Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies
  • C07K16/18—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans
  • C07K16/24—Immunoglobulins [IG], e.g. monoclonal or polyclonal antibodies against material from animals or humans against cytokines, lymphokines or interferons
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P17/00—Drugs for dermatological disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P19/00—Drugs for skeletal disorders
  • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
  • C07K2317/31—Immunoglobulins specific features characterized by aspects of specificity or valency multispecific
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
  • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding

Definitions

• the present invention relates to anti-S100A4 neutralizing antibody molecules and their medical use in the treatment of the disease systemic sclerosis, and more particularly to anti-S100A4 antibody molecules that are capable of inhibiting the biological activity of S100A4, for example in inducing pro-inflammatory signalling and in stimulating TGF-b- induced fibroblast activation and/or collagen synthesis, for the treatment of systemic sclerosis.
• SSc Systemic sclerosis
• S100A4 is a well-known DAMP which is upregulated upon injury, and a strong inducer of both pro-inflammatory and pro-fibrotic pathways through activation of Pattern Recognition Receptors, including RAGE and TLR-4 [4]
• the pro-inflammatory effects are linked to the upregulation of multiple pro- inflammatory cytokines and chemokines such as I L- 1 b , IL-6, TNF-a, serum amyloid A and CCL5; key inflammatory molecules of innate immunity [5,6,7]
• extracellular S100A4 also has a well-documented effect on different elements of the adaptive immune system [8,9,10,11] adding to sustained inflammation, which is important for the pathogenesis of fibrotic disorders [12,13,14]
• TGF-b transforming growth factor b
• TGF-b signaling is persistently activated in both SSc and IPF where it upregulates the synthesis of collagen in fibroblasts and induces fibrosis in vivo
• TGF-b stimulates the expression of S100A4 and in return, S100A4 amplifies TGF ⁇ -induced fibroblast activation and collagen synthesis
• S100A4 plays an important role in inducing, amplifying and sustaining fibrosis through activation of pro-inflammatory and pro-fibrotic pathways.
• TGF-b pathway Directly by the activation of the TGF-b pathway and more indirectly through activation of different immune cells and the secretion of pro-fibrotic cytokines [15]
• anti-S100A4 neutralizing antibodies for the treatment of systemic sclerosis.
• the inventors have tested anti-S100A4 antibodies in two different mouse models for systemic sclerosis and have shown that the antibody treatment is well tolerated and effectively inhibits the pro-fibrotic and pro-inflammatory effects of S100A4 in these disease models.
• the invention additionally provides the first therapeutic approach to selectively inhibit accumulation of extracellular matrix proteins by the pathologically activated fibroblasts in systemic sclerosis.
• the present invention provides an anti-S100A4 antibody for use in the treatment of systemic sclerosis, wherein the antibody is capable of neutralizing a biological activity of S100A4.
• the present invention provides isolated polynucleotides for use in the treatment of systemic sclerosis, which encodes the antibody as described herein.
• the present invention provides a vector comprising the isolated polynucleotide as described herein for use in the treatment of systemic sclerosis.
• the present invention provides an isolated host cell for use in the treatment of systemic sclerosis comprising the isolated polynucleotide or the vector as described herein.
• the present invention provides a pharmaceutical composition for use in the treatment of systemic sclerosis comprising the antibody as described herein together with a pharmaceutically acceptable diluent, carrier and/or excipient.
• the present invention provides a composition for use in the treatment of systemic sclerosis comprising the antibody, and the polynucleotide, the vector and/or the cell as disclosed herein
• the present invention provides a pharmaceutical composition for use in the treatment of systemic sclerosis, comprising the antibody, the polynucleotide, the vector and/or the cell as disclosed herein, further comprising a pharmaceutically- acceptable diluent, carrier and/or excipient.
• Figure 1 shows the relative dermal thickness (and subcutaneous adipose layer thickness) of representative images of tissue samples stained with H&E staining from the 5 different sample groups of example 1. Scale bar: 100 pm. The results are further described in Example 1.
• Figure 2 shows the dermal thickness of samples from the 5 different sample groups of example 1.
• the mean fold change ⁇ SEM of 4 determinations per high power field from 8 mice/group is shown in the graph.
• the value of the NaCI week 1-6 group was set as
• Figure 3 shows the myofibroblast count of samples from the 5 different sample groups of example 1.
• the mean fold change ⁇ SEM is shown in the graph.
• the value of the NaCI week 1-6 group was set as 1.
• the Mann-Whitney U-test was used for selected comparisons. The results are further described in Example 1.
• Figure 4 shows the collagen content of the skin of samples from the 5 different sample groups of example 1.
• the mean fold change ⁇ SEM is shown in the graph.
• the value of the NaCI week 1-6 group was set as 1.
• the Mann-Whitney U-test was used for selected comparisons. The results are further described in Example 1.
• Figure 5 shows CD-3 positive cell count of samples from the 5 different sample groups of example 1.
• the mean fold change ⁇ SEM is shown in the graph.
• the value of the NaCI week 1-6 group was set as 1.
• the Mann-Whitney U-test was used for selected comparisons. The results are further described in Example 1.
• Figure 6 shows the hypodermal thickness of samples from the 4 different sample groups of example 2.
• the mean fold change ⁇ SEM is shown in the graph.
• P-values are expressed as follows: 0.05 > p > 0.01 as *; 0.01 > p > 0.001 as ** as compared to vehicle-treated Tsk1 mice; 0.05 > p > 0.01 as #; 0.01 > p > 0.001 as ## as compared to lgG1-treated Tsk1 mice pa: control mice on the same genetic background not expressing the Tsk1 allele.
• N 10 for all groups.
• Figure 7 shows the myofibroblast counts of samples from the 4 different sample groups of example 2.
• the mean fold change ⁇ SEM is shown in the graph.
• Figure 8 shows the hydroxyproline content of samples from the 4 different sample groups of example 2. The mean fold change ⁇ SEM is shown in the graph.
• Figure 9 shows the number of CD3-positive T cells of samples from the 4 different sample groups of example 2.
• the mean fold change ⁇ SEM is shown in the graph.
• Figure 10 shows the effects of monoclonal humanized anti-S100A4 antibodies on fibrotic readouts in bleomycin-challenged mice. Effects of anti-S100A4 antibodies on dermal thickness (A), myofibroblast counts (B) and hydroxyproline content (C). Representative images of HE stained skin sections are shown in D-E. P-values are expressed as follows: 0.05 > p > 0.01 as *; 0.01 > p > 0.001 as ** as compared to
• FIG. 11 shows IL-6 secretion by monocytes analysed by Luminex analysis. Data shown is the average of 5 donors. Monocytes purified from PBMC were cultured with media, vehicle, LPS, S100A4 in the absence or presence of mouse lgG1 (A), human lgG4 (B), AX-202 (C) or 6B12 (D) for 6 hours. Data shows levels of IL-6 in the supernatant quantified by Luminex assay.
• FIG. 12 shows IL-10 secretion by monocytes analysed by Luminex analysis. Data shown is the average of 5 donors. Monocytes purified from PBMC were cultured with media, vehicle, LPS, S100A4 in the absence or presence of mouse lgG1 (A), human lgG4 (B), AX-202 (C) or 6B12 (D) for 6 hours.
• FIG 13 shows TNF-a secretion by monocytes analysed by Luminex analysis. Data shown is the average of 5 donors. Monocytes purified from PBMC were cultured with media, vehicle, LPS, S100A4 in the absence or presence of mouse lgG1 (A), human lgG4 (B), AX-202 (C) or 6B12 (D) for 6 hours. Data shows levels of TNF-a in the supernatant quantified by Luminex assay. Data presented as mean + SEM arising from five independent donors. indicates at least one donor below the limit of detection for TNF-a (15.20 pg/ml). The results are further described in Example 4.
• anti-S100A4 antibody encompasses any substantially intact antibody, as well as chimeric antibodies, humanised antibodies, isolated human antibodies, single chain antibodies, polyclonal antibodies, monoclonal antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy and/or light chains, and antigen-binding fragments and derivatives of the same, that specifically recognizes an S100A4 antigen.
• Suitable antigen-binding fragments and derivatives include, but are not necessarily limited to, Fv fragments (e.g. single chain Fv and disulphide-bonded Fv), Fab-like fragments (e.g.
• Fab fragments, Fab’ fragments and F(ab’)2 fragments single variable domains (e.g. VH and VL domains) and domain antibodies (dAbs, including single and dual formats [i.e. dAb-linker-dAb]).
• dAbs including single and dual formats [i.e. dAb-linker-dAb]).
• the potential advantages of using antibody fragments, rather than whole antibodies, are several-fold.
• the smaller size of the fragments may lead to improved pharmacological properties, such as better penetration of solid tissue.
• antigen-binding fragments such as Fab, Fv, ScFv and dAb antibody fragments can be expressed in and secreted from E. coli and cell lines, thus allowing the facile production of large amounts of the said fragments.
• the protein S100A4 is also known as 18A2, 42A, CAPL, FSP1, MTS1, P9KA, PEL98 and S100 calcium binding protein A4.
• the singular forms “a”, “an” and “the” include plural referents unless the context clearly states otherwise.
• reference to “an antibody” includes a plurality of such antibodies.
• variant defines either a naturally occurring genetic mutant of a DNA sequence or its encoded RNA or protein product, or a recombinantly prepared variation of a DNA sequence or its encoded RNA or protein product.
• variant may also refer to either a naturally occurring variation of a given peptide or a recombinantly prepared variation of a given peptide or protein in which one or more amino acid residues have been modified by amino acid substitution, addition, or deletion.
• “Inhibition” as used herein means that the presence of the antibody of the invention inhibits, in whole or in part, the binding of ligands to their receptor and/or the disablement of a signal the receptor would elicit upon ligand binding. This includes for example down-stream signalling having effect on cellular behaviour and processes. Also included are other mechanisms of inhibiting the downstream effects of the targeted molecule, such as by blocking dimerization, oligomerization and/or multimerization of the target molecule. “Inhibition”, “blocking” and “neutralizing” are used herein as equivalent terms.
• the present invention relates to anti-S100A4 neutralizing antibodies for use in the treatment of systemic sclerosis.
• an anti-S100A4 antibody for use in the treatment of systemic sclerosis, wherein the antibody is capable of neutralizing a biological activity of S100A4.
• the antibody specifically binds to human the S100A4 polypeptide as set forth in SEQ ID NO: 11, preferably to an epitope contained between amino acids 66 and 89 of SEQ I D NO: 11. In some embodiments, the antibody is capable of binding to an epitope defined by SEQ ID NO: 12. In some embodiments, the antibody is capable of binding to an epitope defined by SEQ ID NO: 13. In some embodiments, the antibody is capable of binding to an epitope defined by SEQ ID NO: 14. In some embodiments, the antibody is capable of binding to the S100A4 protein in its native conformation.
• the antibody is capable of binding to dimeric, oligomeric and/or multimeric forms of S100A4 protein.
• the composition of the anti-S100A4 antibody is as described in US 9,683,032.
• the antibody comprises: a) a heavy chain variable (VH) region comprising: i. a CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1; ii. a CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 2; and iii. a CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 3; and/or b) a light chain variable (VL) region comprising: i. a CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 4; ii.
• VH heavy chain variable
• a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 5; and iii. a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 6; or a CDR variant of any one of SEQ ID NO:s 1 to 6, wherein any one amino acid has been altered for another amino acid, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered in each CDR.
• the heavy chain variable (VH) region of the antibody comprises SEQ ID NO: 7 or a variant thereof having at least 80% identity, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto, and the light chain variable (VL) region of the antibody comprises SEQ ID NO: 9 or variant thereof having at least 80% identity, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 8
• the composition of the anti-S100A4 antibody may be as described in US 9,657,092.
• the antibody comprises: a) a heavy chain variable (VH) region comprising: i. a CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 15; ii. a CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 16; and iii. a CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 17; and/or b) a light chain variable (VL) region comprising: i. a CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 18; ii.
• VH heavy chain variable
• a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 19; and iii. a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 20; or a CDR variant of any one of SEQ ID NO:s 15 to 20, wherein any one amino acid has been altered for another amino acid, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered in each CDR.
• the heavy chain variable (VH) region of the antibody comprises SEQ ID NO: 21 or a variant thereof having at least 80% identity, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto, and the light chain variable (VL) region of the antibody comprises SEQ ID NO: 23 or variant thereof having at least 80% identity, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 8
• the antibody is produced by the ECACC 10022401 hybridoma.
• the antibody is produced by the ECACC 11051801 hybridoma.
• the antibody is produced by the ECACC 11051802 hybridoma.
• the antibody is produced by the ECACC 11051803 hybridoma.
• the antibody is produced by the ECACC 11051804 hybridoma.
• monoclonal antibodies might be useful since they bind to specific binding sites thereby inhibiting access of other molecules to this specific site.
• the antibody is a bispecific antibody.
• the antibody is a complete antibody. In some embodiments, the antibody is a Fab fragment. In some embodiments, the antibody is a F(ab')2 fragment.
• the antibody is a Fab-like fragment. In some embodiments, the antibody is a scFv. In some embodiments, the antibody is a nanobody, also known as a single-domain antibody. In some embodiments, the antibody is a diabody. In some embodiments, the antibody is a triabody.
• the antibody is an lgG1 subclass antibody. In some embodiments, the antibody is an lgG2 subclass antibody. In some embodiments, the antibody is an lgG3 subclass antibody. In some embodiments, the antibody is an lgG4 subclass antibody. Preferably the subclasses are human IgG subclasses. In some embodiments, the antibody comprises an Fc domain with a mutated IgG constant region.
• the antibody may be useful to use an antibody with an immunoglobulin subclass that elicits a weak or no pro-inflammatory response in the host.
• the lgG4 subclass may in particular be useful when reduced effector or cross-linking functions of the antibody are desired.
• the antibody is therefore a human lgG4 subclass antibody.
• the antibody comprises a human heavy chain constant (CH) region comprising or consisting of the sequence as set forth in SEQ ID NO: 27.
• the antibody comprises a CH region comprising or consisting of a variant of SEQ ID NO: 27, said variant having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
• the antibody comprises a human light chain constant (CL) region comprising or consisting of the sequence as set forth in SEQ ID NO: 28.
• the antibody comprises a CL region comprising or consisting of a variant of SEQ ID NO: 28, said variant having at least 80%, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% sequence identity thereto.
• the antibody comprises an Fc domain with a mutated human IgG constant region.
• the antibody comprises a mutant human lgG4 heavy chain constant region.
• said mutant lgG4 heavy chain constant region comprises an S228P substitution, numbering according to EU numbering. Said S228P substitution may prevent in vivo and in vitro lgG4 Fab-arm exchange, which can result in functionally monovalent, bispecific antibodies (bsAbs) with unknown specificity and therefore, potentially, reduced therapeutic effect.
• the terminal lysine of the human lgG4 heavy chain constant region has been removed.
• the antibody is PEGylated.
• the antibody is a humanised antibody. In some embodiments, the antibody is a chimeric antibody. In some embodiments, the antibody is a humaneered antibody. Antibody function and treatment effects
• the antibody is capable of neutralizing a biological activity of S100A4.
• S100A4 is thought to stimulate TGF-b- induced fibroblast activation and collagen synthesis, and to mediate pro-inflammatory effects such as T-cell and/or macrophage recruitment and/or infiltration.
• the antibody is capable of inhibiting T-cell and/or macrophage recruitment and/or infiltration mediated by S100A4.
• the antibody is capable of inhibiting the biological activity of S100A4 protein in stimulating cell invasion.
• treatment with the anti-S100A4 antibody reduces fibrosis. Fibrosis may be assessed by measuring dermal thickness, dermal hydroxyproline content, dermal CD3 + cell count and/or dermal myofibroblast counts by methods known in the art. Thus, in some embodiments, treatment with the anti-S100A4 antibody reduces dermal thickness, dermal collagen or hydroxyproline content, dermal myoblast count and/or T-cell count.
• the antibody is administered for treatment of systemic sclerosis via parenteral administration such as subcutaneously, intramuscularly or intravenously.
• parenteral administration such as subcutaneously, intramuscularly or intravenously.
• the antibody is administered every week, such as every 2 weeks, such as every 3 weeks, for example every 4 weeks.
• the antibody is administered once or twice weekly, with a weekly dosage of 5 mg, such as 10 mg, such as 15 mg, such as 20 mg, such as 25 mg, such as 30 mg, such as 40 mg, such as 50 mg, such as 60 mg, such as 75 mg, such as 100 mg, such as 125 mg, such as 150 mg, such as 175 mg, such as 200 mg, such as 225 mg, such as 250 mg, such as 275 mg, such as 300 mg, such as 325 mg, such as 350 mg, such as 375 mg, such as 400 mg, such as 450 mg, such as 500 mg or more. It may be beneficial to combine treatment with anti-S100A4 antibody together with treatment with other compounds useful for the treatment of systemic sclerosis.
• the antibody is co-administered with another compound for treatment of systemic sclerosis.
• the antibody is co-administered with an angiotensin-converting enzyme inhibitor.
• the antibody is co-administered with an angiotensin receptor blocker.
• the antibody is co-administered with an azathioprine.
• the antibody is co-administered with a calcium channel blocker.
• the antibody is co-administered with a cyclophosphamide.
• the antibody is co- administered with a hydroxychloroquine.
• the antibody is co administered with a mycophenolate. In some embodiments, the antibody is co administered with a 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.
• polynucleotides, vectors and cells encoding or expressing the antibody
• the present invention provides an isolated polynucleotide for use in the treatment of systemic sclerosis, which encodes the antibody as described herein.
• the polynucleotide encoding the heavy chain variable (VH) region of the antibody comprises SEQ ID NO: 8 or a variant thereof having at least 80% identity, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto.
• the polynucleotide encoding the light chain variable (VL) region of the antibody comprises SEQ ID NO: 10 or variant thereof having at least 80% identity, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto.
• the polynucleotide encoding the antibody encodes a VH region defined by SEQ ID NO: 8 and a VL region defined by SEQ ID NO: 10.
• the polynucleotide encoding the heavy chain variable (VH) region of the antibody comprises SEQ ID NO: 22 or a variant thereof having at least 80% identity, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto.
• the polynucleotide encoding the light chain variable (VL) region of the antibody comprises SEQ ID NO: 24 or variant thereof having at least 80% identity, such as at least 81%, such as at least 82%, such as at least 83%, such as at least 84%, such as at least 85%, such as at least 86%, such as at least 87%, such as at least 88%, such as at least 89%, such as at least 90%, such as at least 91%, such as at least 92%, such as at least 93%, such as at least 94%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, such as at least 99% identity thereto.
• the polynucleotide encoding the antibody encodes a VH region defined by SEQ ID NO: 22 and a VL region defined by SEQ ID NO: 24.
• the present invention provides a vector comprising the isolated polynucleotide as described herein for use in the treatment of systemic sclerosis.
• the present invention provides an isolated host cell for use in the treatment of systemic sclerosis comprising the isolated polynucleotide or the vector as described herein.
• compositions comprising the antibody
• the antibody as described herein may be comprised in a pharmaceutical composition for use in the treatment of systemic sclerosis, together with a pharmaceutically acceptable diluent, carrier and/or excipient.
• the present invention provides a pharmaceutical composition for use in the treatment of systemic sclerosis comprising the antibody as described herein together with a pharmaceutically acceptable diluent, carrier and/or excipient.
• the present invention provides a composition for use in the treatment of systemic sclerosis comprising the antibody as disclosed herein, and the polynucleotide, the vector and/or the cell as disclosed herein above in the section “Polynucleotides, vectors and cells encoding or expressing the antibody”.
• the present invention provides a pharmaceutical composition for use in the treatment of systemic sclerosis, comprising the antibody as disclosed herein, and the polynucleotide, the vector and/or the cell as disclosed herein above in the section “Polynucleotides, vectors and cells encoding or expressing the antibody”, and further comprising a pharmaceutically-acceptable diluent, carrier and/or excipient.
• Example 1 Anti-S100A4 antibody efficacy in bleomycin-induced dermal fibrosis
• mice challenged by BLM were intraperitoneally (i.p.) injected each with 100 to 200 mI_ of following concentration of S100A4-neutralizing antibody (SNA): 2.5, or 7.5 mg/kg/d, started on day 22 and continued every third day for 3 weeks.
• SNA S100A4-neutralizing antibody
• 100 mI_ NaCI was injected s.c. every other day for 6 weeks.
• BLM-challenged mice were i.p. injected with 100 mI_ PBS, started on day 22 and continued every third day for 3 weeks.
• a further control was included to determine skin fibrosis upon withdrawal of the BLM- challenge.
• mice were injected by both, (i) 100 pL NaCI s.c. started on day 22 every other day for 3 weeks and (ii) vehiculum control PBS i.p. Mice were sacrificed at day 42, and skin, blood and lungs were collected for analysis. Dermal thickness, collagen content as well as numbers of myofibroblasts and CD3 + cells in the skin tissue were assessed.
• Group 4 BLM (w 1-6) s.c. + SNA (w 4-6) 2.5 mg/kg i.p.
• Group 5 BLM (w 1-6) s.c. + SNA (w 4-6) 7.5 mg/kg i.p.
• BLM was dissolved in 0.9% NaCI in a concentration of 0.5 mg/mL.
• the antibodies were diluted in sterile PBS and injected i.p. in a volume of 100uL for group 4 and 5 and 200 pL for group 6, respectively.
• the anti-S100A4 antibody which was shipped to the study site had following concentration of 2 mg/ml_, batch # Lot 003P10T120116JKL and date 01/2016.
• mice were observed regularly (at every animal house visit) for their vital signs, activity, quality and texture of their fur. In addition, mice were weighted weekly on a calibrated scale on the same day and hour from right before the start of BLM administration, till right before their sacrifice. Upon autopsy of mice, all internal organs were screened for macroscopically visible pathologies (e.g. tumors, hemorrhages etc.). Prior to the commencement of experiments, all 48 mice were randomly allocated into 6 cages by the personnel of the animal house, who was blinded to the aims of this project.
• pathologies e.g. tumors, hemorrhages etc.
• the injected skin areas were excised, subsequently fixed in 4 % formalin for 6 h, thereafter in 70% ethanol, and embedded in paraffin. Skin sections were cut and stained with hematoxylin/eosin. Dermal thickness at the injection sites was analyzed with a BX53 microscope with a DP80 Digital Microscope Camera and CellSens Standard Software (Olympus, Philadelphia, PA, USA) at 100-fold magnification. The dermal thickness was determined by measuring the largest distance between the epidermal-dermal junction and the dermal-subcutaneous fat junction as described [16] The dermal thickness was quantified in 4 different sections from different sites with 4 measurements per section. The analysis was performed in a blinded manner.
• the amount of collagen protein in skin samples was determined via hydroxyproline assay. After digestion of full skin thickness punch biopsies (04 mm, two samples/ animal) derived from the upper back in 6 M HCI for three hours at 120 °C, the pH of the samples was adjusted to 6 with 6 M NaOH. Afterwards, 0.06 M chloramine T was added to each sample and incubated for 20 min at room temperature. Next, 3.15 M perchloric acid and 20 % p-dimethylaminobenzaldehyde were added and samples were incubated for additional 20 min at 60 °C. The absorbance was determined at 557 nm with a Spectra MAX 190 microplate spectrophotometer (Molecular Devices) for each of the two full skin thickness punch biopsies.
• Myofibroblasts are characterized by the expression of a-smooth muscle actin (aSMA). Fibroblasts positive for aSMA were detected in paraffin-embedded skin sections from the upper back by incubation with monoclonal mouse anti-aSMA antibodies (Merck, A5228). The right concentration of the antibody for the staining of the antibody was determined by dilution row experiment and reviled a dilution of 1:1000 with a concentration of 2pg/ml_.
• aSMA smooth muscle actin
• CD3 is a multimeric protein complex serving as a T cell co-receptor. Being a defining feature of the T cell lineage, CD3 can be used as a marker for T cells. Cells positive for CD3 were detected in paraffin-embedded skin sections from the upper back by incubation with polyclonal rabbit anti-CD3 antibodies (Abeam, Ab5690). The right concentration of the antibody for the staining of the antibody was determined by dilution row experiment and reviled a dilution of 1:50 with a concentration of 4 pg/mL.
• the sections were then counterstained with Mayer’s hematoxylin solution and visualized with a BX53 microscope with a DP80 Digital Microscope Camera and CellSens Standard Software (Olympus, Philadelphia, PA, USA) at 100- and 200-fold magnification.
• the analysis was performed by a blinded reviewer counting the number of the mononucleated (with hematoxylin-stained nucleus) circle-shaped cells stained positive for CD3 in the perivascular infiltrates of the subcutaneous tissue in four sections per sample.
• P-values less than 0.05 were considered significant. P-values are expressed as follows: 0.05 > p > 0.01 as *; 0.01 > p > 0.001 as **; p ⁇ 0.001 as ***.
• BLM induced prominent skin fibrosis in the exposed skin area with dermal thickening, myofibroblast differentiation and proliferation and increased hydroxyproline content that progressed during treatment.
• Treatment with 7.5 mg/kg anti-S100A4 antibody reduced the dermal thickness (as seen in Figure 1 and 2), number of myofibroblasts (as seen in Figure 3), hydroxyproline content (as seen in Figure 4) as well as the number of CD3 positive cells to levels below BLM-withdrawal, i.e. mice challenged with BLM for the first three weeks and then injected with NaCI, (as seen in Figure 5), thus suggesting a reduction to the pre treatment levels of fibrosis.
• These observed effects in this group thus suggest therapeutic efficacy of 7.5 mg/kg anti-S100A4 antibody in both prevention of progression of dermal fibrosis and treatment of pre-established dermal fibrosis induced by repetitive subcutaneous injections of bleomycin in mice.
• Tsk-1 Tight-skin-1 (Tsk-1) model
• Tsk-1 mice treated with anti-S100A4 antibodies 7.5 mg/kg, 2mg/ml; injection every Monday, Wednesday and Friday
• Myofibroblasts are characterized by the expression of a-smooth muscle actin (aSMA). Fibroblasts positive for aSMA were detected in paraffin-embedded slides from the upper back by incubation with monoclonal anti-aSMA antibodies (clone 1A4, Sigma- Aldrich, Steinheim, Germany). The expression was visualized with horseradish peroxidase labeled secondary antibodies and 3,3-diaminobenzidine tetrahydrochloride (DAB) (Sigma-Aldrich). Monoclonal mouse IgG antibodies (Calbiochem, San Diego, CA, USA) were used for controls. The analysis was performed by a blinded reviewer (JD) evaluating the myofibroblasts in four sections per sample.
• JD blinded reviewer
• Hydroxyproline assay The amount of collagen protein in skin samples was determined via hydroxyproline assay. After digestion of full skin thickness punch biopsies (03 mm) derived from the upper back in 6 M HCI for three hours at 120 °C, the pH of the samples was adjusted to 6 with 6 M NaOH. Afterwards, 0.06 M chloramine T was added to each sample and incubated for 20 min at room temperature. Next, 3.15 M perchloric acid and 20 % p- dimethylaminobenzaldehyde were added and samples were incubated for additional 20 min at 60 °C. The absorbance was determined at 557 nm with a Spectra MAX 190 microplate spectrophotometer. Staining for CD3/T cells
• Tsk-1 mice developed prominent skin fibrosis with hypodermal thickening, myofibroblast differentiation and increased hydroxyproline content (as seen in Figures 6 to 9).
• Example 3 Humanized anti-S100A4 antibody efficacy in treating bleomycin-induced dermal fibrosis in vivo
• SSc Systemic sclerosis
• SSc Systemic sclerosis
• SSc is a systemic fibrosing orphan disease with high morbidity and mortality.
• SSc is the condition with the highest case specific mortality of any of the autoimmune rheumatic diseases with more than half of cases diagnosed with the condition eventually dying as a direct consequence.
• the hallmark of the disease is accumulation of extracellular matrix proteins by pathologically activated fibroblasts.
• Therapeutic approaches to selectively inhibit the aberrant release of extracellular matrix in SSc are not available to date.
• Bleomycin-induced dermal fibrosis is the most commonly used mouse model of SSc. It resembles in particular early, inflammatory stages of SSc.
• AX-202 a humanized lgG4 mono-clonal anti-S100A4 antibody, was used for this study.
• the antibody comprises a heavy chain sequence as defined in SEQ ID NO: 25 and a light chain sequence as defined in SEQ ID NO: 26.
• the antibody was dissolved in PBS and stored at -20°C.
• 2 mg/ml_ antibody stock solution was diluted in sterile PBS and injected i.p. in a volume of 100 mI_.
• Skin fibrosis was induced by daily subcutaneous injections of bleomycin (2.5 mg/kg, Sigma-Aldrich) in defined and marked areas of the upper back (1 cm 2 ) for up to six weeks. Treatment was commenced after three weeks of pre-challenge with bleomycin, with injections twice weekly intraperitoneally (i.p.) or once every week with intravenous (IV) injection in the tail vein. The outcome was analysed three weeks after the first injection of bleomycin (six weeks after the first bleomycin-injection).
• Group 1 Control / NaCI Group 2: bleomycin 3 weeks and NaCI 3 weeks
• Group 3 bleomycin 6 weeks + NaCL for last 3 weeks (every 3rd day IP)
• Group 4 bleomycin 6 weeks + AX-202 16 mg/kg for the last 3 weeks (twice a week IP)
• Group 5 bleomycin 6 weeks + AX-20224 mg/kg for the last 3 weeks (weekly IV tail vein injection)
• Group 6 bleomycin 6 weeks + AX-202 8 mg/kg for the last 3 weeks (twice a week IP)
• mice were monitored clinically on a daily basis for behavior, activity, texture of the fur and consistency of the stool. After sacrifice, a gross macroscopic evaluation of the lungs and the skin was performed.
• Myofibroblasts are characterized by the expression of a-smooth muscle actin (aSMA). Fibroblasts positive for aSMA were detected in paraffin-embedded slides from the upper back by incubation with monoclonal anti-aSMA antibodies (clone 1A4, Sigma- Aldrich, Steinheim, Germany). The expression was visualized with horseradish peroxidase-labelled secondary antibodies and 3,3-diaminobenzidine tetrahydrochloride (DAB) (Sigma-Aldrich). Monoclonal mouse IgG antibodies (Calbiochem, San Diego,
• mice developed prominent dermal fibrosis upon challenge with bleomycin with more pronounced fibrotic changes in mice challenged with bleomycin for 6 weeks as compared to mice challenged with bleomycin for 3 weeks followed by injections of the solvent of bleomycin, NaCI, for another 3 weeks. Mice injected with NaCI for 6 weeks served as controls.
• PBMCs Peripheral blood mononuclear cells
• FicollPaque PLUS GE Healthcare; 11778538
• monocytes isolated using a monocyte isolation kit (StemCell Technologies; 19359).
• Monocytes were plated (100,000 cells/well) into 96 well plates and cultured for 6 hours in the presence of:
• 6B12 mouse monoclonal lgG1 anti-S100A4 antibody (VH regions and VL regions as defined in SEQ ID NO: 7 and SEQ ID NO: 9, respectively)
• AX-202 humanized monoclonal lgG4 anti-S100A4 antibody as described in Example 3
• cytokines IL-6, TNF-a and IL-10
• Luminex assay according to manufacturer’s instructions
• S100A4 evoked an increase in the levels of IL-6, TNFa and IL-10 (see Figures 2-4).
• S100A4-evoked IL-6 and IL-10 release was reduced by both AX-202 and 6B12 (see Figures 11C-D and 12C-D).
• mouse lgG1 control, human lgG4 control, or AX-202 in combination with S100A4 did not result in significant increases in TNFa levels compared to S100A4 alone (see Figures 13A-B).
• the levels of S100A4-induced pro-inflammatory cytokine TNFa were increased by treatment with the 6B12 antibody in a dose- dependent fashion (see Figures 13C-D).
• the humanized anti-S100A4 antibody does not increase S100A4-induced pro-inflammatory TNFa levels compared to the mouse anti- S100A4 antibody 6B12.
• An anti-human S100A4 antibody for use in the treatment of systemic sclerosis.
• the antibody for the use according to item 1 wherein the antibody specifically binds to human S100A4 polypeptide of SEQ ID NO: 11 , preferably to an epitope contained between amino acids 66 and 89 of SEQ ID NO: 11.
• a heavy chain variable (VH) region comprising: i. a CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 1 ; ii. a CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 2; and iii. a CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 3; and b) a light chain variable (VL) region comprising: i. a CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 4; ii.
• VH heavy chain variable
• a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 5; and iii. a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 6; or a CDR variant of any one of SEQ ID NO:s 1 to 6, wherein any one amino acid has been altered for another amino acid, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered in each CDR.
• the heavy chain variable (VH) region comprises of SEQ ID NO: 7 or a variant thereof having at least 80% identity, such as at least 85%, such as at least 90%, such as at least 95% identity thereto
• the light chain variable (VL) region comprises of SEQ ID NO: 9 or variant thereof having at least 80% identity, such as at least 85%, such as at least 90%, such as at least 95% identity thereto, preferably wherein the antibody has a VH region defined by SEQ ID NO: 7 and a VL region defined by SEQ ID NO: 9.
• the antibody for the use according to any one of the preceding items wherein the antibody comprises: a) a heavy chain variable (VH) region comprising: i. a CDR-H1 comprising or consisting of the amino acid sequence of SEQ ID NO: 15; ii. a CDR-H2 comprising or consisting of the amino acid sequence of SEQ ID NO: 16; and iii. a CDR-H3 comprising or consisting of the amino acid sequence of SEQ ID NO: 17; and b) a light chain variable (VL) region comprising: i. a CDR-L1 comprising or consisting of the amino acid sequence of SEQ ID NO: 18; ii.
• VH heavy chain variable
• a CDR-L2 comprising or consisting of the amino acid sequence of SEQ ID NO: 19; and iii. a CDR-L3 comprising or consisting of the amino acid sequence of SEQ ID NO: 20; or a CDR variant of any one of SEQ ID NO:s 15 to 20, wherein any one amino acid has been altered for another amino acid, with the proviso that no more than 3 amino acids have been so altered, for example wherein 2, or 1 amino acids have been so altered in each CDR.
• the antibody for the use according to any one of the preceding items wherein the antibody is selected from the group consisting of a complete antibody, a Fab fragment, a F(ab')2 fragment, a Fab-like fragment, a scFv, a single-domain antibody, a diabody, and a triabody.
• the antibody molecule is a humanised antibody, a chimeric antibody or a humaneered antibody.
• angiotensin-converting enzyme inhibitor an angiotensin receptor blocker, an azathioprine, a calcium channel blocker, a cyclophosphamide, a hydroxychloroquine, a mycophenolate, a methotrexate, a glucocorticoid, a phosphodiesterase-5 inhibitor, an endothelin receptor antagonist, an alpha blocker, a prostanoid, rituximab, a tyrosine kinase inhibitor such as nintedanib, and tociluzimab.

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