antibody of claim 12, wherein the light chain constant region is a human kappa constant region.

14. The antibody of claim 13, wherein the amino acid sequence of the human kappa light chain constant region consists of or comprises the amino acid sequence of SEQ ID NO: 57 or the amino acid sequence of a variant having at least 95% sequence identity to SEQ ID NO:

57.

15. An isolated antibody that specifically binds to S100A4, the antibody comprising a heavy chain variable region (VH) and a light chain variable region (VL); An antibody wherein the VH and VL comprise the amino acid sequences of SEQ ID NO: 13 and SEQ ID NO: 47, respectively.

16. The antibody of claim 15, wherein the antibody comprises a heavy chain constant region, or an Fc region thereof.

17. 17. The antibody of claim 16, wherein the heavy chain constant region is selected from the group consisting of human IgG1, human IgG2, human IgG3, and human IgG4.

18. The antibody of claim 16, wherein the heavy chain constant region is human IgG4.

19. 19. The antibody of claim 18, wherein the amino acid sequence of the human IgG4 heavy chain constant region comprises a P at position 228 numbered according to the EU numbering system.

20. The antibody of claim 15, wherein the antibody comprises a light chain constant region.

21. 21. The antibody of claim 20, wherein the light chain constant region is a human kappa constant region.

22. An isolated antibody that specifically binds to S100A4, said antibody comprising a heavy chain and a light chain, said heavy chain and said light chain comprising the amino acid sequences of SEQ ID NO: 58 and SEQ ID NO: 59, respectively.

23. A nucleic acid molecule or multiple nucleic acid molecules encoding the VH and / or VL, or heavy and / or light chain, of the antibody of claim 1.

24. 24. A vector or vectors comprising the nucleic acid molecule or molecules of claim 23.

25. 24. A host cell comprising the nucleic acid molecule or molecules of claim 23.

26. A composition comprising an antibody of any one of claims 1, 15, and 22, and a pharmaceutically acceptable diluent, carrier, and / or excipient.

27. 26. A method for producing an antibody, comprising culturing the host cell of claim 25 under suitable conditions so that the nucleic acid molecule or molecules are expressed and the antibody is produced.

28. 27. The antibody of any one of claims 1 to 22, the nucleic acid molecule or molecules of claim 23, the vector or vectors of claim 24, the host cell of claim 25, or the composition of claim 26 for use in a method for inhibiting the activity of S100A4 in a subject, the method comprising administering the antibody, the nucleic acid molecule or molecules, the vector or vectors, the host cell, or the composition to the subject.

29. 27. The antibody of any one of claims 1 to 22, the nucleic acid molecule or molecules of claim 23, the vector or vectors of claim 24, the host cell of claim 25, or the composition of claim 26 for use in a method of treating an S100A4-mediated condition in a subject, the method comprising administering the antibody, the nucleic acid molecule or molecules, the vector or vectors, the host cell, or the composition to the subject.

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