WO2023072405A1 - Ykl-40 antibody and uses thereof - Google Patents

Ykl-40 antibody and uses thereof Download PDF

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Publication number
WO2023072405A1
WO2023072405A1 PCT/EP2021/080177 EP2021080177W WO2023072405A1 WO 2023072405 A1 WO2023072405 A1 WO 2023072405A1 EP 2021080177 W EP2021080177 W EP 2021080177W WO 2023072405 A1 WO2023072405 A1 WO 2023072405A1
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seq
ykl
antibody
cancer
variable region
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PCT/EP2021/080177
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French (fr)
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Peter Kristensen
Peter MINOR
Udo Schumacher
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Bio-Y A/S
Universitätsklinikum Hamburg-Eppendorf
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Priority to PCT/EP2021/080177 priority Critical patent/WO2023072405A1/en
Publication of WO2023072405A1 publication Critical patent/WO2023072405A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
    • A61K33/243—Platinum; Compounds thereof
    • 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
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2803—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the immunoglobulin superfamily
    • 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
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®

Definitions

  • the present invention relates to YKL-40 antibodies capable of binding to YKL-40.
  • the invention relates to the treatment of solid malignant tumours with YKL- 40 antibodies, said treatment resulting in increase in malignant tumour permeability thereby allowing or facilitating access of an anti-cancer agent to the malignant tumour.
  • YKL-40 is a 40 kDa heparin- and chitin-binding glycoprotein also known as human cartilage glycoprotein 39 (HC gp-39), 38-kDa heparin-binding glycoprotein or chitinase- 3-like protein 1 (CHI3L1).
  • the abbreviation YKL-40 is based on the one letter code for the first three N-terminal amino acids, tyrosine (Y), lysine (K) and leucine (L) and the apparent molecular weight of YKL-40.
  • YKL-40 was first identified as a protein secreted in large amounts by a human osteosarcoma cell line MG63 in vitro. Later studies have found that YKL-40 is secreted in vitro by a variety of cells and seems especially involved in activation of the innate immune system and in cell processes in relation to extracellular matrix remodeling.
  • the crystallographic structure of human YKL-40 has been described and the protein contains two globular domains: a big core domain which consists of a (p/a)s domain structure with a triose-phosphase isomerase (TIM) barrel fold and a small a/p domain, composed of five antiparallel p-strands and one a-helix, inserted in the loop between strand p7 and helix a7. This confers the active site of YKL-40 a groove-like character.
  • a big core domain which consists of a (p/a)s domain structure with a triose-phosphase isomerase (TIM) barrel fold
  • TIM triose-phosphase isomerase
  • YKL-40 can also bind to hyaluronan.
  • the folded protein contains two potential hyaluronan binding sites predicted by in silico methods. Binding of short and long oligosaccharides to human YKL-40 are also possible.
  • YKL-40 possesses a number of biological activities. It has been shown that human YKL-40 can acts as a growth factor for cells of connective tissue, such as chondrocytes and synovial cells. YKL-40 also promotes the growths of fibroblasts in a fashion similar to insulin-like growth factor 1 (IGF-1). It has also been demonstrated that YKL-40 can act as a chemoattractant for endothelial cells and stimulates migration of these cells comparable to stimulation by basic fibroblast growth factor. YKL-40 is also found to modulate vascular endothelial cell morphology by promoting formation of branching tubules.
  • IGF-1 insulin-like growth factor 1
  • YKL-40 mRNA in human liver has been shown to be associated with the presence of fibrosis.
  • Immunohistochemical studies of liver biopsies have shown YKL-40 protein expression in areas of the liver with fibrosis, whereas no expression was observed in hepatocytes.
  • Patients with non-malignant diseases characterized by inflammation and fibrosis such as active rheumatoid arthritis, severe bacterial infections, active inflammatory bowel disease, and liver fibrosis have elevated serum YKL-40.
  • YKL-40 is expressed and secreted by several types of human malignancies. Furthermore, YKL-40 is found to be secreted in vitro by the osteoscarcoma cell line MG63, glioblastoma cells and myeloid leukemia cell lines. A number of studies have reported an elevated level of YKL-40 protein in serum of cancer patients.
  • YKL-40 activity is found to be associated with cell growth, survival, differentiation, apoptosis, angiogenesis, extracellular matrix remodeling, development of metastasis, development of liver or tissue fibrosis, development of rheumatoid arthritis and/or development of inflammation.
  • the effectiveness of anticancer drug therapy in treating solid malignant tumours depends on the drug reaching all cancer cells in the malignant tumour.
  • the drug distribution in malignant tumour tissue is dependent on the plasma pharmacokinetics and pharmacodynamics, the structure and function of the malignant tumour vasculature and the ability of the drug to move through microvessel walls and its diffusion and convection across the ECM and the layers of the malignant tumour.
  • the drug For chemotherapy of solid malignant tumours for example, the drug must be able to traverse walls of blood vessels and the surrounding ECM to reach malignant tumour cells.
  • malignant tumours form several layers of malignant tumour and/or matrix cells which all have to be penetrated.
  • Matrix cells may for example be fibrous connective tissue.
  • the structure and flow distribution in the system of microvessels supplying the malignant tumour and the properties of extravascular tissue components such as ECM, normal cells, malignant tumour cells and interstitial spaces play a major role in this regard.
  • solid malignant tumours have a raised IFP due to high vessel permeability, low lymphatic drainage, poor perfusion, and high cell density around the blood vessels (Ferretti et al. 2009).
  • the raised interstitial fluid barrier creates a distribution barrier within solid malignant tumours which limits the access of therapeutic agents towards all malignant cells.
  • Solid malignant tumours form organoid like structures, which may be composed of a variety of tissue components.
  • the malignant tumour cells themselves which in the case of solid malignant tumours, cancers, generally form epithelial structures as the cells are linked to each other by cell to cell junctions including desmosomes, tight junctions, gap junctions. These junctions are responsible for the formation of a “cancer tissue”, which is composed of several cancer cells of the same type.
  • the malignant tumour In addition to the cancer cells themselves the malignant tumour typically builds around its vascular supply which is sheathed in a connective tissue ECM, collectively called the malignant tumour stroma.
  • the stromal component of normal organs contains blood and lymphatic vessels, the latter being absent in cancer tissue stroma.
  • convection is a directed fluid flow from the capillaries to the lymphatic vessels.
  • convection is a directed fluid flow from the capillaries to the lymphatic vessels.
  • This fluid pressure is believed to limit the access of therapeutic antibodies and of classical small molecular weight chemotherapeutic drugs (small molecule drugs) to areas within the malignant tumour which are located 100 pm around the small exchange blood vessels. Thereby most of the malignant tumour cells within solid malignant tumours are not reached by the possible therapeutic drugs and/or antibodies.
  • YKL-40 antibodies useful in treatment of cancer characterised by the presence of at least one solid neoplasm.
  • solid neoplasms are difficult to treat due to reduced access of antibodies or other anti-cancer agents to areas within the solid neoplasms.
  • the present invention provides YKL-40 antibodies, which upon administration to an individual suffering from cancer, are capable of entering into the interior of solid neoplasms.
  • the YKL-40 antibodies of the invention can act in the interior of solid neoplasms.
  • the YKL-40 antibodies of the present invention are preferably capable of facilitating access of other compounds into the inner mass of a malignant tumour, thereby facilitating access of e.g. anti-cancer agents.
  • YKL-40 antibodies may facilitate the access of anti-cancer agents to solid neoplasms, and thus anti-cancer agents may advantageously be administered to an individual suffering from a solid neoplasm either together with or before or subsequent to administration of the YKL-40 antibodies.
  • the invention provides antibodies useful for administration to human beings are provided because said antibodies contains mainly human antibody sequences.
  • the present invention relates to an YKL-40 antibody comprising a variable region comprising a CDR1 , CDR2 and CDR3, wherein
  • CDR1 comprises or consists of an amino acid sequence of SEQ ID NO:20
  • CDR2 comprises or consists of an amino acid sequence of the general formula:
  • Xi is Q, S, N, D, G, E or Y;
  • X 2 is S, G, N, A, D or Y;
  • X 3 is S, E, N, Y, G, D or P;
  • X4 is D, G, S or N.
  • CDR3 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NQ:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NQ:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NQ:50, SEQ ID NO:52, SEQ ID NO:54 and/or SEQ ID NO:57, preferably SEQ ID NO:57.
  • the present invention relates to an YKL-40 antibody comprising or consisting of a variable region comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19.
  • the present invention relates to a method of treating a clinical condition associated with aberrant YKL-40 expression, the method comprising administering a therapeutically effective amount of a YKL-40 antibody as described herein above to a subject in need thereof.
  • kit-of parts and/or a composition comprising a. a YKL-40 antibody; and b. an anti-cancer agent; for use in the treatment of cancer in an individual in need thereof, wherein said cancer is characterised by the presence of at least one solid neoplasm.
  • Figure 1 shows two common antibody formats. Different fragments from the immunoglobulin are often used in recombinant formats, especially the variable domains of the heavy chain VH have been used repeatedly. A) a variable domain of the heavy chain (VH) linked to a constant region of the heavy chain (Fc region). B) a variable domain of the heavy chain ( H).
  • A Entire human FemX-1 primary malignant melanoma xenograft in immunodeficient mice in a histological overview. Animals were treated once with anti-human CEACAM1 mouse monoclonal antibody T84.1 one day prior to sacrifice. The formalin fixed, paraffin wax embedded primary melanoma was stained for mouse immunoglobulins. Note that the immunoreactivity is limited to the region of the necrotic areas (asterisks). Most of the areas of the vital malignant tumour cells (black triangles) remain unstained (low power magnification). The black hollow square indicates the area of the high power magnification B. B: High power magnification of living malignant tumour cells bordering the necrotic area. Note that only a limited area around blood vessels (arrows) is stained indicating the presence of antibodies.
  • necrotic area (asterisk) is also stained. This staining is due to the presence of the intravenously (i. v.) applied monoclonal antibody as shown in Figure 2 A and B.
  • Figure 3 BALB/c SCID mice were injected with human LOX melanoma cells. The mice were treated with either the anti YKL-40 antibody 4A2 or an equivalent volume of PBS as control. Cisplatin was administered to the mice 12 h before mice were sacrificed. A: shows the amount of cisplatin uptake in tumour tissue of the mice.B: shows the weight and the volume of the tumours analysed in Figure 3A.
  • A shows the distribution of the anti-YKL-40 antibody 4A2 in a tumour of a mouse treated with the anti YKL-40 antibody 4A2.
  • Low power magnification scale bar 5mm.
  • High power magnification scale bar 200pm.
  • Low power magnification scale bar 5mm.
  • High power magnification scale bar 500pm.
  • antibody refers to a polypeptide, which is capable of binding a specific antigen via an epitope on the antigen.
  • An antibody comprises at least one antigen binding site, wherein said antigen binding site comprises 3 CDRs, such as a CRD1 , CDR2 and CDR3.
  • the antigen binding site may in particular be a variable region, such as a heavy chain or light chain variable region. Whereas most antibodies comprises a heavy chain comprising an antigen binding site and a light chain comprising another an antigen binding site, some antibodies are single-domain antibodies comprising only one antigen binding site. Single-domain antibodies consisting of heavy chains only are e.g. found in camelids.
  • variable regions include, but are not necessarily limited to Fv fragments, heavy chain variable regions and light chain variable regions.
  • Variable regions may be connected and thus they may form or be part of e.g. single chain Fv (scFv) and disulphide-bonded Fv, Fab-like fragments (e.g. Fab fragments, Fab’ fragments and F (ab) 2 fragments), and domain antibodies (dAbs, including single and dual formats [i.e. dAb-linker-dAb]).
  • scFv single chain Fv
  • Fab-like fragments e.g. Fab fragments, Fab’ fragments and F (ab) 2 fragments
  • dAbs including single and dual formats [i.e. dAb-linker-dAb]
  • Variable regions may be linked to an Fc region.
  • an antibody heavy chain usually consists of a heavy chain variable region and a heavy chain Fc region.
  • Cellular cytotoxicity therapy involves the transfer of immune cells (e.g. T-cells, NK- cells, dendritic cells, granulocytes and/or macrophages) with anti-malignant tumour activity into cancer patients. It is a treatment approach that usually involves the identification of cells with anti-malignant tumour activity, the expansion of these cells to large numbers and their infusion into the cancer-bearing host.
  • immune cells e.g. T-cells, NK- cells, dendritic cells, granulocytes and/or macrophages
  • the term “decreased permeability” refers to malignant tumours having a barrier preventing or reducing the ability of drugs from reaching the interior of said cancers.
  • anti-cancer drug may be prevented from entry into the cancer, e. g from reaching further than 100 pm into said cancer.
  • An effective amount or therapeutically effective amount of a compound is that amount of compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered.
  • the phrase “therapeutically effective amount,” as used herein, may refer to an amount of antibody that is sufficient or effective to treat (delay or prevent the onset of, prevent the progression of, inhibit, decrease or reverse) a clinical condition, e.g. an inflammatory condition or cancer.
  • Fc region is abbreviated form of “fragment crystallisable region”. “Fc region” as used herein refers to the C-terminal constant region of an immune globulin heavy chain.
  • the Fc region may be a “native” or “wild-type” sequence Fc region, or a variant Fc region.
  • the Fc region may be any constant region of IgM, IgD, IgG IgA and IgE.
  • a native Fc region is normally homodimeric and comprises two polypeptide chains.
  • variable domain such as VH
  • scFc region single-chain Fc region
  • immunotherapeutic agent refers to an agent capable of inducing, enhancing and/or suppressing an immune response.
  • an immunotherapeutic agent is an agent useful in the treatment of disease.
  • immunotherapy refers to treatment of disease by activating, augmenting and/or suppressing the natural, humoral and/or adaptive immune system.
  • Immunotherapy may be cell-based therapy such as cellular cytotoxicity therapy or noncell based. Immunotherapy may be antibody therapy with antibodies from either the natural or the adaptive immune system. Immunotherapy may also be therapy with immunomodulators such as cytokines, interleukins and/or chemokines. Immunotherapy may also be treatment aimed at modulating or enhancing interactions with immunomodulators and classical anti-neoplastic agents. Examples of useful types of immunotherapies are described in the figure 3 of the review article by Melero et al. Evolving synergistic combinations of targeted immunotherapies to combat cancer. Nature Reviews 2015, 15:457472. Other useful types of immunotherapies are described below.
  • Naturally occurring antibody refers to an antibody comprising two identical heavy chains and two identical light chains linked to another, wherein the heavy and light chains of the antibody have been made and paired by the immune system of a multi-cellular organism.
  • Spleen, lymph nodes, bone marrow and serum are examples of tissues that produce natural antibodies.
  • the antibodies produced by the antibody producing cells isolated from a first animal immunized with an antigen are natural antibodies.
  • Naturally occurring antibody are heterotetrameric glycoproteins capable of recognising and binding an antigen and comprising two identical heavy (H) chains and two identical light (L) chains inter-connected by disulfide bonds.
  • Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH).
  • Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL).
  • the VH and L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs).
  • Antibodies may comprise several identical heterotetramers.
  • the term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies making up the population are identical except for possible minor differences. Said minor differences may be the result of post-translational modification and/or degradation or they may be caused by naturally occurring mutations that may be present in minor amounts.
  • solid neoplasm solid malignant tumour
  • malignant tumour refers to a disease, which may be characterised by the presence of at least one “solid neoplasm”, “solid malignant tumour” or “malignant tumour”.
  • treatment refers to any kind of treatment, including preventive, ameliorating/ palliative or curative treatment. Treatment may thus result in the prevention, decrease and/or amelioration/palliation of causes and/or symptoms of malignant tumours and cancers. Moreover, the treatment can also stop or slow down disease progression, e.g. it may stop or slow down cancer formation, development, or growth.
  • a YKL-40 antibody may be any antibody specifically binding YKL-40.
  • the YKL-antibody of the present invention may be any YKL-40 antibody, wherein said YKL- 40 antibody comprises a variable region as defined herein below in the section “Variable region”.
  • the present invention provides antibodies capable of binding to YKL-40, and preferably capable of binding human YKL-40.
  • said antibodies are capable of binding to an amino acid sequence of SEQ ID NO:1.
  • the YKL-40 antibody of the invention is capable of penetrating into the interior of a solid tumour or solid neoplasm in vivo.
  • the antibody upon systemic administration of the antibody to an individual affected by a solid neoplasm, it is preferred that the antibody is capable of distributing throughout said solid neoplasm.
  • a non-limiting example of distribution of an antibody of the invention in a solid neoplasm is shown in Fig. 4. Distribution of antibodies within a solid neoplasm may e.g. be determined may obtained a sample (e.g. a biopsy) or a solid neoplasm removed by surgery or after individual is deceased, and staining it for the presence of YKL-40 antibodies, A non-limiting example of a useful method is described in Example 3 below.
  • the YKL-40 antibody of the invention may be an antibody capable of facilitating penetration of an anti-cancer agent into the interior of a solid neoplasm.
  • the YKL-40 antibody of the invention is capable of increasing uptake of an anticancer agent (e.g. cisplatin) into a solid tumour by at least 5%, preferably at least 10%.
  • an anticancer agent e.g. cisplatin
  • the amount of cisplatin taken up into a solid tumour may in particular be determined by graphite-furnace atomic absorption spectrometry as described in Example 3 below.
  • YKL-40 is an extracellular matrix protein, specifically a secretory glycoprotein, which belongs to the mammalian chitinase like family. YKL-40 has been shown to bind collagen, heparin, hyaluronan and chitin. It is mainly produced by macrophages, neutrophils and cancer cells. YKL-40 plasma levels are increased in cancer patients compared to healthy subjects.
  • an antibody that is contemplated for use in the present invention can be in any of a variety of forms, including a whole immunoglobulin, an antibody fragment such as Fv, Fab, Fab’ or F(ab’)2 fragments, a single chain antibody which comprises the variable regions of a heavy and a light chain linked together or single domain antibodies.
  • the antibody of the invention is a single domain antibody.
  • Single domain antibodies usually comprises a variable region optionally linked to an Fc region.
  • the antibody of the invention may consist of a variable region.
  • the antibody may consist of a variable region linked to an Fc region.
  • the variable region may for example consist of either of a VH domain or a VL domain, or another similar variable region.
  • the single domain antibody may comprise a variable region and an Fc region derived from a human antibody, e.g. from a human heavy chain.
  • the antibody comprises a variable region comprising a CRD1, CDR2 and CDR3 as defined herein below.
  • the variable region may be selected from the group consisting of a VH domain, L domain or scFv. It is preferred that the variable region comprises or consists of a VH domain.
  • variable region of said antibody is linked to an Fc region, optionally by a linker.
  • the antibody according to the invention may comprise or consist of a VH domain linked to an Fc region, optionally via a linker.
  • the antibody may comprise or consist of two single domain antibodies.
  • the antibody according to the invention may consist or comprise of two VH domains, wherein each VH domain is linked to an Fc region, wherein said Fc regions are linked to each other by bonds, such as by disulphide bonds.
  • the antibodies according to the present invention are in general monoclonal antibodies.
  • the antibody can be a multispecific antibody (e.g. bispecific antibody) formed from at least two different antibodies, and/or antibody fragments so long as they exhibit binding to YKL-40.
  • a multispecific antibody e.g. bispecific antibody
  • the antibody is a chimeric antibody wherein the variable part is fused with an Fc region of a different species.
  • the antibody is a human antibody or an antibody based on human scaffold.
  • the antibody may be a human single domain antibody.
  • Human single domain antibodies may in particular comprise or consists of a human VH domain optionally linked to a human Fc region.
  • the antibody may also be a single domain antibody based on human sequences, wherein diversity has been generated synthetically. Such antibodies are useful in the treatment of human beings.
  • the antibody may also be a humanised antibody comprising CDR regions (and possibly a few other residues) transferred from another species having the desired specificity, affinity, and capacity.
  • Humanised antibodies may also comprise synthetic CDR regions, e.g. from a synthetic antibody library.
  • the generation of antibodies may be achieved by any standard methods in the art for producing antibodies.
  • YKL-40 protein or a fragment thereof is used.
  • the method comprises use of a natural YKL-40 protein, such as a secreted and optionally purified YKL-40 protein.
  • a recombinant YKL- 40 protein or fragment thereof may also be employed.
  • YKL-40 secreted from MG63 cells may be employed.
  • Medium in which MG63 cells have been cultivated may be used in crude form or YKL-40 may be partly or fully purified from such medium.
  • Recombinant antibodies may be isolated from libraries of genes encoding fragments of antibodies, e.g. using aforementioned YKL-40 protein or fragments thereof for selection.
  • the fragments of antibodies can for example be any of the aforementioned antibody fragments, such as Fab, Fv fragments, single chain fragment of heavy and light chain variable domains or single domain antibodies, such as polypeptides comprising or consisting of VH or VL domains.
  • the libraries of genes may be obtained from natural sources, as in the case of naive or immunised libraries, or they may be created by synthetic means. Isolation of specific antibodies from the libraries can be mediated by panning of phage displayed antibody libraries on specific antigens or complex mixtures, such as described in Mandrup et al., 2013. Alternatively, methods such as yeast display, bacterial display, ribosome display, etc. can be applied in the selection of monoclonal recombinant antibodies.
  • the antibody may be a human single domain antibody or a single domain antibody based on human sequences, wherein diversity has been artificially generated.
  • Several different libraries of useful human single domain antibodies are available.
  • the antibody may be selected by screening any library of human single domain antibodies with YKL-40 protein or fragment(s) thereof.
  • Such libraries include, but are not limited to human domain antibody libraries using the HEL4 scaffold, as well as such libraries, which have been counter-selected for aggregation, wherein the CDR regions of the resulting clones have been sub-cloned and used for generating a new library with diversity in all three CDR regions as described in Christ et al., 2007.
  • a preferred library to be used is the domain antibody library with constant CDR1 and restricted randomizations at 4 and 7 positions in the CDR2 and CDR3 described in Mandrup et al., 2013.
  • This library is based on the aggregation resistant human HEL4 domain antibody scaffold with the inclusion of a hydrophilic mutation at position 29.
  • the diversity of this library is designed to reflect the amino acid composition of CDR regions from known functional human antibody clones.
  • variable domain capable of binding YKL-40
  • said variable domain may be used as YKL-40 antibody per se, but it may also be fused to other domains, e.g. an Fc domain.
  • This may e.g. be achieved by using an expression vector allowing fusion of single chain Fv antibodies or single domains, such as single VH domains to Fc regions of immunoglobulins.
  • This method is useful for the generation of multi-species antibodies and enables fusion of single chain Fv antibodies or single VH domains with human, mouse or rabbit Fc and can be applied to natural monoclonal antibodies cloned as single chain Fv antibodies.
  • the expression vector can be for example a pFuse expression system, such as pFUSE-hFc1 or pFUSE-hFc1 as described by Moutel et al., 2009.
  • an antibody comprising or consisting of a H domain linked to an Fc region can be generated.
  • Such antibodies can then dimerize and form an antibody of two VH domain which are each linked to an Fc region, wherein the two Fc regions are linked by disulphide bonds.
  • said Fc region is a human Fc region.
  • useful Fc regions include the Fc region of SEQ ID NO:58 or SEQ ID NO:59.
  • the antibody according to the present invention comprises a variable region.
  • the variable region can be in any form, comprising a CDR1 , CDR2 and CDR3.
  • the variable region may be a single domain consisting of either H or VL domains, scFv, Fab, Fab’ or F(ab’)2 fragments.
  • the variable region is a H domain, for example a human VH domain or a VH domain based on human sequences, such as a VH domain as described by Mandrup et al., 2013.
  • the YKL-40 antibody comprises a variable region comprising a CDR1 comprising or consisting of an amino acid sequence of SEQ ID NO:20.
  • the antibody may comprise one or more CDR1 s, for example one CDR1 or two CDR1.
  • the YKL-40 antibody comprises a variable region comprising a CDR2 comprising or consisting of an amino acid sequence of the general formula SIX1X2X3X4GSTYYADSV (SEQ ID NO:55) or the general formula SIX1X2X3X4GSTYY wherein:
  • Xi is Q, S, N, D, G, E or Y;
  • X 2 is S, T, G, N, A, D or Y;
  • X 3 is S, E, N, Y, G, D or P;
  • X4 is D, G, S or N.
  • the YKL-40 antibody comprises a variable region comprising a CDR2 comprising or consisting of an amino acid sequence of the general formula SIX1X2X3X4GSTYYADSV (SEQ ID NO:55) or the general formula SIX1X2X3X4GSTYY wherein:
  • Xi is Q, S, N, D, G, E or Y;
  • X 2 is S, T, G, N, D or Y;
  • X 3 is S, N, Y or G
  • X4 is D or N.
  • the YKL-40 antibody comprises a variable region comprising a CDR2 comprising of an amino acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51 , SEQ ID NO:53 and/or SEQ ID NO:56.
  • the YKL-40 antibody of the invention comprises a CDR2 selected from the group consisting of SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53 and/or SEQ ID NO:56.
  • the YKL-40 antibody of the invention comprises a CDR2 of SEQ ID NO: 56.
  • the YKL-40 antibody may comprise one or more CDR2s, for example one CDR2 or two CDR2. Said CDR2 may be the same or different CDR2s.
  • the YKL-40 antibody comprises a variable region comprising a CDR3 comprising or consisting of an amino acid sequence of the general formula Xi X 2 X 3 X 4 X 5 X 6 X7FDY wherein
  • Xi is Q, T, S or V
  • X 2 is Y, S, W, D, P, G or T
  • X 3 is D
  • X 4 is T, S, D, L, W, N or
  • a X 5 G, Y, G, R, N, W, D or E
  • X 6 Y, W, S, T, F or D
  • X 7 S, A, G, Y, V, D, Q or T.
  • the YKL-40 antibody comprises a variable region comprising a CDR3 comprising of an amino acid sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NQ:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NQ:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NQ:50, SEQ ID NO:52, SEQ ID NO:54 and/or SEQ ID NO:57.
  • the YKL-40 antibody of the invention comprises a CDR3 selected from the group consisting of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NQ:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NQ:50, SEQ ID NO:52, SEQ ID NO:54 and/or SEQ ID NO:57.
  • the YKL-40 antibody of the invention comprises a CDR3 of SEQ ID NO:57.
  • the antibody may comprise one or more CDR3s, for example one CDR3 or two CDR3. Said CDR3 may be the same or different CDR3s.
  • CDR1 comprises or consists of an amino acid sequence of SEQ ID NQ:20
  • CDR2 comprises or consists of an amino acid sequence of the general formula:
  • SIX1X2X3X4GSTYYADSV SEQ ID NO:55
  • SIX1X2X3X4GSTYY SEQ ID NO:55
  • Xi is Q, S, N, D, G, E or Y;
  • X 2 is S, T, G, N, A, D or Y;
  • X 3 is S, E, N, Y, G, D or P; preferably X 3 is S, N, Y or G; X4 is D, G, S or N, preferably X D or N is D or N.
  • CDR3 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NQ:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NQ:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NQ:50, SEQ ID NO:52 SEQ ID NO:54 and/or 57, preferably CDR3 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NQ:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID
  • the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:21 and SEQ ID NO:22.
  • the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:23 and SEQ ID NO:24.
  • the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:25 and SEQ ID NO:26.
  • the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:27 and SEQ ID NO:28.
  • the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:29 and SEQ ID NQ:30.
  • the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:31 and SEQ ID NO:32.
  • the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:33 and SEQ ID NO:34.
  • the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:35 and SEQ ID NO:36.
  • the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:37 and SEQ ID NO:38.
  • the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:39 and SEQ ID NQ:40.
  • the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:41 and SEQ ID NO:42. In one embodiment, the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:43 and SEQ ID NO:44.
  • the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NO:20, CDR2 of SEQ ID NO:45 and SEQ ID NO:46.
  • the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:47 and SEQ ID NO:48.
  • the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:49 and SEQ ID NQ:50.
  • the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:51 and SEQ ID NO:52.
  • the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:53 and SEQ ID NO:54.
  • the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:56 and SEQ ID NO:57.
  • the YLK-40 antibody comprises or even consists of a variable region, wherein said variable region comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19.
  • said variable region comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO: 18 and SEQ ID NO: 19. Even more preferably said variable region comprises or consists of SEQ ID NO: 2.
  • the antibody binds to YKL-40, wherein binding to YKL-40 inhibits or prevents binding of YKL-40 to another molecule. Binding of the antibody to YKL-40 can result in activation, stimulation or inhibition of YKL-40. Thus, binding of the antibody to YKL-40 may results in activation, stimulation or inhibition of YKL-40.
  • the antibody is an inhibitor of YKL-40.
  • the YKL-40 antibody may result in inhibition of at least one function of YKL-40.
  • the antibody according to the present invention comprises a variable region, which may be linked to any Fc region.
  • antibodies have been grouped based on the constant domain of their heavy chains, i.e. Fc region, into five classes IgM, IgD, IgG IgA and IgE. Which can be further divided into subclasses (isotypes), e.g. lgG-1 , lgG-2, lgG-3 and lgG-4; IgA-1 and IgA-2.
  • subclasses e.g. lgG-1 , lgG-2, lgG-3 and lgG-4; IgA-1 and IgA-2.
  • the antibody according to the present invention comprises a variable region which may be linked to any constant region of another antibody.
  • the constant region may be identical to a “native” or “wild-type” Fc region, or a variant Fc region with at least one alteration of an amino acid.
  • the Fc region has the amino acid sequence of a human IgG Fc region or a human lgG2 Fc region.
  • a suitable Fc region is described in Moutel et al., 2009.
  • the present invention also concerns a method of treating diseases associated with aberrant YKL-40 expression.
  • Such method comprises administering an YKL-40 antibody as described herein above, to a subject in need thereof.
  • Said treatment may be a curative treatment, but it may also be preventive treatment to delay onset of said disease, or it may be treatment to delay disease progression. Further, it may be ameliorating treatment or treatment of symptoms of said disease.
  • the disease may for example be cancer, such as any of the cancers described herein below.
  • the invention relates to a method of treating cancer, the method comprising administering an YKL-40 antibody according to the invention to a subject in need thereof.
  • the invention in another embodiment, relates to a method for delaying onset of cancer, the method comprising administering a therapeutically effective amount of an YKL-40 antibody according to the invention, to a subject in need thereof
  • the disease to be treated with the YKL-40 antibody according to the invention may also be an inflammatory disease.
  • the disease may be an inflammatory disease on the lungs, such as asthma.
  • the subject to be treated with the antibody of the invention may in particular be a human being, because the antibodies of the invention preferably are based on human antibody sequences.
  • anti-cancer agents may advantageously be administered together with or subsequent or prior to a YKL-40 antibody.
  • anti-cancer agents which otherwise cannot penetrate a solid malignant tumour or which only penetrates solid malignant tumours to a low extend (e.g. only within 100 pm of blood vessels) may advantageously be administered together with or subsequent to a YKL-40 antibody of the invention.
  • the anti-cancer agent may be any anti-cancer agent.
  • the invention is not limited to a particular type of anti-cancer agent, because increasing permeability facilitates penetration of compounds into solid malignant tumour.
  • the anti-cancer agent may be small molecules, biological macro-molecules, cells, hormones or mixtures thereof.
  • the anti-cancer agent is selected from the group consisting of a chemotherapeutic agent, an immunotherapeutic agent, a biologic agent, checkpoint inhibitors, anti-cancer antibodies, a cytostatic small molecule and statins.
  • the anti-cancer agent is a small molecule.
  • Small molecules according to the invention are preferably an organic compound with a molecular weight of ⁇ 900 daltons.
  • said small molecule may be a chemotherapeutic agent.
  • the chemotherapeutic agent may be any chemotherapeutic agent.
  • the chemotherapeutic agent may be selected from the group consisting of antimetabolites, anti-malignant tumour antibiotics, topoisomerase inhibitors, mitotic inhibitors, kinase inhibitors, vinca alkaloids, anthracyclines, aromatase inhibitors, mTor inhibitors and retinoids.
  • the anti-malignant tumour antibiotic is daunorumycin or doxorubicin.
  • the chemotherapeutic agent is cisplatin.
  • the anti-cancer agent is a small molecule inhibitor for example a small molecule kinase inhibitor, a small molecule proteasome inhibitor and a small molecule inhibitor targeting the apoptosis.
  • the chemotherapeutic agent is selected form the group Fluorouracil (5-Fll), Irinotecan, Oxaliplatin, Capecitabin, Gemcitabin, Nab-paclitaxel and cisplatin.
  • the immunotherapeutic agent is a cell-based immunotherapy.
  • the immunotherapeutic agent is T-cell therapy.
  • the T cell therapy can involve adoptive cell transfer where autologous T cells are extracted, cultivated and transfused in the subject. Genetically engineered T cells can also be used as T cell therapy. Harvested T cells are infected with a retrovirus that contains a copy of a T cell receptor (TCR) gene that is specialised to recognise malignant tumour antigens. T cells are expanded and transfused in the subject.
  • TCR T cell receptor
  • the immunotherapeutic agent can also be an autologous enhancement therapy where the subjects own immune cells such as natural killer cells, cytotoxic T cells, mesenchymal stem cells or other immune cells are expanded in vitro and then transfused into the said subject.
  • Immune checkpoints are inhibitory regulators that act as inhibitors on the immune response. Targeting these checkpoints prevents cancers/malignant tumours from evading the immune system.
  • the checkpoint inhibitors are selected from the group consisting of inhibitors of PDL-1, PD1, CTLA4, and/or LAG3.
  • the anti-cancer agent is not an immune checkpoint inhibitor. In one embodiment it is preferred that the anti-cancer agent is not an immunotherapeutic agent.
  • the anti-cancer agent is an anti-cancer antibody.
  • Multiple antibodies are known to be useful in the treatment of cancer.
  • the anti-cancer agent may be any such antibody.
  • the anti-cancer agent may be selected from the group consisting of Trastuzumab, Bevacizumab, Cetuximab, Panitumumab, Ipilimumab, Rituximab, Alemtuzumab and Ofatumumab.
  • the anti-cancer agent is a cytostatic small molecule.
  • the cytostatic small molecule may for example be a tyrosine-kinase inhibitor. It can also be a small molecule kinase inhibitor, a small molecule proteasome inhibitor and a small molecule inhibitor targeting apoptosis. It can also be a serine/threonine-kinase inhibitor such as Temsirolimus, Everolimus, Vemurafenib, Trametinib and Dabrafenib.
  • the cytostatic small molecule is selected from the group consisting of Imatinib, Gefitinib, Erlotinib, Sorafenib, Sunitinib, Dasatinib, Lapatinib, Nilotinib, Bortezomib, tamoxifen, janus kinas inhibitors, ALK inhibitors, Bcl-2 inhibitors, PARP inhibitors, e. g. olaparib, APatinib, Braf inhibitors, MEK inhibitors, CDK inhibitors, Hsp90 inhibitors, and salinomycin.
  • statins are selected from the group consisting of avastatin, lovastatin, rosuvastatin, simvastatin, fluvastatin, pitavastatin, and pravastatin.
  • the anti-cancer agent is an antibody-drug conjugate (ADC).
  • ADC antibody-drug conjugate
  • the ADC may bind a malignant tumour-associated target antigen and deliver a cytotoxic agent to the malignant tumour.
  • the ADC is composed of a monoclonal antibody, a linker and a cytotoxin.
  • the ADC is Trastuzumab emtansine which is an antibody-drug conjugate consisting of the monoclonal antibody trastuzumab linked to the cytotoxic agent emtansine (DM1).
  • the anti-cancer agent is coupled to ADCC thereby utilizing the response of innate immune cells to provide anti-malignant tumour cytotoxicity triggered by the interaction of the Fc portion of an antibody with the Fc receptor on the immune cell.
  • the anti-cancer agent is associated with a nano-particle or a liposome.
  • the anticancer agent may e.g. be protein-bound paclitaxel, also known as nanoparticle albumin-bound paclitaxel or nab-paclitaxel. In this formulation, paclitaxel is bonded to albumin as a delivery vehicle.
  • the anticancer agent may be for example Abraxane ® or a pegylated (polyethylene glycol coated) liposome-encapsulated form of doxorubicin, as e.g. Caelyx®.
  • the anti-cancer agent may be for example liposomal irinotecan. These are liposomal constructs that have been engineered to encapsulate chemotherapy thereby preventing premature metabolism, improving distribution and minimizing toxicity (i.e. Onyvide®).
  • the anti-cancer agent is selected from the therapeutic groups listed in table 1. In another embodiment, the anti-cancer agent is selected from the drug examples listed in table 1.
  • the anti-cancer agent is immunotherapy, such as any of the immunotherapies described in Melero et al. 2015 (see above), such as any of the therapies shown in Fig. 3 of Melero et al. 2015 (see above).
  • the anti-cancer agent is immunotherapy.
  • immunotherapy can be immunostimulatory monoclonal antibodies, neutralizing immune inhibitors, cytokines, adoptive T cell therapy, cancer vaccines and microbial adjuvants.
  • the immunostimulatory monoclonal antibodies are selected from the group consisting of antibodies to CTLA4, PD1 , PDL1 , LAG3, TIM3, CD137, 0X40, GITR and CD40.
  • the neutralizing immune inhibitors are selected from the group consisting of TGFbeta, IL-10 and IDOlln
  • the cytokines are selected from the group consisting of IFN alpha, IL-2 and IL-12.
  • the microbial adjuvants are selected from the group consisting of TLR agonists, alpha-GalCer and STING activators.
  • the anti-cancer agent is not an immune checkpoint inhibitor.
  • the immunotherapy may also be a cell based immunotherapy, for example the immunotherapy may be based on T-cells, NK-cells, dendritic cells, mesenchymal stem cells or modified cells such as CAR-T Cells, CAR-NK cells or T-cells used in conjunction with the BiTE (Bi-specific T cell engager).
  • the immunotherapy may be based on T-cells, NK-cells, dendritic cells, mesenchymal stem cells or modified cells such as CAR-T Cells, CAR-NK cells or T-cells used in conjunction with the BiTE (Bi-specific T cell engager).
  • the anti-cancer agent is selected from the group consisting of radiotherapy, chemotherapy and signal transduction inhibitors.
  • the chemotherapy is selected from the group consisting of alkylating agents, platinum, antimetabolites, tubulin-inhibitors, antibiotics with anti-cancer effect and cytostatic molecules.
  • the signal transduction inhibitors are selected from the group consisting of protein kinase inhibitors, signal inhibitors not affecting the protein kinases, the monoclonal antibodies in cancer therapy, hormones and hormone antagonists, cytokines, antibodies towards immunological checkpoints, oncolytic virus, immunological adjuvants, T-cell or dendritic cell therapy and cancer vaccines.
  • the invention provides antibodies to YKL-40, which may be useful in methods of treating cancer. Furthermore, the invention relates to kits-of-parts and compositions for treatment of cancer characterised by the presence of at least one solid neoplasm. Said solid neoplasm may have an increase in interstitial fluid pressure and/or a decreased permeability.
  • Anti-cancer agents can typically reach single malignant tumour cells or small malignant tumour cell aggregates. This situation changes once tumours grow to an organoid like structure. Without being bound by theory it is believed that the IFP rises with the growth of the malignant tumour until it reaches a plateau resulting in poor permeability of such tumours.
  • Cancers having a decreased permeability refers to cancers having a barrier preventing drugs from reaching the interior of said cancers.
  • anti-cancer treatment may be prevented from entry into the interior of the cancer, e.g. prevented from penetrating more than approx. 100 pm from blood vessels.
  • the decreased permeability may prevent antibodies from penetrating the cancer and thus the antibodies remain in the contour of the malignant tumour.
  • Solid neoplasms frequently consist of malignant cells generally of epithelial origin, in which the cells are linked together by cell adhesions molecules and structurally recognisable cell junctions, namely desmosomes, tight junctions and gap junctions. These hold the cells together in clusters. Between these clusters, connective tissue stroma can frequently be found, in which the tumour blood vessels may be found. Stroma and cell junctions both contribute to the distribution barrier in solid tumours. From about 200 pm onwards, growing tumours start to build up an increased fluid pressure which can reach a plateau.
  • the cancer to be treated may comprise at least one solid neoplasm at least 200 pm in diameter.
  • non-cancer malignancies such as sarcomas and melanomas also build up an increased fluid pressure.
  • diseases can also be treated according to the methods of the invention.
  • the solid neoplasm may be a primary malignant tumour or a metastasis thereof.
  • the methods of the invention are useful both in the treatment of primary malignant tumours as well as metastatic cancers.
  • the cancer may for example be selected from the group consisting of solid neoplasm, metastases and fiber rich Hodgkin type of malignant tumour (lymphomas).
  • the solid neoplasm may be selected from the group consisting of breast-, colorectal-, pancreas-, bile duct-, stomach-, hepatocellular-, other gastrointestinal-, lung-, small cell lung-, ovarian-, uterine-, cervix-, testis-, prostate, bladder-, renal-, thyroid- and head/neck carcinoma, malignant melanoma, other skin cancers, neuroblastoma, glioblastoma, astrocytoma, malignant meningioma and other forms of brain cancer, osteosarcoma, chondrosarcoma, myosarcoma, fibrosarcoma and germ cell malignant tumours.
  • the cancer is malignant melanoma.
  • the present invention provides a method for treatment of cancer in an individual in need thereof.
  • the invention provides a method for treating a cancer characterized by the presence of at least one solid neoplasm. Said method may comprise the steps of
  • the YKL-40 antibody may in particular be any of the antibodies described herein above in the section “YKL-40 antibody”.
  • the YKL-40 antibody may be formulated together with or separately from the anticancer agent.
  • method may comprise administering a therapeutically effective amount of a composition comprising an YKL-40 antibody and an anti-cancer agent.
  • YKL-40 antibody and the anticancer agent may be linked covalently to each other.
  • the YKL-40 antibody and the anti-cancer agent may be administered simultaneously or sequentially.
  • said anti-cancer agent is administered simultaneously with or subsequent to the YKL-40 antibody.
  • the YKL-40 antibody can be administered in combination with an anti-cancer agent.
  • the YKL-40 antibody is administered first to a patient in need thereof followed by administration of a cancer agent at a later time point.
  • the YKL-40 antibody and anti-cancer agents can be administered multiple times in said individual either simultaneously or independently of each other. Thus, when the YKL-40 antibody and anti-cancer agents are administered separately, they may also be administered a different number of times.
  • the individual may be any individual suffering from or at risk of acquiring a cancer characterized by the presence of at least one solid neoplasm.
  • the individual will be a mammal, and more preferably the individual will be a human being.
  • the YKL-40 antibody and anti-cancer agents can be administered by any suitable route known in the art.
  • the YKL-40 antibody is administered parenterally, for example by intravenous infusion.
  • routes of administration include intraperitoneal, intrathecal and intralymphatic.
  • administration may be injection directly to the solid malignant tumour or subcutaneously.
  • the anti-cancer agent may be administered in any manner useful for the particular anti- cancer agent. Suitable modes of administration are known to the skilled person.
  • Many anti-cancer agents are administered parenterally, for example by intravenous infusion.
  • Other examples of routes of administration of anti-cancer agents include intraperitoneal, intrathecal and intralymphatic.
  • administration may be injection directly to the solid malignant tumour or subcutaneously.
  • the dosage of antibody is dependent on several factors, for example the dosage could be in the range of 1 to 50 mg/kg, such as 4 to 10 mg/kg of anti YKL-40 antibody per kg body weight.
  • the antibody is administered to the subject in need thereof once or more than once.
  • the antibody may be administered more than once with an interval of in the range of a couple of days to several weeks.
  • the pharmaceutical formulation comprises the YKL-40 antibody and/or the anti-cancer agents and one or more pharmaceutically acceptable carriers.
  • the pharmaceutical formulations may be prepared by conventional techniques, e.g. as described in Remington: The Science and Practice of Pharmacy 2013, Pharmaceutical Press.
  • the compounds of the present invention may be formulated in a wide variety of formulations for parenteral administration.
  • the formulations may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles.
  • the active ingredient may be in powder form for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water.
  • a suitable vehicle e.g., sterile, pyrogen-free water.
  • the formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules, vials, pre-filled syringes, infusion bags, or can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.
  • the formulations of the present embodiment may also include agents useful for pH maintenance, solution stabilization, or for the regulation of salt content and/or osmotic pressure.
  • the methods of the invention may be performed as the sole therapy, but frequently they are combined with other cancer therapies.
  • the cancer may be treated by surgery and subsequently with the methods of the invention.
  • the cancer may also be treated by irradiation as well as by the methods of the invention.
  • the invention may further be defined by the following items:
  • a YKL-40 antibody comprising a variable region comprising a CDR1 , CDR2 and CDR3, wherein
  • CDR1 comprises or consists of an amino acid sequence of SEQ ID NO:20
  • CDR2 comprises or consists of an amino acid sequence of the general formula:
  • SIX1X2X3X4GSTYYADSV SEQ ID NO: 55
  • SIX1X2X3X4GSTYY SEQ ID NO: 62
  • Xi is Q, S, N, D, G, E or Y;
  • X 2 is S, T, G, N, A, D or Y;
  • X 3 is S, E, N, Y, G, D or P;
  • X4 is D, G, S or N.
  • CDR3 comprises or consists of an amino acid sequence of the general formula
  • Xi is Q, T, S or V
  • X 2 is Y, S, W, D, P, G or T,
  • X 3 is D, Y, P, Q, Y, W or G
  • X 4 is T, S, D, L, W, N or A
  • X 5 G, Y, G, R, N, W, D or E
  • X 6 Y, W, S, T, F or D
  • X 7 S, A, G, Y, V, D, Q or T.
  • variable region comprises or consists of a VH domain.
  • variable region is linked to an Fc region, optionally via a linker.
  • YKL-40 antibody according to any one of the preceding items, wherein the antibody consists of a variable region linked to an Fc region.
  • YKL-40 antibody according to any one of the preceding items, wherein said YKL-40 antibody comprises or consists of two variable regions, wherein each variable region is linked to an Fc region, optionally by a linker, wherein said Fc regions are linked to each other.
  • the YKL-40 antibody according to any one of the preceding items, wherein the antibody is a single domain antibody comprising or consisting of a variable region optionally linked to an Fc region.
  • SIX1X2X3X4GSTYYADSV SEQ ID NO:55
  • SIX1X2X3X4GSTYY SEQ ID NO:55
  • Xi is Q, S, N, D, G, E or Y;
  • X 2 is S, T, G, N, D or Y;
  • X3 is S, N, Y or G
  • X4 is D or N.
  • the YKL-40 antibody according to any one of the preceding items, wherein the antibody comprises a CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:23,
  • the YKL-40 antibody according to any one of the preceding items, wherein the antibody comprises a CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:23,
  • the YKL-40 antibody according to any one of the preceding items, wherein the antibody comprises a CDR2 comprising or consisting of SEQ ID NO: 56.
  • the YKL-40 antibody according to any one of the preceding items, wherein the antibody comprises a CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:24,
  • the YKL-40 antibody according to any one of the preceding items, wherein the antibody comprises a CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:24,
  • SEQ ID NO:26 SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36,
  • the YKL-40 antibody according to any one of the preceding items, wherein the antibody comprises a CDR3 comprising or consisting of SEQ ID NO:57.
  • variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:21 and SEQ ID NO:22.
  • variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:23 and SEQ ID NO:24.
  • variable region comprises a CDR1 of SEQ ID NO:20, CDR2 of SEQ ID NO:25 and SEQ ID NO:26.
  • variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:27 and SEQ ID NO:28.
  • variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:29 and SEQ ID NQ:30.
  • variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:31 and SEQ ID NO:32.
  • variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:33 and SEQ ID NO:34.
  • variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:35 and SEQ ID NO:36.
  • variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:37 and SEQ ID NO:38.
  • variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:39 and SEQ ID NQ:40.
  • variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:41 and SEQ ID NO:42. 26.
  • variable region comprises a CDR1 of SEQ ID NO:20, CDR2 of SEQ ID NO:43 and SEQ ID NO:44.
  • variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:45 and SEQ ID NO:46.
  • variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:47 and SEQ ID NO:48.
  • variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:49 and SEQ ID NQ:50.
  • variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:51 and SEQ ID NO:52.
  • variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:53 and SEQ ID NO:54.
  • variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:56 and SEQ ID NO:57.
  • variable region comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19.
  • variable region comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19.
  • variable region comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:2.
  • said antibody comprises or consists of a variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO: 19, preferably selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:
  • the YLK-40 antibody according to any one of the preceding items, wherein the antibody consists of a variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO: 19, preferably selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID
  • the YKL-40 antibody according to any one of the preceding items, wherein the antibody is capable of distributing throughout a solid neoplasm after systemic administration to an individual affected by a solid neoplasm.
  • the YKL-40 antibody according to any one of the preceding items, wherein the antibody is capable of facilitating penetration of an anti-cancer agent into the interior of a solid neoplasm, e.g. a solid tumour.
  • YKL-40 antibody according to any one of the preceding items, wherein the antibody is capable increasing uptake of an anticancer agent, for example cisplatin, into a solid neoplasm, e.g. a soldi tumour by at least 5%, preferably at least 10%
  • an anticancer agent for example cisplatin
  • a YKL-40 antibody according to any one of the preceding items for use in a method of treating a disease associated with YKL-40 expression.
  • a method of treating a disease associated with YKL-40 expression comprising administering a therapeutically effective amount of the YKL-40 antibody according to any one of items 1 to 43 to a subject in need thereof.
  • the method is a method for delaying onset of cancer.
  • a method for treating cancer in an individual suffering from cancer characterised by the presence of at least one solid neoplasm said method comprising increasing permeability of said solid neoplasm by administration of a YKL-40 antibody to said individual, and administration of an anti-cancer agent to said individual.
  • a kit-of-parts and/or a composition comprising a. a YKL-40 antibody; and b. an anti-cancer agent; for use in the treatment of cancer in an individual in need thereof, wherein said cancer is characterised by the presence of at least one solid neoplasm.
  • kit-of-parts and/or a composition comprising a. a YKL-40 antibody; and b. an anti-cancer agent; for the preparation of a medicament for use in the treatment of a cancer characterised by the presence of at least one solid neoplasm.
  • kits-of-parts, the composition, the method or the use according to item 59 wherein the immunotherapeutic agent is selected from the group consisting of immuno stimulatory monoclonal antibodies, neutralizing immune inhibitors, cytokines, adoptive T cell therapy, cancer vaccines, therapies based on NK- cells or mesenchymal stem cells and microbial adjuvants.
  • the immunotherapeutic agent is selected from the group consisting of immuno stimulatory monoclonal antibodies, neutralizing immune inhibitors, cytokines, adoptive T cell therapy, cancer vaccines, therapies based on NK- cells or mesenchymal stem cells and microbial adjuvants.
  • the anti-cancer agent is a chemotherapeutic agent.
  • kit-of-parts, the composition, the method or the use according to item 65 wherein the signal transduction inhibitors are selected from the group consisting of protein kinase inhibitors, signal inhibitors not affecting the protein kinases, monoclonal antibodies in cancer therapy, hormones and hormone antagonists, cytokines, antibodies towards immunological checkpoints, oncolytic virus, immunological adjuvants, T-cell or dendritic cell or NK-cells or mesenchymal stem cell therapy and cancer vaccines.
  • kit-of-parts, the composition, the method or the use according to any one of items 50 to 71 wherein said cancer is selected from the group consisting of breast-, colorectal-, pancreas-, stomach-, hepatocellular-, bile duct, -other gastrointestinal-, lung-, small cell lung-, ovarian-, uterine-, cervix-, testis-, prostate, bladder-, renal-, thyroid- and head/neck carcinoma, malignant melanoma, other skin cancers, neuroblastoma, glioblastoma, astrocytoma and other forms of brain cancer, osteosarcoma, chondrosarcoma, myosarcoma, and germ cell malignant tumours.
  • said cancer is selected from the group consisting of breast-, colorectal-, pancreas-, stomach-, hepatocellular-, bile duct, -other gastrointestinal-, lung-, small cell lung-, ovarian-, uterine
  • YKL-40 protein was obtained from the supernatant of MG63 cells.
  • MG63 production flasks were seeded at 1.8 x 10 4 cells/cm 2 in RPMI-40 (Irvine Scientific) plus newborn calf serum, 100 mL/L (Irvine Scientific), 0.1 mol/L HEPES, and 50 mg/L vitamin C (complete medium). Flasks were incubated at 37 °C with humidity and CO2-enriched (100 mL/L) atmosphere for 6-8 days, replacing spent medium with fresh every 2-3 days. The cultures were then switched to serum-free medium (complete medium minus the newborn calf serum). The supernatants were harvested and the media replaced every 1-3 days for 30 days.
  • YKL-40 protein was purified from the supernatants by concentrating glass-fiber-filtered material 20-fold with a 30-kDa screen channel cassette with tangential flow (Filtron) and then affinity-purifying over a heparin- Sepharose CL-6B column (Pharmacia Biotech) equilibrated with a solution of 10 mmol/L sodium phosphate and 50 mmol/L sodium chloride, pH 7.5.
  • Bound material was eluted with a sodium chloride gradient (from 50 mmol/L to 2 mol/L) in 10 mmol/L sodium phosphate, pH 7.5, and 4-mL fractions were collected and pooled according to: absorbance at 280 nm, YKL-40 protein concentration by immunoassay and purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
  • phage antibodies recognizing YKL-40 were performed using a predator antibody library, which is a single scaffold domain library with variation in CDR2 and CDR3 (Mandrup, Friis et al. 2013) against the purified YKL-40 protein.
  • the antibodies of this library are single domain antibodies.
  • a plastic immunotube was coated overnight with YKL-40 at 4°C, so the antigen could be partially absorbed by the plastic and anchored to the inner surface of the tube.
  • 100 pl of the phage antibody library pREDATOR was added in 4 ml of 2% BSA in PBS.
  • the phage antibody library was incubated for 60 min at RT rotating using an under- and over turntable and then further 60 min at RT standing on the bench. Unbound phages were washed away with 10 washes in PBS containing 0,1% Tween 20 and 10 washes with PBS.
  • Bound phages were eluted by adding 500 pl of trypsin- PBS (50 pl of 10 mg/ml trypsin stock solution added to 450 pl PBS) and incubating on rotating for 10 min at RT. The eluent was then incubated with 10 ml of TG1 (E.Coli) in exponential growth (at an OD 600 of 0.4) for 30 min at 37°C. The bacteria were then plated on TYE plates containing 100 pg /ml ampicillin and 1% glucose and grown overnight at 30°C.
  • the plate were incubated for 1 hour at 37°C to let the Helper infect the cell, after this media was changed to 2xTY medium containing 100pg/ml Ampicillin and 50pg/ml of Kanamycin, finally the bacteria were incubate at 30°C, on shaking, overnight. The supernatant was used for ELISA.
  • the eluted phages were infected in the E. coli TG-1 and spread on Amp selective agar plates. Around 500 colonies were obtained. These were inoculated in 100 pl 2xTY in 96 well plates. Phage antibodies were produced from the plates using the helper phage KM 13 according to standard procedures.
  • the ELISA plate After coating with used (MG63 conditioned) or fresh media, the ELISA plate were blocked with 2% skimmed milk powder in PBS. After blocking 50 pl of the phage supernatant were added to each well and incubated. The plates were washed and incubated with 1 :5000 diluted anti-M13 antibody (HRP conjugated). After washing the ELISA plate were developed using TMB and the plate read in an ELISA reader.
  • Clones were selected as positive if they had a significant higher signal on the conditioned plate compared to fresh media plate. Clones were named according to plate and position on plate e.g. 3B2, which would be the clone on the third plate in row B column 2 (see table 1 below).
  • Selected clones which were judged potential positive in the preliminary screening were grown in 50 ml culture and rescued using the KM 13 helper phage. After overnight growth the supernatant were PEG precipitated according to standard procedures, and the pellet resuspended in 1 ml PBS. As phage antibody concentration is highly enriched by growing in 50 ml baffled flasks, the phage antibody concentration after PEG precipitation is 100-1000 more concentrated compared to the initial screen.
  • the PEG precipitated phage were used in validating ELISA, were a dilution series of the PEG precipitated phage were applied to ELISA plates coated with MG63 conditioned media or Fresh media. ELISA was performed as above.
  • Table 2 The results of the dilution series is shown in Table 2 below. “Used” as used in Table 2 refers to MG63 conditioned media containing YKL-40. MG63 cells produces YKL-40 and thus medium in which MG63 cells have been grown (MG63 conditioned media) contains YKL-40. “Fresh” as used in Table 2 refers to fresh media which has not been in contact with YKL-40 producing cells (MG63) and thus does not contain YKL-40. Row 1-12 represents a series of dilution. Row 1 represents 100 pl antibody concentrate from 50 ml baffled flasks. Row 2 is a 5x dilution of the concentrate from row 1. Row 3 is a 5x dilution from of the dilution from Row 2 etc.
  • the Predator phage antibody library is a single scaffold domain library with variation in CDR2 and CDR3. Thus, the CDR1 did not vary within the YKL-40 binding clones.
  • the sequence of each clone validated to bind to YKL40 is shown in Table 4.
  • the two clones 4A2 and 4A12 can be cloned into a vector pFuse-hlgG2 as described in Moutel et al., 2009, or a vector containing another immunoglobulin constant part.
  • the vectors can be transfected into a suitable cell, such as ExpiCHO cells and expressed in this cell. This transfection leads to antibodies of VH domains fused to human lgG2 or other Fc regions expressed by the vector.
  • secreted antibodies can be purified from the supernatant.
  • Severe combined immunodeficient mice harbouring human FemX-1 primary melanomas were injected into the tail vein with 10 pg of the murine antihuman CECAM1 antibody T84.1.
  • SCID mice harbouring human FemX-1 primary melanomas were injected into the tail vein with 10 pg of the murine antihuman CECAM1 antibody T84.1.
  • T84.1 murine antihuman CECAM1 antibody
  • the animals were sacrificed after 22.5 hours and perfused with physiological saline solution.
  • the excised malignant tumour samples were fixed in neutral buffered formalin and processed to paraffin wax.
  • Her2/neu overexpressing PT1590 primary malignant tumours were treated for a period of 44 days with twice weekly intraperitoneal injection of 10mg/kg bodyweight trastuzumab antibody.
  • Her2/neu expression was shown both by FACS analysis of the malignant tumour cells in cell culture as well as by immunohistochemistry in primary malignant tumours. Most of the cancer cells in the primary malignant tumours were Her2/neu positive.
  • mice were sacrificed and the malignant tumours were processed as above (for details see Lange et al., 2011).
  • TBS Tris buffered saline
  • a biotinylated polyclonal swine anti rabbit immunoglobulins solid phase absorbed
  • Antibody Diluent was applied for 30 minutes.
  • the Vectastain ABC-AP KIT was used according to the manufacturer's instructions for both anti-human and anti-mouse antibodies. Permanent Red Substrate Chromogen solution was used as a substrate for localization of the alkaline phosphatase.
  • Rabbit polyclonal immunoglobulin fraction appropriately 1:1000 diluted served as a negative control.
  • FIG 2A and 2B show a histological overview of a human FemX-1 primary malignant melanoma xenograft in immunodeficient mice treated with anti-human CEACAM1 mouse monoclonal antibody T84 (see Experiment 1. above).
  • the immunoreactivity is limited to the region of the necrotic areas (asterisks). Most of the areas of the vital malignant tumour cells (black triangles in figure 1A) remain unstained. Only a limited area around blood vessels (arrows) is stained indicating the presence of antibodies.
  • EpCAM immunoreactivity was mainly confined to area adjacent to blood vessels. The necrotic areas or connective tissue areas were not immunoreactive for mouse immunoglobulins.
  • trastuzumab PT 1590 cells practically all PT1590 cancer cells were not labelled by the antibody, while the surrounding connective tissues and the necrotic areas were strongly immunoreactive. This finding indicates that saturation with the trastuzumab antibody exists, which, however, is not reaching the cancer cells within the bulk of the malignant tumour.
  • trastuzumab antibody which was detected in the connective spaces and in the necrotic areas, is really effective in killing PT 1590 cancer cells is demonstrated by the fact, that the number of metastases decreased dramatically (78% of the mice in the control group had developed spontaneous metastases in the lungs, while only 11% did so in the 10 mg/kg bodyweight trastuzumab group), while the primary malignant tumour weight decreased much less (3,16 gram control versus 2,36 gram treatment group)( Lange et al., 2011).
  • the finding that small metastatic deposits can be treated successfully while larger primary malignant tumour masses cannot be treated argues for the limited access of the antibody to the large primary malignant tumour mass in comparison to the small metastatic deposits.
  • mice were 8-16 weeks old and weigh 20-30 g at the beginning of the experiments. They were housed in filter top cages and provided with sterile water and food ad libitum.
  • human LOX melanoma cells were harvested by trypsinisation and viable cells (5x10 6 ) were suspended in 1 ml of cell culture medium. An aliquot of 200 ml of this suspension was injected subcutaneously between the scapulae of each SCID mouse. Human LOX cells have been shown to express YKL-40 by flow cytometry.
  • mice carried tumours, 10 g antibody dissolved in PBS was injected intraperitoneally into LOX melanoma bearing mice.
  • Five mice received anti-YKL-40 monoclonal antibody, consisting of the variable region of antibody 4A2 (SEQ ID NO: 2) linked to an Fc region by cloning into a pFUSE expression system as described in Moutel et al., 2009.
  • Five mice received an equivalent volume of PBS as control.
  • mice were sacrificed, intracardially perfused with NaCI and tumours were removed. Tumor size during experiments was measured with calipers, and the tumour weight at the end of treatment was determined (Figure 3B). Parts of tumours were then embedded in paraffin, other parts were processed for cryostat sections.
  • test antibodies were single-domain antibodies comprising the variable region of antibody 4A2 (SEQ ID NO:2) linked to human Fc, and for detection a 1 : 50 diluted polyclonal rabbit anti-human IgG specific for gamma chains is used. Representative examples of the results are shown in Figure 4 A-C.
  • Formalin-fixed and paraffin-embedded sections were deparaffinized in two changes of xylene (5 min each) and rehydrated in a series of graded ethanol (100, 96, 70 and 50% for 5 min each). Sections were then washed in aqua dest for 2 min. The following incubation steps were carried out in a moist chamber. For epitope retrieval, samples were treated for 5 min with Fast Enzyme (Zytomed Systems, Bargteheide, Germany) at room temperature, following two 5 min washes in Tris-buffered saline/0.1% Tween20 (TBS-T) and one 5 min wash in TBS (pH 7.6).
  • Fast Enzyme Zytomed Systems, Bargteheide, Germany
  • Blocking with 4% BSA in TBS was performed for 30 min to prevent nonspecific antibody binding. Afterwards, sections were incubated with primary rat anti-Pt-[GpG] monoclonal antibody diluted 1 :1000 in antibody diluent (medac, Wedel, Germany) or rat lgG2a kappa at a dilution of 1 :500 (eBioscience, San Diego, USA) for 80 min at room temperature and then rinsed twice with TBS-T as well as with TBS for 5 min each.
  • the secondary biotin- conjugated rabbit anti-rat antibody (Dako, Glostrup, Denmark) was incubated at a dilution of 1 :100 in antibody diluent for 30 min at room temperature, followed by rinsing twice with TBS-T and once with TBS for 5 min each. Sections were treated with Vectastain® ABC-AP Kit (Vector Laboratories, Burlingame, CA, US) according to the manufacturer’s recommendations for 30 min at RT and again washed in TBS-T and TBS as described above.
  • alkaline phosphatase enzyme activity was visualized by incubating the sections with Permanent Red solution (Dako, Glostrup Denmark) for 20 min and counterstained with hematoxylin for 4 s, with intermediate washes under running tap water (3 min) and in aqua dest (2 min). Slides were dehydrated in a series of graded ethanol (70% for 15 s, 96 and 100% for 5 min each) and three changes of xylene (5 min each) and finally covered with Eukitt® Mounting Medium (Sigma-Aldrich, Steinheim, Germany) and coverslips.
  • HC-processed sections were first evaluated using a ZEISS Axiophot 2 microscope (Carl Zeiss, Jena, Germany). Digital images were obtained with a ZEISS Axio Scan Z1 slide scanner equipped with a ZEISS EC Plan-Neofluar 20x/0.50 Pol M27 objective (Carl Zeiss, Jena, Germany) and a Hitachi HV-F20SCL camera with 1600 x 1200 pixels (Hitachi Kokusai Electric America Ltd., New York, USA). For image acquisition, ZEISS ZEN 2.3 software was used (Carl Zeiss, Jena, Germany). Images were further processed with netScope Viewer software (Net-Base Software, Freiburg, Germany).
  • the cisplatin concentration in the tumours was determined by graphitefurnace atomic absorption spectrometry as described in Bernhard Welz, Michael Sperling: Atomabsorptionsspektrometrie, WILEY-VCH-Verlag Weinheim, 1997, pages 572 - 573. The results are shown in Figure 3A.
  • a GF-AAS- (Graphite Furnace - Atomic Absorption Spectrometry) machine from Perkin-Elmer, Model AAnalyst 600 was used.
  • Ferretti S Allegrini PR, Becquet MM, McSheehy PM. Tumor interstitial fluid pressure as an early-response marker for anticancer therapeutics. Neoplasia. 2009 Sep; 11 (9):874-81.
  • trastuzumab has anti- metastatic and anti-angiogenic activity in a spontaneous metastasis xenograft model of esophageal adenocarcinoma. Cancer Lett. 2011 Sep 1;308(1):54-61.

Abstract

Provided herein are antibodies capable of binding to YKL-40. These YKL-40 antibodies are suitable for the treatment of cancer or for delaying the progression of cancer if applied with an additional anti-cancer drug.

Description

YKL-40 antibody and uses thereof
Technical field
The present invention relates to YKL-40 antibodies capable of binding to YKL-40.
These antibodies are useful for multiple purposes, including for detection of YKL-40, for treatment of cancers, or delaying the onset of cancers or for treatment of other diseases associated with aberrant YKL-40 expression, such as inflammatory diseases. In addition, the invention relates to the treatment of solid malignant tumours with YKL- 40 antibodies, said treatment resulting in increase in malignant tumour permeability thereby allowing or facilitating access of an anti-cancer agent to the malignant tumour.
Background
YKL-40 is a 40 kDa heparin- and chitin-binding glycoprotein also known as human cartilage glycoprotein 39 (HC gp-39), 38-kDa heparin-binding glycoprotein or chitinase- 3-like protein 1 (CHI3L1). The abbreviation YKL-40 is based on the one letter code for the first three N-terminal amino acids, tyrosine (Y), lysine (K) and leucine (L) and the apparent molecular weight of YKL-40.
YKL-40 was first identified as a protein secreted in large amounts by a human osteosarcoma cell line MG63 in vitro. Later studies have found that YKL-40 is secreted in vitro by a variety of cells and seems especially involved in activation of the innate immune system and in cell processes in relation to extracellular matrix remodeling.
The crystallographic structure of human YKL-40 has been described and the protein contains two globular domains: a big core domain which consists of a (p/a)s domain structure with a triose-phosphase isomerase (TIM) barrel fold and a small a/p domain, composed of five antiparallel p-strands and one a-helix, inserted in the loop between strand p7 and helix a7. This confers the active site of YKL-40 a groove-like character.
Besides binding to heparin and chitin, YKL-40 can also bind to hyaluronan. The folded protein contains two potential hyaluronan binding sites predicted by in silico methods. Binding of short and long oligosaccharides to human YKL-40 are also possible.
YKL-40 possesses a number of biological activities. It has been shown that human YKL-40 can acts as a growth factor for cells of connective tissue, such as chondrocytes and synovial cells. YKL-40 also promotes the growths of fibroblasts in a fashion similar to insulin-like growth factor 1 (IGF-1). It has also been demonstrated that YKL-40 can act as a chemoattractant for endothelial cells and stimulates migration of these cells comparable to stimulation by basic fibroblast growth factor. YKL-40 is also found to modulate vascular endothelial cell morphology by promoting formation of branching tubules. A strong expression of YKL-40 mRNA in human liver has been shown to be associated with the presence of fibrosis. Immunohistochemical studies of liver biopsies have shown YKL-40 protein expression in areas of the liver with fibrosis, whereas no expression was observed in hepatocytes. Patients with non-malignant diseases characterized by inflammation and fibrosis such as active rheumatoid arthritis, severe bacterial infections, active inflammatory bowel disease, and liver fibrosis have elevated serum YKL-40.
YKL-40 is expressed and secreted by several types of human malignancies. Furthermore, YKL-40 is found to be secreted in vitro by the osteoscarcoma cell line MG63, glioblastoma cells and myeloid leukemia cell lines. A number of studies have reported an elevated level of YKL-40 protein in serum of cancer patients.
Thus, YKL-40 activity is found to be associated with cell growth, survival, differentiation, apoptosis, angiogenesis, extracellular matrix remodeling, development of metastasis, development of liver or tissue fibrosis, development of rheumatoid arthritis and/or development of inflammation.
The effectiveness of anticancer drug therapy in treating solid malignant tumours depends on the drug reaching all cancer cells in the malignant tumour. The drug distribution in malignant tumour tissue is dependent on the plasma pharmacokinetics and pharmacodynamics, the structure and function of the malignant tumour vasculature and the ability of the drug to move through microvessel walls and its diffusion and convection across the ECM and the layers of the malignant tumour. For chemotherapy of solid malignant tumours for example, the drug must be able to traverse walls of blood vessels and the surrounding ECM to reach malignant tumour cells. Often malignant tumours form several layers of malignant tumour and/or matrix cells which all have to be penetrated. Matrix cells may for example be fibrous connective tissue. The structure and flow distribution in the system of microvessels supplying the malignant tumour and the properties of extravascular tissue components such as ECM, normal cells, malignant tumour cells and interstitial spaces play a major role in this regard.
Treatment of metastatic solid malignant tumours with classical chemotherapeutic antineoplastic drugs, formulations of chemotherapeutic drugs increasing accessibility to the matrix surrounding the malignant tumour as Caleux®, Onyvide® or abraxane®, kinase inhibitors, small molecules and/or therapeutic monoclonal antibodies is increasingly used in clinical medicine. However, a cure of widespread distant metastases has not been achieved; still more than 90% of the cancer patients die from them. The reason for this failure might reside in the increased interstitial fluid pressure (IFP) observed in solid malignant tumours which hampers the access of the antibodies to cancer cells. Indeed, solid malignant tumours have a raised IFP due to high vessel permeability, low lymphatic drainage, poor perfusion, and high cell density around the blood vessels (Ferretti et al. 2009). The raised interstitial fluid barrier creates a distribution barrier within solid malignant tumours which limits the access of therapeutic agents towards all malignant cells.
Solid malignant tumours form organoid like structures, which may be composed of a variety of tissue components. First there are the malignant tumour cells themselves, which in the case of solid malignant tumours, cancers, generally form epithelial structures as the cells are linked to each other by cell to cell junctions including desmosomes, tight junctions, gap junctions. These junctions are responsible for the formation of a “cancer tissue”, which is composed of several cancer cells of the same type. In addition to the cancer cells themselves the malignant tumour typically builds around its vascular supply which is sheathed in a connective tissue ECM, collectively called the malignant tumour stroma. The stromal component of normal organs contains blood and lymphatic vessels, the latter being absent in cancer tissue stroma. This absence of functioning lymphatic vessels has considerable consequences for the flow of fluids within malignant tumours. In order to understand this process, the normal fluid transport within tissues has to be understood. In the arterial part of the capillary, fluid is pressed out (exudate) into the ECM surrounding the capillary. While the fluid and with it small molecules are pressed out into the ECM the large molecules such as proteins remain partially in the capillaries. These remaining osmotically active macromolecules suck the fluid of the ECM back into the venous part of the capillaries. However, about 10% of the exsudated fluid is not sucked back into the venous part of the capillary but remains in the extracellular matrix. There it is transported away by a process called convection, which is a directed fluid flow from the capillaries to the lymphatic vessels. Thereby the fluid exchange in normal organs is balanced resulting in a slightly negative to zero interstitial fluid pressure. In contrast to normal organs, organoid like malignant tumours do not contain functioning lymphatic vessels, thereby this excess fluid is not drained away and accumulates within the malignant tumour thus building up the interstitial fluid pressure.
This fluid pressure is believed to limit the access of therapeutic antibodies and of classical small molecular weight chemotherapeutic drugs (small molecule drugs) to areas within the malignant tumour which are located 100 pm around the small exchange blood vessels. Thereby most of the malignant tumour cells within solid malignant tumours are not reached by the possible therapeutic drugs and/or antibodies.
Summary
There is an unmet need of additional YKL-40 antibodies, and in particularly for YKL-40 antibodies useful in treatment of cancer characterised by the presence of at least one solid neoplasm. As outlined above, in general solid neoplasms are difficult to treat due to reduced access of antibodies or other anti-cancer agents to areas within the solid neoplasms. The present invention provides YKL-40 antibodies, which upon administration to an individual suffering from cancer, are capable of entering into the interior of solid neoplasms. Thus, the YKL-40 antibodies of the invention can act in the interior of solid neoplasms. Furthermore, the YKL-40 antibodies of the present invention are preferably capable of facilitating access of other compounds into the inner mass of a malignant tumour, thereby facilitating access of e.g. anti-cancer agents. Thus, YKL-40 antibodies may facilitate the access of anti-cancer agents to solid neoplasms, and thus anti-cancer agents may advantageously be administered to an individual suffering from a solid neoplasm either together with or before or subsequent to administration of the YKL-40 antibodies.
Furthermore, the invention provides antibodies useful for administration to human beings are provided because said antibodies contains mainly human antibody sequences. In one aspect, the present invention relates to an YKL-40 antibody comprising a variable region comprising a CDR1 , CDR2 and CDR3, wherein
• CDR1 comprises or consists of an amino acid sequence of SEQ ID NO:20
• CDR2 comprises or consists of an amino acid sequence of the general formula:
SIX1X2X3X4GSTYYADSV (SEQ ID NO:55) wherein:
Xi is Q, S, N, D, G, E or Y;
X2 is S, G, N, A, D or Y;
X3 is S, E, N, Y, G, D or P;
X4 is D, G, S or N.
• CDR3 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NQ:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NQ:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NQ:50, SEQ ID NO:52, SEQ ID NO:54 and/or SEQ ID NO:57, preferably SEQ ID NO:57.
In one aspect, the present invention relates to an YKL-40 antibody comprising or consisting of a variable region comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19.
In one aspect, the present invention relates to a method of treating a clinical condition associated with aberrant YKL-40 expression, the method comprising administering a therapeutically effective amount of a YKL-40 antibody as described herein above to a subject in need thereof.
It is also an aspect of the invention to provide kit-of parts and/or a composition comprising a. a YKL-40 antibody; and b. an anti-cancer agent; for use in the treatment of cancer in an individual in need thereof, wherein said cancer is characterised by the presence of at least one solid neoplasm.
The invention is furthermore defined by the claims attached hereto.
Description of Drawings
Figure 1. shows two common antibody formats. Different fragments from the immunoglobulin are often used in recombinant formats, especially the variable domains of the heavy chain VH have been used repeatedly. A) a variable domain of the heavy chain (VH) linked to a constant region of the heavy chain (Fc region). B) a variable domain of the heavy chain ( H).
Figure 2
A: Entire human FemX-1 primary malignant melanoma xenograft in immunodeficient mice in a histological overview. Animals were treated once with anti-human CEACAM1 mouse monoclonal antibody T84.1 one day prior to sacrifice. The formalin fixed, paraffin wax embedded primary melanoma was stained for mouse immunoglobulins. Note that the immunoreactivity is limited to the region of the necrotic areas (asterisks). Most of the areas of the vital malignant tumour cells (black triangles) remain unstained (low power magnification). The black hollow square indicates the area of the high power magnification B. B: High power magnification of living malignant tumour cells bordering the necrotic area. Note that only a limited area around blood vessels (arrows) is stained indicating the presence of antibodies.
To demonstrate that the lack of CEACAM1 immunoreactivity in the melanoma depicted in Fig. 2 A and B is not due to a lack of its expression, a parallel section of the malignant tumour used in 2 A and B was immunohistochemically stained with another anti- CEACAM1 monoclonal antibody. C: In the overview section the necrotic areas (asterisks) are intensively stained but now the vital malignant tumour tissue (black triangles) is stained as well (compare with 2A in which this area remains unstained (light regions)). The black hollow square indicates the area of the high power magnification D. D: High power magnification of Fig. 2 C. Note the cell membrane bound CEACAM1 immunoreactivity in the vital malignant tumour areas (black triangles). Note that the necrotic area (asterisk) is also stained. This staining is due to the presence of the intravenously (i. v.) applied monoclonal antibody as shown in Figure 2 A and B. Figure 3. BALB/c SCID mice were injected with human LOX melanoma cells. The mice were treated with either the anti YKL-40 antibody 4A2 or an equivalent volume of PBS as control. Cisplatin was administered to the mice 12 h before mice were sacrificed. A: shows the amount of cisplatin uptake in tumour tissue of the mice.B: shows the weight and the volume of the tumours analysed in Figure 3A.
Figure 4.
Shows the distribution of the anti-YKL-40 antibody 4A2 in the tumours analysed in Figure 3.
A: shows the distribution of the anti-YKL-40 antibody 4A2 in a tumour of a mouse treated with the anti YKL-40 antibody 4A2. Low power magnification scale bar = 5mm. High power magnification scale bar = 200pm.
B: shows the distribution of the anti-YKL-40 antibody 4A2 in (I) a tumour from the treatment group, scale bar = 5mm. (II) a tumour from the control group without any antibody, scale bar = 2mm.
C: shows the distribution of the anti-YKL-40-Antibody 4A2 in a tumour of a mouse treated with the anti YKL-40 antibody 4A2. Scale bar = 200pm.
Figure 5.
Shows the distribution of cisplatin in the tumours analysed in Figure 3.
A: Formation and distribution of cisplatin Pt-GG-Adducts in a tumor from the control group. Low power magnification scale bar = 5mm. High power magnification scale bar = 500pm.
B: Formation and distribution of cisplatin Pt-GG-Adducts in a tumor from the treatment group. Low power magnification scale bar = 5mm. High power magnification scale bar = 500pm
Detailed description
Definitions
The term “antibody” as used herein refers to a polypeptide, which is capable of binding a specific antigen via an epitope on the antigen. An antibody comprises at least one antigen binding site, wherein said antigen binding site comprises 3 CDRs, such as a CRD1 , CDR2 and CDR3. The antigen binding site may in particular be a variable region, such as a heavy chain or light chain variable region. Whereas most antibodies comprises a heavy chain comprising an antigen binding site and a light chain comprising another an antigen binding site, some antibodies are single-domain antibodies comprising only one antigen binding site. Single-domain antibodies consisting of heavy chains only are e.g. found in camelids. Typically, the antigen binding site of an antibody is positioned within an antibody variable region. Suitable variable regions include, but are not necessarily limited to Fv fragments, heavy chain variable regions and light chain variable regions. Variable regions may be connected and thus they may form or be part of e.g. single chain Fv (scFv) and disulphide-bonded Fv, Fab-like fragments (e.g. Fab fragments, Fab’ fragments and F (ab) 2 fragments), and domain antibodies (dAbs, including single and dual formats [i.e. dAb-linker-dAb]). Variable regions may be linked to an Fc region. Thus, an antibody heavy chain usually consists of a heavy chain variable region and a heavy chain Fc region. Similarly, an antibody light chain usually consists of a light chain variable region and a light chain Fc region. Single-domain antibodies typically consists of variable region comprising 3 CDRs and a constant region of a heavy chain in one polypeptide. Various antibody chains may be linked to each other, e.g. by disulphide bonds. Thus, a heavy chain and a light chain may be linked to each other. Furthermore, a heavy chain and a light chain pair, may be linked to another (frequently identical) heavy chain/light chain pair. The heavy chain of a single domain antibody may also be linked to another (frequently identical) heavy chain, e.g. by disulphide bonds, thereby forming an antibody made up of two constant domains of the heavy chains and two variable domains of the heavy chain, wherein said Fc regions are joined by disulphide bonds (see Fig. 1).
“Cellular cytotoxicity therapy” involves the transfer of immune cells (e.g. T-cells, NK- cells, dendritic cells, granulocytes and/or macrophages) with anti-malignant tumour activity into cancer patients. It is a treatment approach that usually involves the identification of cells with anti-malignant tumour activity, the expansion of these cells to large numbers and their infusion into the cancer-bearing host.
The term “decreased permeability” refers to malignant tumours having a barrier preventing or reducing the ability of drugs from reaching the interior of said cancers. For example, anti-cancer drug may be prevented from entry into the cancer, e. g from reaching further than 100 pm into said cancer. An effective amount or therapeutically effective amount of a compound is that amount of compound which is sufficient to provide a beneficial effect to the subject to which the compound is administered. The phrase “therapeutically effective amount,” as used herein, may refer to an amount of antibody that is sufficient or effective to treat (delay or prevent the onset of, prevent the progression of, inhibit, decrease or reverse) a clinical condition, e.g. an inflammatory condition or cancer.
The term “Fc region” is abbreviated form of “fragment crystallisable region”. “Fc region” as used herein refers to the C-terminal constant region of an immune globulin heavy chain. The Fc region may be a “native” or “wild-type” sequence Fc region, or a variant Fc region. The Fc region may be any constant region of IgM, IgD, IgG IgA and IgE. A native Fc region is normally homodimeric and comprises two polypeptide chains. In respect of preparing YKL-40 antibodies a variable domain, such as VH, may be linked to a “single-chain” Fc region (scFc region), which may later form a dimeric antibody comprising two VH domains and two scFc regions.
The term “Immunotherapeutic agent” refers to an agent capable of inducing, enhancing and/or suppressing an immune response. In particular, an immunotherapeutic agent is an agent useful in the treatment of disease.
The term “immunotherapy” refers to treatment of disease by activating, augmenting and/or suppressing the natural, humoral and/or adaptive immune system.
Immunotherapy may be cell-based therapy such as cellular cytotoxicity therapy or noncell based. Immunotherapy may be antibody therapy with antibodies from either the natural or the adaptive immune system. Immunotherapy may also be therapy with immunomodulators such as cytokines, interleukins and/or chemokines. Immunotherapy may also be treatment aimed at modulating or enhancing interactions with immunomodulators and classical anti-neoplastic agents. Examples of useful types of immunotherapies are described in the figure 3 of the review article by Melero et al. Evolving synergistic combinations of targeted immunotherapies to combat cancer. Nature Reviews 2015, 15:457472. Other useful types of immunotherapies are described below.
The term ’’naturally occurring antibody " refers to an antibody comprising two identical heavy chains and two identical light chains linked to another, wherein the heavy and light chains of the antibody have been made and paired by the immune system of a multi-cellular organism. Spleen, lymph nodes, bone marrow and serum are examples of tissues that produce natural antibodies. For example, the antibodies produced by the antibody producing cells isolated from a first animal immunized with an antigen are natural antibodies. Naturally occurring antibody are heterotetrameric glycoproteins capable of recognising and binding an antigen and comprising two identical heavy (H) chains and two identical light (L) chains inter-connected by disulfide bonds. Each heavy chain comprises a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region (abbreviated herein as CH). Each light chain comprises a light chain variable region (abbreviated herein as VL) and a light chain constant region (abbreviated herein as CL). The VH and L regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs). Antibodies may comprise several identical heterotetramers.
The term “monoclonal antibody” as used herein refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies making up the population are identical except for possible minor differences. Said minor differences may be the result of post-translational modification and/or degradation or they may be caused by naturally occurring mutations that may be present in minor amounts.
The terms “solid neoplasm”, “solid malignant tumour”, and “malignant tumour” are herein used interchangeably. The term “cancer” refers to a disease, which may be characterised by the presence of at least one “solid neoplasm”, “solid malignant tumour” or “malignant tumour”.
The term “treatment” refers to any kind of treatment, including preventive, ameliorating/ palliative or curative treatment. Treatment may thus result in the prevention, decrease and/or amelioration/palliation of causes and/or symptoms of malignant tumours and cancers. Moreover, the treatment can also stop or slow down disease progression, e.g. it may stop or slow down cancer formation, development, or growth. YKL-40 antibody
A YKL-40 antibody may be any antibody specifically binding YKL-40. For example, the YKL-antibody of the present invention may be any YKL-40 antibody, wherein said YKL- 40 antibody comprises a variable region as defined herein below in the section “Variable region”. Thus, the present invention provides antibodies capable of binding to YKL-40, and preferably capable of binding human YKL-40. In particular, said antibodies are capable of binding to an amino acid sequence of SEQ ID NO:1.
It is preferred that the YKL-40 antibody of the invention is capable of penetrating into the interior of a solid tumour or solid neoplasm in vivo. Thus, upon systemic administration of the antibody to an individual affected by a solid neoplasm, it is preferred that the antibody is capable of distributing throughout said solid neoplasm. A non-limiting example of distribution of an antibody of the invention in a solid neoplasm is shown in Fig. 4. Distribution of antibodies within a solid neoplasm may e.g. be determined may obtained a sample (e.g. a biopsy) or a solid neoplasm removed by surgery or after individual is deceased, and staining it for the presence of YKL-40 antibodies, A non-limiting example of a useful method is described in Example 3 below.
In particular, the YKL-40 antibody of the invention may be an antibody capable of facilitating penetration of an anti-cancer agent into the interior of a solid neoplasm. Thus, in preferred embodiments of the invention, the YKL-40 antibody of the invention is capable of increasing uptake of an anticancer agent (e.g. cisplatin) into a solid tumour by at least 5%, preferably at least 10%. The amount of cisplatin taken up into a solid tumour may in particular be determined by graphite-furnace atomic absorption spectrometry as described in Example 3 below.
YKL-40 is an extracellular matrix protein, specifically a secretory glycoprotein, which belongs to the mammalian chitinase like family. YKL-40 has been shown to bind collagen, heparin, hyaluronan and chitin. It is mainly produced by macrophages, neutrophils and cancer cells. YKL-40 plasma levels are increased in cancer patients compared to healthy subjects.
An antibody that is contemplated for use in the present invention can be in any of a variety of forms, including a whole immunoglobulin, an antibody fragment such as Fv, Fab, Fab’ or F(ab’)2 fragments, a single chain antibody which comprises the variable regions of a heavy and a light chain linked together or single domain antibodies. In a preferred embodiment, the antibody of the invention is a single domain antibody. Single domain antibodies usually comprises a variable region optionally linked to an Fc region. Thus, the antibody of the invention may consist of a variable region.
Alternatively, the antibody may consist of a variable region linked to an Fc region. The variable region may for example consist of either of a VH domain or a VL domain, or another similar variable region. In particular, the single domain antibody may comprise a variable region and an Fc region derived from a human antibody, e.g. from a human heavy chain.
In one embodiment, the antibody comprises a variable region comprising a CRD1, CDR2 and CDR3 as defined herein below. The variable region may be selected from the group consisting of a VH domain, L domain or scFv. It is preferred that the variable region comprises or consists of a VH domain.
In another embodiment, the variable region of said antibody is linked to an Fc region, optionally by a linker. Thus, the antibody according to the invention may comprise or consist of a VH domain linked to an Fc region, optionally via a linker. Hereby forming a single chain antibody which comprises the CDRs of a variable domain of the heavy chain and the constant region of the heavy chain in one polypeptide.
In yet another embodiment, the antibody may comprise or consist of two single domain antibodies. Thus, the antibody according to the invention may consist or comprise of two VH domains, wherein each VH domain is linked to an Fc region, wherein said Fc regions are linked to each other by bonds, such as by disulphide bonds.
The antibodies according to the present invention are in general monoclonal antibodies.
The antibody can be a multispecific antibody (e.g. bispecific antibody) formed from at least two different antibodies, and/or antibody fragments so long as they exhibit binding to YKL-40.
In one embodiment, the antibody is a chimeric antibody wherein the variable part is fused with an Fc region of a different species. In one embodiment, the antibody is a human antibody or an antibody based on human scaffold. For example, the antibody may be a human single domain antibody. Human single domain antibodies may in particular comprise or consists of a human VH domain optionally linked to a human Fc region. The antibody may also be a single domain antibody based on human sequences, wherein diversity has been generated synthetically. Such antibodies are useful in the treatment of human beings. The antibody may also be a humanised antibody comprising CDR regions (and possibly a few other residues) transferred from another species having the desired specificity, affinity, and capacity. Humanised antibodies may also comprise synthetic CDR regions, e.g. from a synthetic antibody library.
The generation of antibodies may be achieved by any standard methods in the art for producing antibodies.
For generation and/or selection of YKL-40 antibodies, YKL-40 protein or a fragment thereof is used. Preferably the method comprises use of a natural YKL-40 protein, such as a secreted and optionally purified YKL-40 protein. Alternatively, a recombinant YKL- 40 protein or fragment thereof may also be employed. In particular, YKL-40 secreted from MG63 cells may be employed. Medium in which MG63 cells have been cultivated may be used in crude form or YKL-40 may be partly or fully purified from such medium. Recombinant antibodies may be isolated from libraries of genes encoding fragments of antibodies, e.g. using aforementioned YKL-40 protein or fragments thereof for selection. The fragments of antibodies can for example be any of the aforementioned antibody fragments, such as Fab, Fv fragments, single chain fragment of heavy and light chain variable domains or single domain antibodies, such as polypeptides comprising or consisting of VH or VL domains. The libraries of genes may be obtained from natural sources, as in the case of naive or immunised libraries, or they may be created by synthetic means. Isolation of specific antibodies from the libraries can be mediated by panning of phage displayed antibody libraries on specific antigens or complex mixtures, such as described in Mandrup et al., 2013. Alternatively, methods such as yeast display, bacterial display, ribosome display, etc. can be applied in the selection of monoclonal recombinant antibodies. The antibody may be a human single domain antibody or a single domain antibody based on human sequences, wherein diversity has been artificially generated. Several different libraries of useful human single domain antibodies are available. Thus, the antibody may be selected by screening any library of human single domain antibodies with YKL-40 protein or fragment(s) thereof. Such libraries include, but are not limited to human domain antibody libraries using the HEL4 scaffold, as well as such libraries, which have been counter-selected for aggregation, wherein the CDR regions of the resulting clones have been sub-cloned and used for generating a new library with diversity in all three CDR regions as described in Christ et al., 2007. Other useful libraries include Pansri et al., 2009, Rothe et al., 2008, Fellouse et al., 2007, Hust et la., 2004, Silacci et al., 2005 and Brockmann et al., 2011.
A preferred library to be used is the domain antibody library with constant CDR1 and restricted randomizations at 4 and 7 positions in the CDR2 and CDR3 described in Mandrup et al., 2013. This library is based on the aggregation resistant human HEL4 domain antibody scaffold with the inclusion of a hydrophilic mutation at position 29. The diversity of this library is designed to reflect the amino acid composition of CDR regions from known functional human antibody clones.
Once a variable domain capable of binding YKL-40 has been selected, said variable domain may be used as YKL-40 antibody per se, but it may also be fused to other domains, e.g. an Fc domain.
This may e.g. be achieved by using an expression vector allowing fusion of single chain Fv antibodies or single domains, such as single VH domains to Fc regions of immunoglobulins. This method is useful for the generation of multi-species antibodies and enables fusion of single chain Fv antibodies or single VH domains with human, mouse or rabbit Fc and can be applied to natural monoclonal antibodies cloned as single chain Fv antibodies. The expression vector can be for example a pFuse expression system, such as pFUSE-hFc1 or pFUSE-hFc1 as described by Moutel et al., 2009. Thus, an antibody comprising or consisting of a H domain linked to an Fc region can be generated. Such antibodies can then dimerize and form an antibody of two VH domain which are each linked to an Fc region, wherein the two Fc regions are linked by disulphide bonds. Preferably, said Fc region is a human Fc region. Non-limiting examples of useful Fc regions include the Fc region of SEQ ID NO:58 or SEQ ID NO:59. Variable region
The antibody according to the present invention comprises a variable region. The variable region can be in any form, comprising a CDR1 , CDR2 and CDR3. Thus, the variable region may be a single domain consisting of either H or VL domains, scFv, Fab, Fab’ or F(ab’)2 fragments. It is preferred that the variable region is a H domain, for example a human VH domain or a VH domain based on human sequences, such as a VH domain as described by Mandrup et al., 2013.
The variable domains are for binding and determine the specificity of each particular antibody for its particular antigen. However, the variable region is not normally evenly distributed thought the variable region of antibodies. It is concentrated in CDRs also known as hyper variable regions. The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions connected by three CDRs. It is preferred that the FR regions are human FR regions, or at least highly identical to human FR regions.
In one embodiment, the YKL-40 antibody comprises a variable region comprising a CDR1 comprising or consisting of an amino acid sequence of SEQ ID NO:20. The antibody may comprise one or more CDR1 s, for example one CDR1 or two CDR1.
In another embodiment, the YKL-40 antibody comprises a variable region comprising a CDR2 comprising or consisting of an amino acid sequence of the general formula SIX1X2X3X4GSTYYADSV (SEQ ID NO:55) or the general formula SIX1X2X3X4GSTYY wherein:
Xi is Q, S, N, D, G, E or Y;
X2 is S, T, G, N, A, D or Y;
X3 is S, E, N, Y, G, D or P;
X4 is D, G, S or N.
In another embodiment, the YKL-40 antibody comprises a variable region comprising a CDR2 comprising or consisting of an amino acid sequence of the general formula SIX1X2X3X4GSTYYADSV (SEQ ID NO:55) or the general formula SIX1X2X3X4GSTYY wherein:
Xi is Q, S, N, D, G, E or Y;
X2 is S, T, G, N, D or Y;
X3 is S, N, Y or G;
X4 is D or N.
In another embodiment, the YKL-40 antibody comprises a variable region comprising a CDR2 comprising of an amino acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51 , SEQ ID NO:53 and/or SEQ ID NO:56. In a preferred embodiment, the YKL-40 antibody of the invention comprises a CDR2 selected from the group consisting of SEQ ID NO:21, SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53 and/or SEQ ID NO:56. In a very preferred embodiment, the YKL-40 antibody of the invention comprises a CDR2 of SEQ ID NO: 56. The YKL-40 antibody may comprise one or more CDR2s, for example one CDR2 or two CDR2. Said CDR2 may be the same or different CDR2s.
In one embodiment, the YKL-40 antibody comprises a variable region comprising a CDR3 comprising or consisting of an amino acid sequence of the general formula Xi X2 X3 X4 X5 X6 X7FDY wherein
Xi is Q, T, S or V,
X2 is Y, S, W, D, P, G or T, X3 is D, Y, P, Q, Y, W or G X4 is T, S, D, L, W, N or A X5 = G, Y, G, R, N, W, D or E X6 = Y, W, S, T, F or D X7 = S, A, G, Y, V, D, Q or T.
In yet another embodiment, the YKL-40 antibody comprises a variable region comprising a CDR3 comprising of an amino acid sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NQ:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NQ:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NQ:50, SEQ ID NO:52, SEQ ID NO:54 and/or SEQ ID NO:57. In a preferred embodiment, the YKL-40 antibody of the invention comprises a CDR3 selected from the group consisting of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NQ:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NQ:50, SEQ ID NO:52, SEQ ID NO:54 and/or SEQ ID NO:57. In a very preferred embodiment, the YKL-40 antibody of the invention comprises a CDR3 of SEQ ID NO:57. The antibody may comprise one or more CDR3s, for example one CDR3 or two CDR3. Said CDR3 may be the same or different CDR3s.
In one embodiment, the YKL-40 antibody comprises a variable region comprising a CDR1 , CDR2 and CDR3, wherein
• CDR1 comprises or consists of an amino acid sequence of SEQ ID NQ:20
• CDR2 comprises or consists of an amino acid sequence of the general formula:
SIX1X2X3X4GSTYYADSV (SEQ ID NO:55) or the general formula SIX1X2X3X4GSTYY wherein:
Xi is Q, S, N, D, G, E or Y;
X2 is S, T, G, N, A, D or Y;
X3 is S, E, N, Y, G, D or P; preferably X3 is S, N, Y or G; X4 is D, G, S or N, preferably X D or N is D or N.
• CDR3 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NQ:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NQ:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NQ:50, SEQ ID NO:52 SEQ ID NO:54 and/or 57, preferably CDR3 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID NO:38, SEQ ID NQ:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID NO:48, SEQ ID NQ:50, SEQ ID NO:52, SEQ ID NO:54 and/or SEQ ID NO:57, even more preferably CDR3 comprises or consists of SEQ ID NO:57.
In one embodiment, the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:21 and SEQ ID NO:22.
In one embodiment, the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:23 and SEQ ID NO:24.
In one embodiment, the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:25 and SEQ ID NO:26.
In one embodiment, the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:27 and SEQ ID NO:28.
In one embodiment, the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:29 and SEQ ID NQ:30.
In one embodiment, the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:31 and SEQ ID NO:32.
In one embodiment, the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:33 and SEQ ID NO:34.
In one embodiment, the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:35 and SEQ ID NO:36.
In one embodiment, the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:37 and SEQ ID NO:38.
In one embodiment, the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:39 and SEQ ID NQ:40.
In one embodiment, the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:41 and SEQ ID NO:42. In one embodiment, the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:43 and SEQ ID NO:44.
In one embodiment, the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NO:20, CDR2 of SEQ ID NO:45 and SEQ ID NO:46.
In one embodiment, the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:47 and SEQ ID NO:48.
In one embodiment, the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:49 and SEQ ID NQ:50.
In one embodiment, the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:51 and SEQ ID NO:52.
In one embodiment, the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:53 and SEQ ID NO:54.
In one embodiment, the YKL-40 antibody comprises a variable region comprising a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:56 and SEQ ID NO:57.
In one embodiment, the YLK-40 antibody comprises or even consists of a variable region, wherein said variable region comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19. More preferably, said variable region comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO: 18 and SEQ ID NO: 19. Even more preferably said variable region comprises or consists of SEQ ID NO: 2.
In one embodiment, the antibody binds to YKL-40, wherein binding to YKL-40 inhibits or prevents binding of YKL-40 to another molecule. Binding of the antibody to YKL-40 can result in activation, stimulation or inhibition of YKL-40. Thus, binding of the antibody to YKL-40 may results in activation, stimulation or inhibition of YKL-40.
In one embodiment the antibody is an inhibitor of YKL-40. Thus, the YKL-40 antibody may result in inhibition of at least one function of YKL-40.
Fc region
The antibody according to the present invention comprises a variable region, which may be linked to any Fc region.
Traditionally, antibodies have been grouped based on the constant domain of their heavy chains, i.e. Fc region, into five classes IgM, IgD, IgG IgA and IgE. Which can be further divided into subclasses (isotypes), e.g. lgG-1 , lgG-2, lgG-3 and lgG-4; IgA-1 and IgA-2.
In some embodiments, the antibody according to the present invention comprises a variable region which may be linked to any constant region of another antibody. The constant region may be identical to a “native” or “wild-type” Fc region, or a variant Fc region with at least one alteration of an amino acid.
In one embodiment, the Fc region has the amino acid sequence of a human IgG Fc region or a human lgG2 Fc region. A suitable Fc region is described in Moutel et al., 2009.
Method of treatment
The present invention also concerns a method of treating diseases associated with aberrant YKL-40 expression. Such method comprises administering an YKL-40 antibody as described herein above, to a subject in need thereof.
Said treatment may be a curative treatment, but it may also be preventive treatment to delay onset of said disease, or it may be treatment to delay disease progression. Further, it may be ameliorating treatment or treatment of symptoms of said disease.
The disease may for example be cancer, such as any of the cancers described herein below. In one embodiment, the invention relates to a method of treating cancer, the method comprising administering an YKL-40 antibody according to the invention to a subject in need thereof.
In another embodiment, the invention relates to a method for delaying onset of cancer, the method comprising administering a therapeutically effective amount of an YKL-40 antibody according to the invention, to a subject in need thereof
The disease to be treated with the YKL-40 antibody according to the invention may also be an inflammatory disease. For example the disease may be an inflammatory disease on the lungs, such as asthma.
The subject to be treated with the antibody of the invention may in particular be a human being, because the antibodies of the invention preferably are based on human antibody sequences.
Anti-cancer agent
The present invention YKL-40 antibodies, which may facilitate the penetration of an anti-cancer agent into the interior of a solid malignant tumour. Thus, anti-cancer agents may advantageously be administered together with or subsequent or prior to a YKL-40 antibody. In particular, anti-cancer agents, which otherwise cannot penetrate a solid malignant tumour or which only penetrates solid malignant tumours to a low extend (e.g. only within 100 pm of blood vessels) may advantageously be administered together with or subsequent to a YKL-40 antibody of the invention.
Thus, the anti-cancer agent may be any anti-cancer agent. The invention is not limited to a particular type of anti-cancer agent, because increasing permeability facilitates penetration of compounds into solid malignant tumour. Thus, the anti-cancer agent may be small molecules, biological macro-molecules, cells, hormones or mixtures thereof.
In one embodiment, the anti-cancer agent is selected from the group consisting of a chemotherapeutic agent, an immunotherapeutic agent, a biologic agent, checkpoint inhibitors, anti-cancer antibodies, a cytostatic small molecule and statins. In one embodiment the anti-cancer agent is a small molecule. Small molecules according to the invention are preferably an organic compound with a molecular weight of <900 daltons. In particular, said small molecule may be a chemotherapeutic agent.
The chemotherapeutic agent may be any chemotherapeutic agent. For example, the chemotherapeutic agent may be selected from the group consisting of antimetabolites, anti-malignant tumour antibiotics, topoisomerase inhibitors, mitotic inhibitors, kinase inhibitors, vinca alkaloids, anthracyclines, aromatase inhibitors, mTor inhibitors and retinoids.
In one embodiment, the anti-malignant tumour antibiotic is daunorumycin or doxorubicin. In another embodiment, the chemotherapeutic agent is cisplatin.
In one embodiment, the anti-cancer agent is a small molecule inhibitor for example a small molecule kinase inhibitor, a small molecule proteasome inhibitor and a small molecule inhibitor targeting the apoptosis.
In one embodiment, the chemotherapeutic agent is selected form the group Fluorouracil (5-Fll), Irinotecan, Oxaliplatin, Capecitabin, Gemcitabin, Nab-paclitaxel and cisplatin.
In one embodiment, the immunotherapeutic agent is a cell-based immunotherapy. In a preferred embodiment the immunotherapeutic agent is T-cell therapy. The T cell therapy can involve adoptive cell transfer where autologous T cells are extracted, cultivated and transfused in the subject. Genetically engineered T cells can also be used as T cell therapy. Harvested T cells are infected with a retrovirus that contains a copy of a T cell receptor (TCR) gene that is specialised to recognise malignant tumour antigens. T cells are expanded and transfused in the subject. The immunotherapeutic agent can also be an autologous enhancement therapy where the subjects own immune cells such as natural killer cells, cytotoxic T cells, mesenchymal stem cells or other immune cells are expanded in vitro and then transfused into the said subject.
Immune checkpoints are inhibitory regulators that act as inhibitors on the immune response. Targeting these checkpoints prevents cancers/malignant tumours from evading the immune system. In one embodiment, the checkpoint inhibitors are selected from the group consisting of inhibitors of PDL-1, PD1, CTLA4, and/or LAG3.
In one embodiment it is preferred that the anti-cancer agent is not an immune checkpoint inhibitor. In one embodiment it is preferred that the anti-cancer agent is not an immunotherapeutic agent.
In one embodiment, the anti-cancer agent is an anti-cancer antibody. Multiple antibodies are known to be useful in the treatment of cancer. The anti-cancer agent may be any such antibody. For example, the anti-cancer agent may be selected from the group consisting of Trastuzumab, Bevacizumab, Cetuximab, Panitumumab, Ipilimumab, Rituximab, Alemtuzumab and Ofatumumab.
In one embodiment, the anti-cancer agent is a cytostatic small molecule. The cytostatic small molecule may for example be a tyrosine-kinase inhibitor. It can also be a small molecule kinase inhibitor, a small molecule proteasome inhibitor and a small molecule inhibitor targeting apoptosis. It can also be a serine/threonine-kinase inhibitor such as Temsirolimus, Everolimus, Vemurafenib, Trametinib and Dabrafenib. In another embodiment, the cytostatic small molecule is selected from the group consisting of Imatinib, Gefitinib, Erlotinib, Sorafenib, Sunitinib, Dasatinib, Lapatinib, Nilotinib, Bortezomib, tamoxifen, janus kinas inhibitors, ALK inhibitors, Bcl-2 inhibitors, PARP inhibitors, e. g. olaparib, APatinib, Braf inhibitors, MEK inhibitors, CDK inhibitors, Hsp90 inhibitors, and salinomycin.
In one embodiment, the statins are selected from the group consisting of avastatin, lovastatin, rosuvastatin, simvastatin, fluvastatin, pitavastatin, and pravastatin.
In one embodiment, the anti-cancer agent is an antibody-drug conjugate (ADC). For example the ADC may bind a malignant tumour-associated target antigen and deliver a cytotoxic agent to the malignant tumour. The ADC is composed of a monoclonal antibody, a linker and a cytotoxin. For example, the ADC is Trastuzumab emtansine which is an antibody-drug conjugate consisting of the monoclonal antibody trastuzumab linked to the cytotoxic agent emtansine (DM1). In another embodiment, the anti-cancer agent is coupled to ADCC thereby utilizing the response of innate immune cells to provide anti-malignant tumour cytotoxicity triggered by the interaction of the Fc portion of an antibody with the Fc receptor on the immune cell.
In another embodiment the anti-cancer agent is associated with a nano-particle or a liposome. Thus the anticancer agent may e.g. be protein-bound paclitaxel, also known as nanoparticle albumin-bound paclitaxel or nab-paclitaxel. In this formulation, paclitaxel is bonded to albumin as a delivery vehicle. In another embodiment, the anticancer agent may be for example Abraxane ® or a pegylated (polyethylene glycol coated) liposome-encapsulated form of doxorubicin, as e.g. Caelyx®. In yet another embodiment, the anti-cancer agent may be for example liposomal irinotecan. These are liposomal constructs that have been engineered to encapsulate chemotherapy thereby preventing premature metabolism, improving distribution and minimizing toxicity (i.e. Onyvide®).
In one embodiment, the anti-cancer agent is selected from the therapeutic groups listed in table 1. In another embodiment, the anti-cancer agent is selected from the drug examples listed in table 1.
In one embodiment, the anti-cancer agent is immunotherapy, such as any of the immunotherapies described in Melero et al. 2015 (see above), such as any of the therapies shown in Fig. 3 of Melero et al. 2015 (see above). In one embodiment, the anti-cancer agent is immunotherapy. For example, immunotherapy can be immunostimulatory monoclonal antibodies, neutralizing immune inhibitors, cytokines, adoptive T cell therapy, cancer vaccines and microbial adjuvants.
In one embodiment, the immunostimulatory monoclonal antibodies are selected from the group consisting of antibodies to CTLA4, PD1 , PDL1 , LAG3, TIM3, CD137, 0X40, GITR and CD40. In another embodiment, the neutralizing immune inhibitors are selected from the group consisting of TGFbeta, IL-10 and IDOlln yet another embodiment, the cytokines are selected from the group consisting of IFN alpha, IL-2 and IL-12. In one embodiment, the microbial adjuvants are selected from the group consisting of TLR agonists, alpha-GalCer and STING activators. However, as noted above in some embodiments it is preferred that the anti-cancer agent is not an immune checkpoint inhibitor.
The immunotherapy may also be a cell based immunotherapy, for example the immunotherapy may be based on T-cells, NK-cells, dendritic cells, mesenchymal stem cells or modified cells such as CAR-T Cells, CAR-NK cells or T-cells used in conjunction with the BiTE (Bi-specific T cell engager).
In one embodiment, the anti-cancer agent is selected from the group consisting of radiotherapy, chemotherapy and signal transduction inhibitors. In another embodiment, the chemotherapy is selected from the group consisting of alkylating agents, platinum, antimetabolites, tubulin-inhibitors, antibiotics with anti-cancer effect and cytostatic molecules. In another embodiment, the signal transduction inhibitors are selected from the group consisting of protein kinase inhibitors, signal inhibitors not affecting the protein kinases, the monoclonal antibodies in cancer therapy, hormones and hormone antagonists, cytokines, antibodies towards immunological checkpoints, oncolytic virus, immunological adjuvants, T-cell or dendritic cell therapy and cancer vaccines.
Table 1 : Synergistic therapeutic groups, chemical groups and drug examples
Figure imgf000026_0001
Figure imgf000027_0001
Figure imgf000028_0001
Figure imgf000029_0001
Figure imgf000030_0001
Figure imgf000031_0001
Figure imgf000032_0001
Solid neoplasm and cancer
The invention provides antibodies to YKL-40, which may be useful in methods of treating cancer. Furthermore, the invention relates to kits-of-parts and compositions for treatment of cancer characterised by the presence of at least one solid neoplasm. Said solid neoplasm may have an increase in interstitial fluid pressure and/or a decreased permeability.
Anti-cancer agents can typically reach single malignant tumour cells or small malignant tumour cell aggregates. This situation changes once tumours grow to an organoid like structure. Without being bound by theory it is believed that the IFP rises with the growth of the malignant tumour until it reaches a plateau resulting in poor permeability of such tumours.
Cancers having a decreased permeability refers to cancers having a barrier preventing drugs from reaching the interior of said cancers. For example, anti-cancer treatment may be prevented from entry into the interior of the cancer, e.g. prevented from penetrating more than approx. 100 pm from blood vessels. The decreased permeability may prevent antibodies from penetrating the cancer and thus the antibodies remain in the contour of the malignant tumour.
Solid neoplasms frequently consist of malignant cells generally of epithelial origin, in which the cells are linked together by cell adhesions molecules and structurally recognisable cell junctions, namely desmosomes, tight junctions and gap junctions. These hold the cells together in clusters. Between these clusters, connective tissue stroma can frequently be found, in which the tumour blood vessels may be found. Stroma and cell junctions both contribute to the distribution barrier in solid tumours. From about 200 pm onwards, growing tumours start to build up an increased fluid pressure which can reach a plateau. Thus, in one embodiment the cancer to be treated may comprise at least one solid neoplasm at least 200 pm in diameter.
In addition to classical cancers, non-cancer malignancies such as sarcomas and melanomas also build up an increased fluid pressure. Thus such diseases can also be treated according to the methods of the invention. The solid neoplasm may be a primary malignant tumour or a metastasis thereof. Thus, the methods of the invention are useful both in the treatment of primary malignant tumours as well as metastatic cancers.
The cancer may for example be selected from the group consisting of solid neoplasm, metastases and fiber rich Hodgkin type of malignant tumour (lymphomas). For example, the solid neoplasm may be selected from the group consisting of breast-, colorectal-, pancreas-, bile duct-, stomach-, hepatocellular-, other gastrointestinal-, lung-, small cell lung-, ovarian-, uterine-, cervix-, testis-, prostate, bladder-, renal-, thyroid- and head/neck carcinoma, malignant melanoma, other skin cancers, neuroblastoma, glioblastoma, astrocytoma, malignant meningioma and other forms of brain cancer, osteosarcoma, chondrosarcoma, myosarcoma, fibrosarcoma and germ cell malignant tumours.
In a particular embodiment, the cancer is malignant melanoma.
Method of treatment of cancer
The present invention provides a method for treatment of cancer in an individual in need thereof.
In one embodiment, the invention provides a method for treating a cancer characterized by the presence of at least one solid neoplasm. Said method may comprise the steps of
• administering a therapeutically effective an YKL-40 antibody to an individual in need thereof thereby increasing the permeability of said solid neoplasm; and
• administering a therapeutically effective amount of an anti-cancer agent to said individual, thereby treating the cancer. The YKL-40 antibody may in particular be any of the antibodies described herein above in the section “YKL-40 antibody”.
The YKL-40 antibody may be formulated together with or separately from the anticancer agent. Thus, in one embodiment method may comprise administering a therapeutically effective amount of a composition comprising an YKL-40 antibody and an anti-cancer agent.
It is also comprised within the present invention that YKL-40 antibody and the anticancer agent may be linked covalently to each other.
In embodiments of the invention where the YKL-40 antibody and the anti-cancer agent are formulated separately they may be administered simultaneously or sequentially. Typically, it is preferred that said anti-cancer agent is administered simultaneously with or subsequent to the YKL-40 antibody.
In one embodiment, the YKL-40 antibody can be administered in combination with an anti-cancer agent. In another embodiment, the YKL-40 antibody is administered first to a patient in need thereof followed by administration of a cancer agent at a later time point. The YKL-40 antibody and anti-cancer agents can be administered multiple times in said individual either simultaneously or independently of each other. Thus, when the YKL-40 antibody and anti-cancer agents are administered separately, they may also be administered a different number of times.
The individual may be any individual suffering from or at risk of acquiring a cancer characterized by the presence of at least one solid neoplasm. In general the individual will be a mammal, and more preferably the individual will be a human being.
The YKL-40 antibody and anti-cancer agents can be administered by any suitable route known in the art. In general, the YKL-40 antibody is administered parenterally, for example by intravenous infusion. Other examples of routes of administration include intraperitoneal, intrathecal and intralymphatic. In some embodiments, administration may be injection directly to the solid malignant tumour or subcutaneously. The anti-cancer agent may be administered in any manner useful for the particular anti- cancer agent. Suitable modes of administration are known to the skilled person. Many anti-cancer agents are administered parenterally, for example by intravenous infusion. Other examples of routes of administration of anti-cancer agents include intraperitoneal, intrathecal and intralymphatic. In some embodiments, administration may be injection directly to the solid malignant tumour or subcutaneously.
The dosage of antibody is dependent on several factors, for example the dosage could be in the range of 1 to 50 mg/kg, such as 4 to 10 mg/kg of anti YKL-40 antibody per kg body weight. In one embodiment, the antibody is administered to the subject in need thereof once or more than once. For example, the antibody may be administered more than once with an interval of in the range of a couple of days to several weeks.
Whilst it is possible for the YKL-40 antibody and the anti-cancer agents to be administered as raw compounds, it is preferred to present them in the form of a pharmaceutical formulation. The pharmaceutical formulation comprises the YKL-40 antibody and/or the anti-cancer agents and one or more pharmaceutically acceptable carriers. The pharmaceutical formulations may be prepared by conventional techniques, e.g. as described in Remington: The Science and Practice of Pharmacy 2013, Pharmaceutical Press.
The compounds of the present invention may be formulated in a wide variety of formulations for parenteral administration. For injections and infusions the formulations may take such forms as suspensions, solutions, or emulsions in oily or aqueous vehicles. Alternatively, the active ingredient may be in powder form for constitution before use with a suitable vehicle, e.g., sterile, pyrogen-free water. The formulations can be presented in unit-dose or multi-dose sealed containers, such as ampoules, vials, pre-filled syringes, infusion bags, or can be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use.
The formulations of the present embodiment may also include agents useful for pH maintenance, solution stabilization, or for the regulation of salt content and/or osmotic pressure. The methods of the invention may be performed as the sole therapy, but frequently they are combined with other cancer therapies. The cancer may be treated by surgery and subsequently with the methods of the invention. The cancer may also be treated by irradiation as well as by the methods of the invention.
Items
The invention may further be defined by the following items:
1. A YKL-40 antibody comprising a variable region comprising a CDR1 , CDR2 and CDR3, wherein
• CDR1 comprises or consists of an amino acid sequence of SEQ ID NO:20
• CDR2 comprises or consists of an amino acid sequence of the general formula:
SIX1X2X3X4GSTYYADSV (SEQ ID NO: 55) or the general formula SIX1X2X3X4GSTYY (SEQ ID NO: 62) wherein:
Xi is Q, S, N, D, G, E or Y;
X2 is S, T, G, N, A, D or Y;
X3 is S, E, N, Y, G, D or P;
X4 is D, G, S or N.
• CDR3 comprises or consists of an amino acid sequence of the general formula
Xi X2 X3 X4 X5 X6 X7FDY (SEQ ID NO: 61) wherein
Xi is Q, T, S or V,
X2 is Y, S, W, D, P, G or T,
X3 is D, Y, P, Q, Y, W or G
X4 is T, S, D, L, W, N or A
X5 = G, Y, G, R, N, W, D or E
X6 = Y, W, S, T, F or D
X7 = S, A, G, Y, V, D, Q or T.
2. The YKL-40 antibody according to item 1 , wherein said variable region comprises or consists of a VH domain. 3. The YKL-40 antibody according to any one of the preceding items, wherein said variable region is linked to an Fc region, optionally via a linker.
4. The YKL-40 antibody according to any one of the preceding items, wherein the antibody consists of a variable region linked to an Fc region.
5. The YKL-40 antibody according to any one of the preceding items, wherein said YKL-40 antibody comprises or consists of two variable regions, wherein each variable region is linked to an Fc region, optionally by a linker, wherein said Fc regions are linked to each other.
6. The YKL-40 antibody according to any one of the preceding items, wherein said Fc regions are linked together by disulfide bonds.
7. The YKL-40 antibody according to any one of the preceding items, wherein the antibody is a single domain antibody comprising or consisting of a variable region optionally linked to an Fc region.
8. The YKL-40 antibody according to any one of the preceding items, wherein the antibody comprises a CDR2 comprising or consisting of an amino acid sequence of the general formula
SIX1X2X3X4GSTYYADSV (SEQ ID NO:55) or the general formula SIX1X2X3X4GSTYY wherein:
Xi is Q, S, N, D, G, E or Y;
X2 is S, T, G, N, D or Y;
X3 is S, N, Y or G; and
X4 is D or N.
9. The YKL-40 antibody according to any one of the preceding items, wherein the antibody comprises a CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:23,
SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29, SEQ ID NO:31 , SEQ ID NO:33,
SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 , SEQ ID NO:43,
SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51 , SEQ ID NO:53 and/or SEQ ID NO:56. 10. The YKL-40 antibody according to any one of the preceding items, wherein the antibody comprises a CDR2 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:21, SEQ ID NO:23,
SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:31, SEQ ID NO:33, SEQ ID NO:35,
SEQ ID NO:37, SEQ ID NO:39, SEQ ID NO:41 , SEQ ID NO:43, SEQ ID NO:45,
SEQ ID NO:47, SEQ ID NO:49, SEQ ID NO:51, SEQ ID NO:53 and/or SEQ ID
NO:56.
11. The YKL-40 antibody according to any one of the preceding items, wherein the antibody comprises a CDR2 comprising or consisting of SEQ ID NO: 56.
12. The YKL-40 antibody according to any one of the preceding items, wherein the antibody comprises a CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:24,
SEQ ID NO:26, SEQ ID NO:28, SEQ ID NQ:30, SEQ ID NO:32, SEQ ID NO:34,
SEQ ID NO:36, SEQ ID NO:38, SEQ ID NQ:40, SEQ ID NO:42, SEQ ID NO:44,
SEQ ID NO:46, SEQ ID NO:48, SEQ ID NQ:50, SEQ ID NO:52, SEQ ID NO:54 and SEQ ID NO:57.
13. The YKL-40 antibody according to any one of the preceding items, wherein the antibody comprises a CDR3 comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:24,
SEQ ID NO:26, SEQ ID NO:28, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36,
SEQ ID NO:38, SEQ ID NQ:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46,
SEQ ID NO:48, SEQ ID NQ:50, SEQ ID NO:52, SEQ ID NO:54 and/or SEQ ID
NO:57.
14. The YKL-40 antibody according to any one of the preceding items, wherein the antibody comprises a CDR3 comprising or consisting of SEQ ID NO:57.
15. The YKL-40 antibody according to any one of the preceding items, wherein the variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:21 and SEQ ID NO:22.
16. The YKL-40 antibody according to any one of the preceding items, wherein the variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:23 and SEQ ID NO:24. The YKL-40 antibody according to any one of the preceding items, wherein the variable region comprises a CDR1 of SEQ ID NO:20, CDR2 of SEQ ID NO:25 and SEQ ID NO:26. The YKL-40 antibody according to any one of the preceding items, wherein the variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:27 and SEQ ID NO:28. The YKL-40 antibody according to any one of the preceding items, wherein the variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:29 and SEQ ID NQ:30. The YKL-40 antibody according to any one of the preceding items, wherein the variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:31 and SEQ ID NO:32. The YKL-40 antibody according to any one of the preceding items, wherein the variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:33 and SEQ ID NO:34. The YKL-40 antibody according to any one of the preceding items, wherein the variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:35 and SEQ ID NO:36. The YKL-40 antibody according to any one of the preceding items, wherein the variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:37 and SEQ ID NO:38. The YKL-40 antibody according to any one of the preceding items, wherein the variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:39 and SEQ ID NQ:40. The YKL-40 antibody according to any one of the preceding items, wherein the variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:41 and SEQ ID NO:42. 26. The YKL-40 antibody according to any one of the preceding items, wherein the variable region comprises a CDR1 of SEQ ID NO:20, CDR2 of SEQ ID NO:43 and SEQ ID NO:44.
27. The YKL-40 antibody according to any one of the preceding items, wherein the variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:45 and SEQ ID NO:46.
28. The YKL-40 antibody according to any one of the preceding items, wherein the variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:47 and SEQ ID NO:48.
29. The YKL-40 antibody according to any one of the preceding items, wherein the variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:49 and SEQ ID NQ:50.
30. The YKL-40 antibody according to any one of the preceding items, wherein the variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:51 and SEQ ID NO:52.
31. The YKL-40 antibody according to any one of the preceding items, wherein the variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:53 and SEQ ID NO:54.
32. The YKL-40 antibody according to any one of the preceding items, wherein the variable region comprises a CDR1 of SEQ ID NQ:20, CDR2 of SEQ ID NO:56 and SEQ ID NO:57.
33. The YLK-40 antibody according to any one of the preceding items, wherein said variable region comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19. The YLK-40 antibody according to any one of the preceding items, wherein said variable region comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19. The YLK-40 antibody according to any one of the preceding items, wherein said variable region comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:2. The YLK-40 antibody according to any one of the preceding items, wherein said antibody comprises or consists of a variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO: 19, preferably selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO: 19, more preferably SEQ ID NO: 2. The YLK-40 antibody according to any one of the preceding items, wherein the antibody consists of a variable region consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO: 19, preferably selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO: 19, more preferably SEQ ID NO:2, wherein the variable region is linked to an Fc region. The YLK-40 antibody according to item 27, wherein said antibody forms dimers. 39. The YKL-40 antibody according to any one of the preceding items, wherein the antibody comprises an Fc region, wherein the Fc region is a human Fc region.
40. The YKL-40 antibody according to any one of the preceding items, wherein the antibody comprises an Fc region comprising or consisting of SEQ ID NO: 58 or SEQ ID NO:59.
41. The YKL-40 antibody according to any one of the preceding items, wherein the antibody is capable of distributing throughout a solid neoplasm after systemic administration to an individual affected by a solid neoplasm.
42. The YKL-40 antibody according to any one of the preceding items, wherein the antibody is capable of facilitating penetration of an anti-cancer agent into the interior of a solid neoplasm, e.g. a solid tumour.
43. The YKL-40 antibody according to any one of the preceding items, wherein the antibody is capable increasing uptake of an anticancer agent, for example cisplatin, into a solid neoplasm, e.g. a soldi tumour by at least 5%, preferably at least 10%
44. A YKL-40 antibody according to any one of the preceding items for use in a method of treating a disease associated with YKL-40 expression.
45. Use of a YKL-40 antibody according to any one of items 1 to 43 in the preparation of a medicament for use in a method of treating a disease associated with YKL-40 expression.
46. A method of treating a disease associated with YKL-40 expression comprising administering a therapeutically effective amount of the YKL-40 antibody according to any one of items 1 to 43 to a subject in need thereof.
47. The antibody for use, the use or the method according to any one of items 44 or
46, wherein the disease is cancer.
48. The antibody for use, the use or the method according to any one of items 44 to
47, wherein the method is a method for delaying onset of cancer. The antibody for use, the use or the method according to any one of items 44 or 46, wherein the disease is an inflammatory disease. A method for treating cancer in an individual suffering from cancer characterised by the presence of at least one solid neoplasm, said method comprising increasing permeability of said solid neoplasm by administration of a YKL-40 antibody to said individual, and administration of an anti-cancer agent to said individual. A kit-of-parts and/or a composition comprising a. a YKL-40 antibody; and b. an anti-cancer agent; for use in the treatment of cancer in an individual in need thereof, wherein said cancer is characterised by the presence of at least one solid neoplasm. Use of a kit-of-parts and/or a composition comprising a. a YKL-40 antibody; and b. an anti-cancer agent; for the preparation of a medicament for use in the treatment of a cancer characterised by the presence of at least one solid neoplasm. The kit-of-parts, the composition, the method or the use according to any one of items 50 to 52, wherein YKL-40 is YKL-40 of SEQ ID NO: 1 or a functional homologue thereof sharing at least 95% sequence identity therewith. The kit-of-parts, the composition, the method or the use according to any one of items 50to 53, wherein the antibody is a single domain antibody optionally linked to an Fc region, such as a human Fc region. The kit-of-parts, the composition, the method or the use according to any one of items 50to 54, wherein the antibody is the antibody according to any one of claims 1 to 49. The kit-of-parts, the composition, the method or the use according to any one of items 50to 55, wherein said individual is a human being. 57. The kit-of-parts, the composition, the method or the use according to any one of items 50 to 56, wherein the anti-cancer agent is selected from the group consisting of a chemotherapeutic agent, an immunotherapeutic agent, checkpoint inhibitors, anti-cancer antibodies, and cytostatic small molecule.
58. The kit-of-parts, the composition, the method or the use according to any one of items 50 to 57, wherein the anti-cancer agent is a small molecule, for example an organic compound having a molecular weight <900 dalton.
59. The kit-of-parts, the composition, the method or the use according to any one of items 50to 57, wherein the anti-cancer agent is immunotherapeutic agent.
60. The kit-of-parts, the composition, the method or the use according to item 59, wherein the immunotherapeutic agent is selected from the group consisting of immuno stimulatory monoclonal antibodies, neutralizing immune inhibitors, cytokines, adoptive T cell therapy, cancer vaccines, therapies based on NK- cells or mesenchymal stem cells and microbial adjuvants.
61. The kit-of-parts, the composition, the method or the use according to item 60, wherein the immuno stimulatory monoclonal antibodies are selected from the group consisting of CTLA4, PD1, PDL1 , LAG3, TIM3, CD137, 0X40, GITR, CD40.
62. The kit-of-parts, the composition, the method or the use according to item 60, wherein the neutralizing immune inhibitors are selected from the group consisting of TGF-beta, IL-10 and IDO1.
63. The kit-of-parts, the composition, the method or the use according to item 60, wherein the cytokines are selected from the group consisting of IFN alpha, IL-2 and IL-12.
64. The kit-of-parts, the composition, the method or the use according to item 60, wherein the microbial adjuvants are selected from the group consisting of TLR agonists, alpha-GalCer and STING activators. The kit-of-parts, the composition, the method or the use according to any one of items 50 to 58, wherein the anti-cancer agent is selected from the group consisting of radiotherapy, chemotherapeutic agents and signal transduction inhibitors. The kit-of-parts, the composition, the method or the use according to any one of items 50 to 58, wherein the anti-cancer agent is a chemotherapeutic agent. The kit-of-parts, the composition, the method or the use according item 66, wherein the chemotherapeutic agent is selected from the group consisting of alkylating agents, platinum compounds, antimetabolites, tubulin-inhibitors, antibiotics with anti-cancer effect and cytostatic molecules. The kit-of-parts, the composition, the method or the use according to item 65, wherein the signal transduction inhibitors are selected from the group consisting of protein kinase inhibitors, signal inhibitors not affecting the protein kinases, monoclonal antibodies in cancer therapy, hormones and hormone antagonists, cytokines, antibodies towards immunological checkpoints, oncolytic virus, immunological adjuvants, T-cell or dendritic cell or NK-cells or mesenchymal stem cell therapy and cancer vaccines. The kit-of-parts, the composition, the method or the use according to any one of items 50 to 68, wherein the anti-cancer agent is selected from the group consisting of the drug examples listed in table 1. The kit-of-parts, the composition, the method or the use according to any one of items 50 to 69, wherein said cancer is characterised by the presence of at least one solid neoplasm with increased interstitial fluid pressure and/or decreased permeability. The kit-of-parts, the composition, the method or the use according to any one of items 50 to 70, wherein said cancer is selected from the group consisting of solid neoplasms, metastasis and fiber rich Hodgkin type of malignant tumour (lymphomas). 72. The kit-of-parts, the composition, the method or the use according to any one of items 50 to 71 , wherein said cancer is selected from the group consisting of breast-, colorectal-, pancreas-, stomach-, hepatocellular-, bile duct, -other gastrointestinal-, lung-, small cell lung-, ovarian-, uterine-, cervix-, testis-, prostate, bladder-, renal-, thyroid- and head/neck carcinoma, malignant melanoma, other skin cancers, neuroblastoma, glioblastoma, astrocytoma and other forms of brain cancer, osteosarcoma, chondrosarcoma, myosarcoma, and germ cell malignant tumours.
73. The kit-of-parts, the composition, the method or the use according to any of the items 50 to 72, wherein said cancer is melanoma.
74. The kit-of-parts, the composition, the method or the use according to any one of items 50 to 58 and 65 to 73, wherein the anti-cancer agent is not anti-cancer agent is not an immune checkpoint inhibitor.
Sequences
SEQ ID NO: 1. Human YKL-40 also known as Chitinase-3-like protein 1 (CHI3L1)
MGVKASQTGFVVLVLLQCCSAYKLVCYYTSWSQYREGDGSCFPDALDRFLCTHIIYS
FANISNDHIDTWEWNDVTLYGMLNTLKNRNPNLKTLLSVGGWNFGSQRFSKIASNTQ
SRRTFIKSVPPFLRTHGFDGLDLAWLYPGRRDKQHFTTLIKEMKAEFIKEAQPGKKQL
LLSAALSAGKVTIDSSYDIAKISQHLDFISIMTYDFHGAWRGTTGHHSPLFRGQEDASP
DRFSNTDYAVGYMLRLGAPASKLVMGIPTFGRSFTLASSETGVGAPISGPGIPGRFTK
EAGTLAYYEICDFLRGATVHRILGQQVPYATKGNQWVGYDDQESVKSKVQYLKDRQL
AGAMVWALDLDDFQGSFCGQDLRFPLTNAIKDALAAT
SEQ ID NO: 2. 4A2 predator
LLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFRDSDEDMGWVRQAPGKGL
EWVSSISTSDGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASQYDT
GYSFDYWGQGTLVTVSSAAAEQKLISEEDLNGAATVESCLAKPHTENSFTNV
SEQ ID NO: 3. 4G1 predator
LLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFRDSDEDMGWVRQAPGKGL
EWVSSIGDESGSTYYADSVKGRFTISRDNSKNTLYLQNSLRAEDTAVYYCASTSDSY WSFDYWGQGTLVTVSSAAAEQKLISEEDLNGAATVESCLAKPHTENSFTNV
SEQ ID NO: 4. 4H3 predator
LLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFRDSDEDMGWVRQAPGKGL
EWVSSINAPDGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASQWDD
GYAFDYWGQGTLVTVSSAAAEQKLISEEDLNGAATVESCLAKPHTENSFTNVW
SEQ ID NO: 5.4D6 predator
LLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFRDSDEDMGWVRQAPGKGL
EWVSSISGSDGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASTDYL
RSSFDYWGQGTLVTVSSAAAEQKLISEEDLNGAATVESCLAKPHTENSFTNV
SEQ ID NO: 6. 4B6 predator
LLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFRDSDEDMGWVRQAPGKGL
EWVSSINNSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASTYDW
NYSFDYWGQGTLVTVSSAAAEQKLISEEDLNGAATVESCLAKPHTENSFTNV
SEQ ID NO: 7. 4A 12 predator
LLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFRDSDEDMGWVRQAPGKGL
EWVSSISAESGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASQYGA
YHDFDYWGQGTLVTVSSAAAEQKLISEEDLNGAATVESCLAKPHTENSFTNV
SEQ ID NO: 8. 1X2 predator
LLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFRDSDEDMGWVRQAPGKGL
EWVSSINYNSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASTPNL
NSSFDYWGQGTLVTVSSAAAEQKLISEEDLNGAATVESCLAKPHTENSFTNV
SEQ ID NO: 9. 2F6 predator
LLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFRDSDEDMGWVRQAPGKGL
EWVSSISDEDGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSGDW
WYGFDYWGQGTLVTVSSAAAEQKLISEEDLNGAATVESCLAKPHTENSFTNV
SEQ ID NO: 10. 2D7 predator LLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFRDSDEDMGWVRQAPGKGL
EWVSSISGESGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASVDPL
DTYFDYWGQGTLVTVSSAAAEQKLISEEDLNGAATVESCLAKPHTENSFTNV
SEQ ID NO: 11. 4F3 predator
LLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFRDSDEDMGWVRQAPGKGL
EWVSSIQSSDGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSSQN
GYVFDYWGQGTLVTVSSAAAEQKLISEEDLNGAATVESCLAKPHTENSFTNV
SEQ ID NO: 12. 2H12 predator
LLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFRDSDEDMGWVRQAPGKGL
EWVSSINNESGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASTSYY
GFDFDYWGQGTLVTVSSAAAEQKLISEEDLNGAATVESCLAKPHTENSFTNV
SEQ ID NO: 13. 2B8 predator
LLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFRDSDEDMGWVRQAPGKGL
EWVSSIYAPNGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASNTYD
AFDYWGQGTLVTVSSAAAEQKLISEEDLNGAATVESCLAKPHTENSFTNVWK
SEQ ID NO: 14. 4D9 predator
LLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFRDSDEDMGWVRQAPGKGL
EWVSSIASDSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSGYA
GTVFDYWGQGTLVTVSSAAAEQKLISEEDLNGAATVESCLAKPHTENSFTNVWKDDK
TLDRYANYEGCLWNATGVVVCT
SEQ ID NO: 15. 2F3 predator
LLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFRDSDEDMGWVRQAPGKGL
EWVSSIGAGSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSTDA
RWQFDYWGQGTLVTVSSAAAEQKLISEEDLNGAATVESCLAKPHTENSFTNV
SEQ ID NO: 16. 3F12 predator
LLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFRDSDEDMGWVRQAPGKGL
EWVSSINANDGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASQYDD
EFAFDYWGQGTLVTVSSAAAEQKLISEEDLNGAATVESCLAKPHTENSFTNV SEQ ID NO: 17. 4D5 predator
LLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFRDSDEDMGWVRQAPGKGL
EWVSSIGNYNGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASVSDS
GFSFDYWGQGTLVTVSSAAAEQKLISEEDLNGAATVESCLAKPHTENSFTNV
SEQ ID NO: 18. 4E10 predator
LLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFRDSDEDMGWVRQAPGKGL
EWVSSIYGPSGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASSDWD
GYSFDYWGQGTLVTVSSAAAEQKLISEEDLNGAATVESCLAKPHTENSFTNV
SEQ ID NO: 19. 2D3 predator
LLLLAAQPAMAEVQLLESGGGLVQPGGSLRLSCAASGFRDSDEDMGWVRQAPGKGL
EWVSSIDAEDGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCASQDGA
YYTFDYWGQGTLVTVSSAAAEQKLISEEDLNGAATVESCLAKPHTENSFTNV
SEQ ID NO: 20. CDR1
FRDSDEDMG
SEQ ID NO: 21. 4G1 CDR2
SIGDESGSTYYADSV
SEQ ID NO: 22. 4G1 CDR3
TSDSYWSFDY
SEQ ID NO: 23. 4H3 CDR2
SINAPDGSTYYADSV
SEQ ID NO: 24. 4H3 CDR3
QWDDGYAFDY
SEQ ID NO: 25. 4D6 CDR2
SISGSDGSTYYADSV
SEQ ID NO: 26. 4D6 CDR3
TDYLRSSFDY SEQ ID NO: 27. 4B6 CDR2 SINNSGGSTYYADSV
SEQ ID NO: 28. 4B6 CDR3 TYDWNYSFDY
SEQ ID NO: 29. 4A12 CDR2 SISAESGSTYYADSV
SEQ ID NO: 30. 4A12 CDR3 QYGAYHDFDY
SEQ ID NO: 31. 1X2 CDR2 SINYNSGSTYYADSV
SEQ ID NO: 32. 1X2 CDR3 TPNLNSSFDY
SEQ ID NO: 33. 2F6 CDR2 SISDEDGSTYYADSV
SEQ ID NO: 34. 2F6 CDR3 SGDWWYGFDY
SEQ ID NO: 35. 2D7 CDR2 SISGESGSTYYADSV
SEQ ID NO: 36. 2D7 CDR3 VDPLDTYFDY
SEQ ID NO: 37. 4F3 CDR2 SIQSSDGSTYYADSV
SEQ ID NO: 38. 4F3 CDR3 SSQNGYVFDY
SEQ ID NO: 39. 2H12 CDR2 SINNESGSTYYADSV
SEQ ID NO: 40. 2H12 CDR3 TSYYGFDFDY
SEQ ID NO: 41. 2B8 CDR2 SIYAPNGSTYYADSV
SEQ ID NO: 42. 2B8 CDR3 NTYDAFDY
SEQ ID NO: 43. 4D9 CDR2 SIASDSGSTYYADSV
SEQ ID NO: 44. 4D9 CDR3 SGYAGTVFDY
SEQ ID NO: 45. 2F3 CDR2 SIGAGSGSTYYADSV
SEQ ID NO: 46. 2F3 CDR3 STDARWQFDY
SEQ ID NO: 47. 3F12 CDR2 SINANDGSTYYADSV
SEQ ID NO: 48. 3F12 CDR3 QYDDEFAFDY
SEQ ID NO: 49. 4D5 CDR2
SIGNYNGSTYYADSV SEQ ID NO: 50. 4D5 CDR3
VSDSGFSFDY
SEQ ID NO: 51. 4E10 CDR2
SIYGPSGSTYYADSV
SEQ ID NO: 52. E1O CDR3
SDWDGYSFDY
SEQ ID NO: 53. 2D3 CDR2
SIDAEDGSTYYADSV
SEQ ID NO: 54. 2D3 CDR3
QDGAYYTFDY
SEQ ID NO: 55. CDR2 general formula
SIX1X2X3X4GSTYYADSV
SEQ ID NO: 56. 4A2 CDR2
SISTSDGSTYY
SEQ ID NO: 57. 4A2 CDR3
QYDTGYSFDY
SEQ ID NO:58
Amino acid sequence of hFc1
DKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSHEDPEVKFNW
YVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEK
TISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNY
KTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO:59
Amino acid sequence of hFc2
VECPPCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVQFNWYVDG
MEVHNAKTKPREEQFNSTFRVVSVLTVVHQDWLNGKEYKCKVSNKGLPAPIEKTISKT
KGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPP
MLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK
SEQ ID NO: 60. CDR2 general formula 2 SIX1X2X3X4GSTYY
SEQ ID NO: 61. CDR3 general formula
Xi X2 X3 X4 X5 X6 X7FDY
SEQ ID NO: 62. CDR2 general formula 3
SIX1X2X3X4GSTYY
Examples
Example 1 Selection of phage antibodies recognizing YKL-40
YKL-40 protein
YKL-40 protein was obtained from the supernatant of MG63 cells. MG63 production flasks were seeded at 1.8 x 104 cells/cm2 in RPMI-40 (Irvine Scientific) plus newborn calf serum, 100 mL/L (Irvine Scientific), 0.1 mol/L HEPES, and 50 mg/L vitamin C (complete medium). Flasks were incubated at 37 °C with humidity and CO2-enriched (100 mL/L) atmosphere for 6-8 days, replacing spent medium with fresh every 2-3 days. The cultures were then switched to serum-free medium (complete medium minus the newborn calf serum). The supernatants were harvested and the media replaced every 1-3 days for 30 days. YKL-40 protein was purified from the supernatants by concentrating glass-fiber-filtered material 20-fold with a 30-kDa screen channel cassette with tangential flow (Filtron) and then affinity-purifying over a heparin- Sepharose CL-6B column (Pharmacia Biotech) equilibrated with a solution of 10 mmol/L sodium phosphate and 50 mmol/L sodium chloride, pH 7.5. Bound material was eluted with a sodium chloride gradient (from 50 mmol/L to 2 mol/L) in 10 mmol/L sodium phosphate, pH 7.5, and 4-mL fractions were collected and pooled according to: absorbance at 280 nm, YKL-40 protein concentration by immunoassay and purity by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
Phage rescue
Selection of phage antibodies recognizing YKL-40 were performed using a predator antibody library, which is a single scaffold domain library with variation in CDR2 and CDR3 (Mandrup, Friis et al. 2013) against the purified YKL-40 protein. Thus, the antibodies of this library are single domain antibodies.
First a plastic immunotube was coated overnight with YKL-40 at 4°C, so the antigen could be partially absorbed by the plastic and anchored to the inner surface of the tube. The day after the tube was washed 5 times with PBS and incubated with 2% BSA in PBS for 2 hours at room temperature (RT). After another round of washing, 100 pl of the phage antibody library pREDATOR was added in 4 ml of 2% BSA in PBS. The phage antibody library was incubated for 60 min at RT rotating using an under- and over turntable and then further 60 min at RT standing on the bench. Unbound phages were washed away with 10 washes in PBS containing 0,1% Tween 20 and 10 washes with PBS.
Bound phages were eluted by adding 500 pl of trypsin- PBS (50 pl of 10 mg/ml trypsin stock solution added to 450 pl PBS) and incubating on rotating for 10 min at RT. The eluent was then incubated with 10 ml of TG1 (E.Coli) in exponential growth (at an OD 600 of 0.4) for 30 min at 37°C. The bacteria were then plated on TYE plates containing 100 pg /ml ampicillin and 1% glucose and grown overnight at 30°C. The following day individual colonies from the plates were picked up with a toothpick and put in a well of a 96 cell-well plate containing 2xTY medium with 100 pg/ml ampicillin and 1% glucose and grown on a shaker overnight at 37°C. The day after a transfer device was used to transfer a small inoculum from this plate (the master plate) to a second 96 cell- well plate containing the same medium. Bacteria were grown shaking at 37°C for 2 hours, new medium containing Helper phage, KM 13 was added. The plate were incubated for 1 hour at 37°C to let the Helper infect the cell, after this media was changed to 2xTY medium containing 100pg/ml Ampicillin and 50pg/ml of Kanamycin, finally the bacteria were incubate at 30°C, on shaking, overnight. The supernatant was used for ELISA.
Selection and amplification rounds
One round of selection was performed before screening the individual clones (to preserve diversity of binders regardless of affinity). The selection was run on all phage antibodies.
Screening rounds
Preliminary screening
After selection the eluted phages were infected in the E. coli TG-1 and spread on Amp selective agar plates. Around 500 colonies were obtained. These were inoculated in 100 pl 2xTY in 96 well plates. Phage antibodies were produced from the plates using the helper phage KM 13 according to standard procedures.
Example of Preliminary screening
After coating with used (MG63 conditioned) or fresh media, the ELISA plate were blocked with 2% skimmed milk powder in PBS. After blocking 50 pl of the phage supernatant were added to each well and incubated. The plates were washed and incubated with 1 :5000 diluted anti-M13 antibody (HRP conjugated). After washing the ELISA plate were developed using TMB and the plate read in an ELISA reader.
76 clones were selected as positive if they had a significant higher signal on the conditioned plate compared to fresh media plate. Clones were named according to plate and position on plate e.g. 3B2, which would be the clone on the third plate in row B column 2 (see table 1 below).
Table 1
Figure imgf000056_0001
Dilution series of the PEG precipitated phages
Selected clones which were judged potential positive in the preliminary screening were grown in 50 ml culture and rescued using the KM 13 helper phage. After overnight growth the supernatant were PEG precipitated according to standard procedures, and the pellet resuspended in 1 ml PBS. As phage antibody concentration is highly enriched by growing in 50 ml baffled flasks, the phage antibody concentration after PEG precipitation is 100-1000 more concentrated compared to the initial screen.
The PEG precipitated phage were used in validating ELISA, were a dilution series of the PEG precipitated phage were applied to ELISA plates coated with MG63 conditioned media or Fresh media. ELISA was performed as above.
The results of the dilution series is shown in Table 2 below. “Used” as used in Table 2 refers to MG63 conditioned media containing YKL-40. MG63 cells produces YKL-40 and thus medium in which MG63 cells have been grown (MG63 conditioned media) contains YKL-40. “Fresh” as used in Table 2 refers to fresh media which has not been in contact with YKL-40 producing cells (MG63) and thus does not contain YKL-40. Row 1-12 represents a series of dilution. Row 1 represents 100 pl antibody concentrate from 50 ml baffled flasks. Row 2 is a 5x dilution of the concentrate from row 1. Row 3 is a 5x dilution from of the dilution from Row 2 etc.
Table 2.
Figure imgf000057_0001
Figure imgf000058_0001
The clone indicated as “#NAME?” in table 2 corresponds to clone 1X2.
All positive clones were collected for validating the binding to YKL-40.
Secondary screening - validation of binding to YKL-40 After phage rescue supernatants containing selected single-domain antibodies were applied in ELISA to test for binding to YKL40. Serum free media (RPMI 1640 with 1 % Non-essential amino acids) were conditioned by growing MG63 cell line for 4 days. The supernatant was coated in ELISA plates. Fresh media without conditioning with MG63 were used as controls.
An ELISA was performed with the PEG precipitated phage from above (obtained under the section “Dilution series of the PEG precipitated phages’y. one plate were coated using conditioned MG63 media containing YKL-40 (“used”) one plate was coated with fresh media (“fresh media”) one plate was coated with 0,5 g purified YKL-40 from a different preparation compared to the preparation used for the screening (“YKL-40”) The ELISA was performed as above. The results are shown in Table 3 below. The first plate indicates the tested phages and their corresponding well. Row G column 5 to row H column 12 do not contain any phages and can be used as control wells.
Table 3.
Figure imgf000060_0001
The clone indicated as “#NAME?” in table 3 corresponds to clone 1X2. Phage antibodies with a signal higher than the background signal in the empty wells (Row G column 5 to row H column 12) were considered positive. A number of phage antibodies, indicated in gray, showed significant binding to purified YKL40 and were selected as the best candidates. Sequencing of antibodies
All the clones validated to bind to YKL40 were next sequenced. The Predator phage antibody library is a single scaffold domain library with variation in CDR2 and CDR3. Thus, the CDR1 did not vary within the YKL-40 binding clones. The sequence of each clone validated to bind to YKL40 is shown in Table 4.
Table 4:
Figure imgf000061_0001
Figure imgf000062_0001
Figure imgf000063_0001
Cloning
The two clones 4A2 and 4A12 can be cloned into a vector pFuse-hlgG2 as described in Moutel et al., 2009, or a vector containing another immunoglobulin constant part.
The vectors can be transfected into a suitable cell, such as ExpiCHO cells and expressed in this cell. This transfection leads to antibodies of VH domains fused to human lgG2 or other Fc regions expressed by the vector.
After transient transfection of suitable cells with the expression plasmid, secreted antibodies can be purified from the supernatant.
Example 2
Antibodies distribution in solid tumours
At least three primary malignant tumour tissue blocks from each of the following experiments were analysed:
1) Severe combined immunodeficient (SCID) mice harbouring human FemX-1 primary melanomas were injected into the tail vein with 10 pg of the murine antihuman CECAM1 antibody T84.1. (For details see Heine et al. , 2011). In histological sections of excised primary FEMX-1 melanomas, nearly all melanoma cells in the primary malignant tumours are CEACAM1 positive. The animals were sacrificed after 22.5 hours and perfused with physiological saline solution. The excised malignant tumour samples were fixed in neutral buffered formalin and processed to paraffin wax. 2) SCID mice harbouring human colon HT29 primary malignant tumours were injected with 10 pg of the anti EpCAM antibody MOC31 (for details see Heine et al., 2012). In histological sections of excised primary HT29 colon cancers, nearly all HT29 colon cancer cells in the primary malignant tumours are EpCAM positive. After 24 hours the mice were sacrificed and the malignant tumours were processed as above.
3) Her2/neu overexpressing PT1590 primary malignant tumours were treated for a period of 44 days with twice weekly intraperitoneal injection of 10mg/kg bodyweight trastuzumab antibody. Her2/neu expression was shown both by FACS analysis of the malignant tumour cells in cell culture as well as by immunohistochemistry in primary malignant tumours. Most of the cancer cells in the primary malignant tumours were Her2/neu positive. At the end of the experiment, mice were sacrificed and the malignant tumours were processed as above (for details see Lange et al., 2011).
Immunohistochemical demonstration of the antibodies applied
Four micron thick sections were cut from wax blocks of all these experiments and adhered to HistoBond® Adhesion Slides. After rehydration, sections were treated for 5 minutes with Fast Enzyme in a moist chamber at room temperature. After careful washes, the sections were incubated with the primary antibodies for one hour in a moist chamber. For the murine monoclonal antibodies a biotinylated goat anti mouse IgG polyclonal antibody diluted 1 :200 in Antibody Diluent was used. The humanized trastuzumab antibody was detected by a 1: 50 diluted polyclonal rabbit anti-human IgG specific for gamma chains. After three careful washes in Tris buffered saline (TBS), a biotinylated polyclonal swine anti rabbit immunoglobulins (solid phase absorbed) diluted 1 :200 in Antibody Diluent was applied for 30 minutes. After three further careful washes in TBS the Vectastain ABC-AP KIT was used according to the manufacturer's instructions for both anti-human and anti-mouse antibodies. Permanent Red Substrate Chromogen solution was used as a substrate for localization of the alkaline phosphatase. Rabbit polyclonal immunoglobulin fraction appropriately 1:1000 diluted served as a negative control.
A representative result is shown in figure 2A and 2B, which show a histological overview of a human FemX-1 primary malignant melanoma xenograft in immunodeficient mice treated with anti-human CEACAM1 mouse monoclonal antibody T84 (see Experiment 1. above). The immunoreactivity is limited to the region of the necrotic areas (asterisks). Most of the areas of the vital malignant tumour cells (black triangles in figure 1A) remain unstained. Only a limited area around blood vessels (arrows) is stained indicating the presence of antibodies.
To demonstrate that the lack of CEACAM1 immunoreactivity in the melanoma depicted in Fig. 2 A and B is not due to a lack of its expression, a parallel section of the malignant tumour used in 2 A and B was immunohistochemically stained with another anti- CEACAM1 monoclonal antibody. The results are shown in figures 1C and D showing expression of CEACAM in both the necrotic areas (asterisks) as well as in the vital malignant tumour tissue (black triangles).
Similar observations were made with the other tested antibodies (results not shown). Thus, in tumours from mice treated with EpCAM mouse monoclonal antibody MOC31 according to Experiment 2, the antibody is detected only in the areas around the blood vessels. More than 80% of the malignant tumour mass remains unstained indicating that the antibody did not reach the malignant tumour cells within the bulk of the malignant tumour. PT 1590 adenocarcinomas from mice treated with anti-human HER- 2/neu humanized mouse monoclonal antibody trastuzumab (Herceptin®) according to Experiment 3, the antibody is detected only in necrotic areas, while the area of the vital malignant tumour cells area remains unstained indicating the antibody has not penetrated into this region. Thus, the antibody is detected only in a small rim around the blood vessels.
Results& Conclusions
After a single injection of an anti EpCAM monoclonal antibody, most of the HT29 cancer cells in the primary xenograft malignant tumour were not decorated by the antibody. EpCAM immunoreactivity was mainly confined to area adjacent to blood vessels. The necrotic areas or connective tissue areas were not immunoreactive for mouse immunoglobulins.
A similar distribution immunoreactivity pattern was observed with anti-CEACAM1 antibody in FEMX malignant melanoma cells in the primary xenograft malignant tumour (Figure 2).
In another experiment, after multiple injections of the trastuzumab antibody in mice harbouring the Her-2/neu overexpressing cell line PT1590 as a primary malignant tumour, its immunoreactivity was confined to connective tissue and to the necrotic areas of the malignant tumour which were all heavily labelled. However, the PT1590 cells in the mass of the malignant tumour remained completely unlabelled.
In the case of the trastuzumab PT 1590 cells, practically all PT1590 cancer cells were not labelled by the antibody, while the surrounding connective tissues and the necrotic areas were strongly immunoreactive. This finding indicates that saturation with the trastuzumab antibody exists, which, however, is not reaching the cancer cells within the bulk of the malignant tumour. That the trastuzumab antibody, which was detected in the connective spaces and in the necrotic areas, is really effective in killing PT 1590 cancer cells is demonstrated by the fact, that the number of metastases decreased dramatically (78% of the mice in the control group had developed spontaneous metastases in the lungs, while only 11% did so in the 10 mg/kg bodyweight trastuzumab group), while the primary malignant tumour weight decreased much less (3,16 gram control versus 2,36 gram treatment group)( Lange et al., 2011). The finding that small metastatic deposits can be treated successfully while larger primary malignant tumour masses cannot be treated argues for the limited access of the antibody to the large primary malignant tumour mass in comparison to the small metastatic deposits.
Example 3 Penetration of antibodies into interior of solid tumours
To investigate penetration of YKL-40 antibodies in vivo, pathogen free BALB/c SCID (scid/scid) mice were used. The mice were 8-16 weeks old and weigh 20-30 g at the beginning of the experiments. They were housed in filter top cages and provided with sterile water and food ad libitum. For injection, human LOX melanoma cells were harvested by trypsinisation and viable cells (5x106) were suspended in 1 ml of cell culture medium. An aliquot of 200 ml of this suspension was injected subcutaneously between the scapulae of each SCID mouse. Human LOX cells have been shown to express YKL-40 by flow cytometry. Once mice carried tumours, 10 g antibody dissolved in PBS was injected intraperitoneally into LOX melanoma bearing mice. Five mice received anti-YKL-40 monoclonal antibody, consisting of the variable region of antibody 4A2 (SEQ ID NO: 2) linked to an Fc region by cloning into a pFUSE expression system as described in Moutel et al., 2009. Five mice received an equivalent volume of PBS as control.
Treatment with antibody started on day 3 and continued for 3 times per week for a total of 7 injections. 12 hours before sacrifice mice were injected 7,5 mg Cisplatin I kg body weight intraperitoneally.
Mice were sacrificed, intracardially perfused with NaCI and tumours were removed. Tumor size during experiments was measured with calipers, and the tumour weight at the end of treatment was determined (Figure 3B). Parts of tumours were then embedded in paraffin, other parts were processed for cryostat sections.
Cryostat or paraffin sections were prepared and stained for determination of antibody distribution in the melanoma tumours essentially as described in Example 2. The test antibodies were single-domain antibodies comprising the variable region of antibody 4A2 (SEQ ID NO:2) linked to human Fc, and for detection a 1 : 50 diluted polyclonal rabbit anti-human IgG specific for gamma chains is used. Representative examples of the results are shown in Figure 4 A-C.
In addition, the paraffin sections were stained for the distribution of cisplatin/DNA adducts as described below. Representative examples of the results are shown in Figure 5 A-B.
Cisplatin immunohistochemistry
Formalin-fixed and paraffin-embedded sections were deparaffinized in two changes of xylene (5 min each) and rehydrated in a series of graded ethanol (100, 96, 70 and 50% for 5 min each). Sections were then washed in aqua dest for 2 min. The following incubation steps were carried out in a moist chamber. For epitope retrieval, samples were treated for 5 min with Fast Enzyme (Zytomed Systems, Bargteheide, Germany) at room temperature, following two 5 min washes in Tris-buffered saline/0.1% Tween20 (TBS-T) and one 5 min wash in TBS (pH 7.6). Blocking with 4% BSA in TBS was performed for 30 min to prevent nonspecific antibody binding. Afterwards, sections were incubated with primary rat anti-Pt-[GpG] monoclonal antibody diluted 1 :1000 in antibody diluent (medac, Wedel, Germany) or rat lgG2a kappa at a dilution of 1 :500 (eBioscience, San Diego, USA) for 80 min at room temperature and then rinsed twice with TBS-T as well as with TBS for 5 min each. Subsequently, the secondary biotin- conjugated rabbit anti-rat antibody (Dako, Glostrup, Denmark) was incubated at a dilution of 1 :100 in antibody diluent for 30 min at room temperature, followed by rinsing twice with TBS-T and once with TBS for 5 min each. Sections were treated with Vectastain® ABC-AP Kit (Vector Laboratories, Burlingame, CA, US) according to the manufacturer’s recommendations for 30 min at RT and again washed in TBS-T and TBS as described above. Finally, alkaline phosphatase enzyme activity was visualized by incubating the sections with Permanent Red solution (Dako, Glostrup Denmark) for 20 min and counterstained with hematoxylin for 4 s, with intermediate washes under running tap water (3 min) and in aqua dest (2 min). Slides were dehydrated in a series of graded ethanol (70% for 15 s, 96 and 100% for 5 min each) and three changes of xylene (5 min each) and finally covered with Eukitt® Mounting Medium (Sigma-Aldrich, Steinheim, Germany) and coverslips.
HC-processed sections were first evaluated using a ZEISS Axiophot 2 microscope (Carl Zeiss, Jena, Germany). Digital images were obtained with a ZEISS Axio Scan Z1 slide scanner equipped with a ZEISS EC Plan-Neofluar 20x/0.50 Pol M27 objective (Carl Zeiss, Jena, Germany) and a Hitachi HV-F20SCL camera with 1600 x 1200 pixels (Hitachi Kokusai Electric America Ltd., New York, USA). For image acquisition, ZEISS ZEN 2.3 software was used (Carl Zeiss, Jena, Germany). Images were further processed with netScope Viewer software (Net-Base Software, Freiburg, Germany).
Microscopy of immunohistochemical stainings
Furthermore, the cisplatin concentration in the tumours was determined by graphitefurnace atomic absorption spectrometry as described in Bernhard Welz, Michael Sperling: Atomabsorptionsspektrometrie, WILEY-VCH-Verlag Weinheim, 1997, pages 572 - 573. The results are shown in Figure 3A. To perform the analysis a GF-AAS- (Graphite Furnace - Atomic Absorption Spectrometry) machine from Perkin-Elmer, Model AAnalyst 600 was used.
Results& Conclusions Furnace atomic absorption spectrometry revealed that tumours from mice injected with with YKL-40 antibody (4A2) showed a significant increase uptake of cisplatin compared to the control group (Figure 3). While the tumour weight was not affected, tumours from mice that received the antibody treatment displayed an increased volume (Figure 3). It is noted that cisplatin was administrated shortly before sacrificing the mice, and thus an impact of cisplatin on tumour weight or volume, was not expected. Taken together this data shows that the treatment with YKL-40 antibody 4A2 facilitated the permeability of cisplatin in the tumour. Without being bound by theory it is speculated that this is caused by the YKL-40 antibody A42 reducing interstitial fluid pressure.
To explore this hypothesis, the distribution of YKL-40 was investigated by Immunohistochemistry. The analysis of sections from tumours revealed that 4A2 YKL- 40 antibody administered by intraperitoneally distributed homogenously within the tumour (Figure 4). This homogenous distribution is in contrast to the distribution pattern observed for other antibodies as described in Example 2. Thus, in general, antibodies are detected only in the areas around the blood vessels. This data suggests that the YKL-40 antibody 4A2 enters and is distributed homogenously throughout the inner mass of the tumour.
Immunohistochemistry detection of cisplatin Pt-GG-Adducts is shown in figure 5, where the cisplatin Pt-GG-Adducts are visible as black spots. In the treatment group there is a visible increase in the amount of adducts in the inner tumour mass compared to the control group (Figure 5). While in the control group, adducts where detected mainly in regions easily accessible by the drug, within the treatment group adducts were detected homogeneusly within the tumor, confirming facilitated access of cisplatin to the inner mass of the malignant tumours (Figure 5).
References
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Ferretti S, Allegrini PR, Becquet MM, McSheehy PM. Tumor interstitial fluid pressure as an early-response marker for anticancer therapeutics. Neoplasia. 2009 Sep; 11 (9):874-81.
Heine M, Nollau P, Masslo C, Nielsen P, Freund B, Bruns OT, Reimer R, Hohenberg H, Peldschus K, Ittrich H, Schumacher II. Investigations on the usefulness of CEACAMs as potential imaging targets for molecular imaging purposes. PLoS One. 2011;6 (12):e28030.
Heine M, Freund B, Nielsen P, Jung C, Reimer R, Hohenberg H, Zangemeister-Wittke II, Wester HJ, Luers GH, Schumacher II. High interstitial fluid pressure is associated with low tumor penetration of diagnostic monoclonal antibodies applied for molecular imaging purposes. PLoS One. 2012;7(5):e36258.
Hust M, Dubel S (2004) Mating antibody phage display with proteomics. Trends Biotechnol 22: 8-14. doi:10.1016/j.tibtech.2003.10.011. PubMed: 14690617.
Johansen et al., 1992. Identifications of proteins secreted by human osteoblastic cells in culture. J Bone Miner Res, 7:501-512, 1992.
Lange T, Nentwich MF, Luth M, Yekebas E, Schumacher U. Trastuzumab has anti- metastatic and anti-angiogenic activity in a spontaneous metastasis xenograft model of esophageal adenocarcinoma. Cancer Lett. 2011 Sep 1;308(1):54-61.
Mandrup, O. A., N. A. Friis, S. Lykkemark, J. Just and P. Kristensen (2013). "A novel heavy domain antibody library with functionally optimized complementarity determining regions." PLoS One 8(10): e76834.
Moutel, S., A. El Marjou, O. Vielemeyer, C. Nizak, P. Benaroch, S. Dubel and F. Perez (2009). "A multi-Fc-species system for recombinant antibody production." BMC Biotechnol 9: 14.
Pansri P, Jaruseranee N, Rangnoi K, Kristensen P, Yamabhai M (2009) A compact phage display human scFv library for selection of antibodies to a wide variety of antigens. BMC Biotechnol 9: 6. doi:10.1186/1472-6750-9-6. PubMed: 19175944.
Rothe C, Urlinger S, Ldhning C, Prassler J, Stark Y et al. (2008) The human combinatorial antibody library HuCAL GOLD combines diversification of all six CDRs according to the natural immune system with a novel display method for efficient selection of high-affinity antibodies. J Mol Biol 376: 1182-1200. doi:10.1016/j.jmb.2007.12.018. PubMed: 18191144. Silacci M, Brack S, Schirru G, Marlind J, Ettorre A et al. (2005) Design, construction, and characterization of a large synthetic human antibody phage display library. Proteomics 5: 2340-2350. doi:10.1002/pmic. 200401273. PubMed: 15880779.

Claims

Claims
1. A YKL-40 antibody comprising a variable region comprising a CDR1 , CDR2 and CDR3, wherein
• CDR1 comprises or consists of an amino acid sequence of SEQ ID NO:20
• CDR2 comprises or consists of an amino acid sequence of the general formula:
SIX1X2X3X4GSTYYADSV (SEQ ID NO:55) or the general formula SIX1X2X3X4GSTYY wherein:
Xi is Q, S, N, D, G, E or Y;
X2 is S, T, G, N, A, D or Y;
X3 is S, E, N, Y, G, D or P;
X4 is D, G, S or N; and
• CDR3 comprises or consists of an amino acid sequence of the general formula
Xi X2 X3 X4 X5 X6 X7FDY wherein
Xi is Q, T, S or V,
X2 is Y, S, W, D, P, G or T,
X3 is D, Y, P, Q, Y, W or G
X4 is T, S, D, L, W, N or A
X5 is G, Y, G, R, N, W, D or E
X6 is Y, W, S, T, F or D
X7 is S, A, G, Y, V, D, Q or T.
2. The YKL-40 antibody according to claim 1, wherein said variable region comprises or consists of a VH domain.
3. The YKL-40 antibody according to any one of the preceding claims, wherein said variable region is linked to an Fc region, optionally via a linker.
4. The YKL-40 antibody according to any one of the preceding claims, wherein said YKL-40 antibody comprises or consists of two variable regions, wherein each variable region is linked to an Fc region, optionally by a linker, wherein said Fc regions are linked to each other.
5. The YKL-40 antibody according to any one of the preceding claims, wherein said Fc regions are linked together by disulphide bonds.
6. The YKL-40 antibody according to any one of the preceding claims, wherein the antibody comprises a CDR2 comprising or consisting of an amino acid sequence of the general formula
SIX1X2X3X4GSTYYADSV (SEQ ID NO:55) or the general formula SIX1X2X3X4GSTYY wherein:
Xi is Q, S, N, D, G, E or Y;
X2 is S, T, G, N, D or Y;
X3 is S, N, Y or G; and X4 is D or N.
7. The YKL-40 antibody according to any one of the preceding claims, wherein the
CDR2 comprises an amino acid sequence selected from the group consisting of SEQ ID NO:21 , SEQ ID NO:23, SEQ ID NO:25, SEQ ID NO:27, SEQ ID NO:29,
SEQ ID NO:31 , SEQ ID NO:33, SEQ ID NO:35, SEQ ID NO:37, SEQ ID NO:39,
SEQ ID NO:41 , SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, SEQ ID NO:49,
SEQ ID NO:51, SEQ ID NO:53 and/or SEQ ID NO:56.
8. The YKL-40 antibody according to any one of the preceding claims, wherein the CDR2 comprises or consists of SEQ ID NO: 56.
9. The YKL-40 antibody according to any one of the preceding claims, wherein the
CDR3 comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO:22, SEQ ID NO:24, SEQ ID NO:26, SEQ ID NO:28, SEQ ID NQ:30, SEQ ID NO:32, SEQ ID NO:34, SEQ ID NO:36, SEQ ID
NO:38, SEQ ID NQ:40, SEQ ID NO:42, SEQ ID NO:44, SEQ ID NO:46, SEQ ID
NO:48, SEQ ID NQ:50, SEQ ID NO:52, SEQ ID NO:54 and SEQ ID NO:57.
10. The YKL-40 antibody according to any one of the preceding claims, wherein the
CDR3 comprises or consists of SEQ ID NO:57.
11. The YLK-40 antibody according to any one of the preceding claims, wherein the antibody comprises or consists of a variable region comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NQ:10, SEQ ID NO:11 , SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19.
12. The YLK-40 antibody according to any one of the preceding claims, wherein the antibody comprises a variable region consisting of the amino acid sequence SEQ ID NO:2.
13. The YLK-40 antibody according to any one of the preceding claims, wherein the antibody consists of a variable region consisting of the amino acid sequence SEQ ID NO:2 linked to an Fc region, wherein said antibody optionally forms dimers.
14. The YKL-40 antibody according to claim 13, wherein the Fc region is a human Fc region.
15. The YKL-40 antibody according to claim 13, wherein the Fc region comprises or consists of amino acid sequence SEQ ID NO:58 or SEQ ID NO:59.
16. A method of treating disease associated with expression of YKL-40, the method comprising administering a therapeutically effective amount of the YKL-40 antibody according to any one of the preceding claims to a subject in need thereof.
17. A YKL-40 antibody according to any one of claims 1 to 15 for use in a method of treating a disease associated with YKL-40 expression.
18. Use of a YKL-40 antibody according to any one of claims 1 to 15 in the preparation of a medicament for use in a method of treating a disease associated with YKL-40 expression.
19. The method, antibody for use or the use according to any one of claims 16 to 18, wherein the disease is cancer.
20. A method for treating cancer in an individual suffering from cancer characterised by the presence of at least one solid neoplasm, said method comprising increasing permeability of said solid neoplasm by administration of a YKL-40 antibody to said individual, and administration of an anti-cancer agent to said individual.
21. A kit-of-parts and/or a composition comprising a. a YKL-40 antibody; and b. an anti-cancer agent; for use in the treatment of cancer in an individual in need thereof, wherein said cancer is characterised by the presence of at least one solid neoplasm.
22. Use of a kit-of-parts and/or a composition comprising a. a YKL-40 antibody; and b. an anti-cancer agent; for the preparation of a medicament for use in the treatment of a cancer characterised by the presence of at least one solid neoplasm.
23. The method, the kit-of-parts, the composition or the use according to any one of claims 20 to 22, wherein the method is a method for inhibiting the progression of cancer.
24. The method, the kit-of-parts, the composition or the use according to any one of claims 20 to 23, wherein the anti-cancer agent is selected from the group consisting of a chemotherapeutic agent, an immunotherapeutic agent, checkpoint inhibitors, anti-cancer antibodies, and cytostatic small molecule.
25. The method, the kit-of-parts, the composition or the use according to any one of claims 20 to 24, wherein the YKL-40 antibody is the YKL-40 antibody according to any one of claims 1 to 15.
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