WO2015067277A1 - Timp-1 binding partner - Google Patents

Timp-1 binding partner Download PDF

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WO2015067277A1
WO2015067277A1 PCT/DK2014/050371 DK2014050371W WO2015067277A1 WO 2015067277 A1 WO2015067277 A1 WO 2015067277A1 DK 2014050371 W DK2014050371 W DK 2014050371W WO 2015067277 A1 WO2015067277 A1 WO 2015067277A1
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cancer
timp
composition
use according
patient
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PCT/DK2014/050371
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French (fr)
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Nils Aage Brünner
José Moreira
Lena Vinther JENSEN
Birgitte Martine VIUFF
Mikkel HØEBERG
Anja Thoe FUGLSANG
Ulrik Axel Lademann
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University Of Copenhagen
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    • 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
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C12N15/1138—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against receptors or cell surface proteins
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.

Definitions

  • the present invention relates to a method of targeting cancer cells having an intracellular and/or extracellular environment of increased TIMP-1 levels by inhibition of TIMP-1 in order to increase the effect (increase sensitivity or reduce resistance) of a chemotherapeutic compound administered to said cancer cells.
  • TIMP-1 tissue inhibitor of metalloproteases-1 glycoprotein
  • MMP matrix metalloproteinases
  • TIMP-1 The cancer and/or blood levels of TIMP-1 have also been shown to correlate inversely with the response rate to some chemotherapeutics.
  • One MMP-independent function of TIMP-1 is inhibition of apoptosis, which may be involved in resistance to
  • TIMP-1 protein Some cancer cells have high intracellular levels of TIMP-1 protein but no
  • TIMP-1 is taken up by cancer cells such as by internalization.
  • Known cell surface binding partners for TIMP-1 include CD63 (Jung et al. EMBO J. 2006 Sep 6;25(17):3934-42). However, cells that do not express CD63 also internalizes TIMP-1 suggesting that additional cell surface binding partners for TIMP-1 exist.
  • the response rate to chemotherapeutics may be increased if the high levels of TIMP associated with poor response to chemotherapeutics are inhibited. Specifically the response rate to chemotherapeutics may be increased if the internalization of TIMP-1 into cancer cells is reduced or inhibited, such as by inhibiting the newly identified interaction between TIMP-1 and the cell surface protein CD74.
  • Disclosed herein is a method of treating a patient with cancer, such as by improving the response rate of one or more chemotherapeutic agents, said method comprising administering a TIMP-1 inhibitor as defined herein, whereby the sensitivity towards said one or more chemotherapeutic agents is increased and the response rate improved.
  • the TIMP-1 inhibitor may be administered alone or in combination with said one or more chemotherapeutic agents.
  • the TIMP-1 inhibitor targets TIMP-1 directly; interferes with TIMP-1 binding to cell surface proteins; interferes with TIMP-1 mediated activation of intracellular signalling.
  • the TIMP-1 inhibitor is a compound capable of inhibiting the interaction between TIMP-1 and CD74. In one embodiment the TIMP-1 inhibitor is capable of decreasing cellular internalization of TIMP-1 in a cell. In one embodiment said cell is a cancer cell, such as a CD74-positive cancer cell.
  • said patient with cancer has high or increased TIMP-1 levels, such as in bodily fluids including plasma, and/or in the cancer cells of said patient, and/or CD74-positive cancer cells.
  • FIG. 1 Western Blot. Protein complex formation of TIMP-1 and CD74 as revealed by Co-lmmunoprecipitation.
  • FIG. 1 western blot. Uptake of TIMP-1 is visualized in MDA-MB-231 cells +/- CD74 expression at 4 and 24 hours.
  • Chemotherapy is a category of cancer treatment that uses chemical substances, especially one or more anti-cancer drugs (chemotherapeutic agents). Chemotherapy may be given with a curative intent or it may aim to prolong life or to palliate symptoms. It is often used in conjunction with other cancer treatments, such as radiation therapy or surgery. Traditional chemotherapeutic agents act by killing cells that divide rapidly. Some newer anti-cancer agents (for example, various monoclonal antibodies) are not indiscriminately cytotoxic, but rather target proteins that are abnormally expressed in cancer cells and that are essential for their growth. Such treatments are often referred to as targeted therapy (as distinct from classic chemotherapy) and are often used alongside traditional chemotherapeutic agents in anti-neoplastic treatment regimens.
  • CD74 has a role in TIMP-1 internalization, causing an increased intracellular level of TIMP-1 in CD74 expressing cells. Increased intracellular levels of TIMP-1 may subsequently decrease the sensitivity towards certain anti-cancer agents, such as by affecting anti-apoptotic intracellular signaling.
  • the invention relates to improving the effect of anti-cancer agents such as anti- hormones, classic chemotherapeutic drugs and biologicals, such as antibodies and kinase inhibitors. More specifically, the invention relates to inhibition of the biological activities/functions of TIMP-1 in patients suffering from a cancer disease and being treated with said anti-cancer agent, in order to target the cancer and/or increase the sensitivity towards said anti-cancer agent.
  • the inhibition of TIMP-1 activities/functions can be accomplished by directly targeting the TIMP-1 protein or by inhibiting TIMP-1 protein interaction with its binding partners, e.g. cell surface binding proteins.
  • the inventors have identified herein CD74 as a new cell surface binding partner for TIMP-1 , which is shown to be involved in TIMP-1 internalization into cancer cells.
  • TIMP-1 inhibition examples include use of a neutralizing drug, e.g. an anti-TIMP-1 antibody; inhibition of the mRNA expression of TIMP-1 ; inhibition of the interaction between TIMP-1 protein and one or more binding partners located at cancer cell surfaces, such as by targeting at least CD74 or targeting CD74 and CD63; inhibiting TIMP-1 cellular internalization through prevention of its binding to one or more specific cell surface interaction partners, preferably comprising a compound capable of inhibiting the interaction between TIMP-1 and CD74; inhibiting TIMP-1 -induced intracellular signaling through its binding to cell surface proteins;
  • a neutralizing drug e.g. an anti-TIMP-1 antibody
  • inhibition of the mRNA expression of TIMP-1 inhibition of the interaction between TIMP-1 protein and one or more binding partners located at cancer cell surfaces, such as by targeting at least CD74 or targeting CD74 and CD63
  • inhibiting TIMP-1 cellular internalization through prevention of its binding to one or more specific cell surface interaction partners, preferably comprising a compound capable of inhibiting the interaction between TIMP-1 and
  • TIMP-1 mediated reduction in cellular sensitivity to anti-cancer drugs through inhibition of one or more of TIMP-1 cell surface binding, TIMP-1 internalization, TIMP-1 intracellular signaling or any way by which TIMP-1 mediates resistance to chemotherapeutics.
  • TIMP-1 (UniProt entry P01033; Metalloproteinase inhibitor 1 ) is one among four known endogenous inhibitors of the matrix metalloproteinases (MMPs). It is a 24 kD highly glycosylated protein produced and secreted by many different cell types. The main function of TIMP-1 is believed to be extracellular inhibition of the MMPs. However, more recent research has pointed to MMP-independent biological functions of TIMP-1 , including inhibition of apoptosis. TIMP-1 functions as a growth factor that regulates cell differentiation, migration and cell death and activates cellular signaling cascades via CD63 and ITGB1 .
  • MMPs matrix metalloproteinases
  • TIMP-1 protein With respect to treatment of cancer, an association between high tumor cell and/or high patient with cancer blood levels of TIMP-1 protein and a reduced effect of certain types of chemotherapy, including biological anticancer therapy and endocrine therapy, has been observed and validated in the clinical setting. For example, it is shown that while patients with primary breast cancer and TIMP-1 positive cancer cells do not gain additional benefit from exchanging methotrexate with epirubicin when given in combination with 5-Fluorouracil and cyclophosphamide, in patients with no or little TIMP-1 tumor cell protein, the epirubicin based treatment resulted in a significant benefit compared to the non-epirubicin treatment.
  • TIMP-1 is involved in determining the sensitivity (high levels conferring resistance) to epirubicin in clinical adjuvant breast cancer treatment.
  • Results from the present inventors show that patients with metastatic breast cancer, receiving a combination of docetaxel and gemcitabin versus docetaxel alone, and having no or little TIMP-1 protein expression in their cancer cells had a doubling of their survival while this difference was not observed among patients with TIMP-1 protein expressing cancer cells.
  • TIMP-1 Binding of TIMP-1 to the transmembrane protein CD63 with subsequent activation of the Akt survival pathway has been described. Moreover, the literature indicates that cell surface binding of TIMP-1 can also induce signaling though Erk (reviewed in Sci Signal. 2008 Jul 8;1 (27):re6. doi: 10.1 126/scisignal.127re6).
  • TIMP-1 protein produced by a mesenchymal stem cell culture can be internalized by human breast cancer cells in vitro when co-cultured (Kuvaja et al., Exp Cell Res. 2012 Jun 10;318(10):1094-103. doi: 10.1016/j.yexcr.2012.03.009)
  • the present inventors have performed extensive immunohistochemistry and in situ hybridization for TIMP-1 in formalin fixed paraffin embedded breast cancer tissue and observed that some patients have TIMP-1 positive tumor cells by
  • cancer cells can bind and internalize TIMP-1 .
  • cancer cells may internalize or take up TIMP-1 from the surroundings, rather than producing TIMP-1 by intracellular protein synthesis (transcription/translation).
  • Known cell surface binding partners for TIMP-1 include CD63 (Jung et al. EMBO J. 2006 Sep 6;25(17):3934-42).
  • the inventors have stained human breast cancer tissue biopsies for the known TIMP-1 binding partner CD63, and surprisingly have observed that some of the tumors that are TIMP-1 protein positive but TIMP-1 mRNA negative did not express CD63. This lead the inventors to propose that other cell surface binding partners for TIMP-1 than CD63 could be in play. The inventors therefore initiated research with the aim of identifying new TIMP-1 cell surface binding partners.
  • CD74 (also known as HLA-DR antigens-associated invariant chain; la antigen- associated invariant chain; li, p33) is a transmembrane protein with a short cytoplasmic tail. It participates in several key processes of the immune system, including antigen presentation, B-cell differentiation and inflammatory signaling. CD74 is a protein trafficking regulator and a cell membrane receptor for the pro-inflammatory cytokine MIF (Macrophage migration inhibitory factor), D-dopachrome tautomerase (D-DT/MIF- 2) and bacterial proteins. Cell surface CD74 is very rapidly internalized both with and without CD74 antibodies bound.
  • cytokine MIF Macrophage migration inhibitory factor
  • D-DT/MIF- 2 D-dopachrome tautomerase
  • CD74 is highly expressed in inflammatory disorders and some cancer types including hematopoietic cancers and some solid tumors (see e.g. Berkova et al.: "Milatuzumab - a promising new immunotherapeutic agent”. Expert Opin Investig Drugs. 2010 Jan;19(1 ):141 -9 and WO12104344A1 ).
  • Various formats of a humanized CD74-specific monoclonal antibody, hLLI have been proposed for treatment of CD74- positive tumors.
  • CD74-specific antibodies are currently studied for the treatment of multiple myeloma, non-Hodgkin's lymphoma and chronic lymphocytic leukemia.
  • CD74 and CD74 antigen are used interchangeably herein. Unless specified otherwise, the terms include any variants, isoforms and species homologs of human CD74 which are naturally expressed by cells or are expressed on cells transfected with the CD74 gene. At least four human isoforms are known to exist; p43, p41 , p35 and p33 (Borghese F et al., Expert Opin Ther Targets 201 1 ; 15(3) : 237-251 ). These result from alternative transcript splicing and two translation start sites.
  • p43 also known as CD74 isoform 1 , isoform a, or "long”; see UniProt entry P04233-1
  • p35 also known as CD74 isoform 2, isoform b or "short”; see Uniprot entry P04233-2
  • p41 and p33 arise from an alternative translation start site (48 bp downstream; 16 amino acids shorter protein) leading to variants lacking the endoplasmic reticulum (ER) retention signal that is present within these 16 amino acids, but having an identical extracellular part as p43 and p35, respectively.
  • TIMP-1 internalization
  • internalization when used in the context of TIMP-1 includes any mechanism by which TIMP-1 is internalized from the cell-surface into a CD74- expressing cell.
  • a TIMP-1 inhibitor in the present context in one embodiment refers to an antibody, preferably an antibody that selectively targets TIMP-1 or CD74.
  • An antibody targeting TIMP-1 or CD74 in one embodiment prevents cellular internalization of TIMP-1 .
  • Immunoglobulins are a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four inter-connected by disulfide bonds.
  • the structure of immunoglobulins has been well characterized. Briefly, each heavy chain typically is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH) typically comprised of three domains, CH1 , CH2, and CH3.
  • Each light chain typically is comprised of a light chain variable region (VL) and a light chain constant region, typically comprised of one domain (CL).
  • VH and VL regions may be further subdivided into regions of hypervariability also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs).
  • CDRs complementarity determining regions
  • antibody refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen under typical physiological conditions.
  • the variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen.
  • An antibody may also be multispecific, having specificities for two or more different epitopes, typically non-overlapping. Examples of multispecific antibodies include bispecific antibodies, diabodies, and similar antibody molecules.
  • the term antibody herein includes fragments of an antibody that retain the ability to specifically bind to the antigen.
  • the antigen-binding function of an antibody may be performed by fragments of a full-length antibody, e.g., Fab and F(ab')2 fragments.
  • antibody includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides such as chimeric antibodies and humanized antibodies.
  • An antibody as generated can possess any isotype.
  • epipe means a protein determinant capable of specific binding to an antibody.
  • human antibody include antibodies having variable and constant regions derived from human germline immunoglobulin sequences.
  • the human antibodies of the invention may include amino acid residues not encoded by human germline
  • immunoglobulin sequences e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo.
  • human antibody as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
  • mAb monoclonal antibody
  • a monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.
  • human monoclonal antibody refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences.
  • the human monoclonal antibodies may be produced by a hybridoma which includes a B cell obtained from a transgenic or transchromosomal non-human animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene, fused to an immortalized cell.
  • isotype refers to the immunoglobulin class (for instance IgGI, lgG2, lgG3, lgG4, IgD, IgA, IgE, or IgM) that is encoded by heavy chain constant region genes.
  • full-length antibody when used herein, refers to an antibody which contains all heavy and light chain constant and variable domains that are normally found in an antibody of that isotype.
  • chemotherapeutic agent comprises all conceivable anti-cancer agents including the traditional chemotherapeutics as well as targeted therapy.
  • a composition comprising a TIMP-1 inhibitor for use in a method of increasing the sensitivity of a cancer cell towards one or more chemotherapeutic agents, in a patient with cancer.
  • the patient with cancer may be defined as specified elsewhere herein. It is understood that increasing the sensitivity towards a chemotherapeutic agent is synonymous to increasing the response rate, reducing resistance and enhancing the effect to said chemotherapeutic agents.
  • Increasing the sensitivity of a cancer cell means that the cancer cell will become responsive to the chemotherapeutic agent where it was more or less non-responsive; in one embodiment effectively enabling use of a lower dosage of the chemotherapeutic agents to achieve a sufficient response rate.
  • composition comprising a TIMP-1 inhibitor for the manufacture of a medicament for use in a method of increasing the sensitivity of a cancer cell towards one or more chemotherapeutic agents in a patient with cancer.
  • Also disclosed is a method of reducing cellular TIMP-1 internalization comprising administering to a patient in need thereof a composition comprising a TIMP- 1 inhibitor.
  • the method reduces the cellular TIMP-1 internalization in a cancer cell, such as a CD74-positive cancer cell.
  • Reducing TIMP-1 internalization in cancer cells in one embodiment increases TIMP-1 concentration in the tumor microenvironment, which may reduce the propensity of the tumor to metastasize.
  • composition comprising, separately or together, i) a TIMP-1 inhibitor and ii) one or more chemotherapeutic agents for use in a method of treating cancer.
  • composition comprising, separately or together, i) a TIMP-1 inhibitor and ii) one or more chemotherapeutic agents for use in a method of increasing the sensitivity of a cancer cell towards said one or more chemotherapeutic agents.
  • compositions comprising i) a compound capable of inhibiting the interaction between TIMP-1 and CD74 alone or in combination with ii) one or more chemotherapeutic agents for use in the treatment of cancer.
  • compositions comprising i) a compound capable of inhibiting the interaction between TIMP-1 and CD74 alone or in combination with ii) one or more chemotherapeutic agents for use in a method of increasing the sensitivity of a cancer cell towards said one or more chemotherapeutic agents.
  • composition comprises two (or more)
  • said two or more compounds/agents may be administered simultaneous, separately or sequentially.
  • Also disclosed herein is a method of inhibiting the interaction between TIMP-1 and CD74, said method comprising administration of a TIMP-1 inhibitor to a patient in need thereof, such as to a cancer patient.
  • a method of inhibiting the interaction between TIMP-1 and CD74 said method comprising administration of a compound capable of inhibiting the interaction between TIMP-1 and CD74 to a patient in need thereof, such as to a cancer patient.
  • said patient with cancer has high or increased TIMP-1 levels, such as in in bodily fluids including plasma, and/or in the cancer cells of said patient, and/or has CD74-positive cancer cells.
  • a composition comprising a TIMP-1 inhibitor comprises two or more TIMP-1 inhibitors as defined herein.
  • the two TIMP-1 inhibitors comprises a compound that inhibits TIMP-1 directly, and a compound that inhibits CD74 directly.
  • An individual in need as referred to herein is in one embodiment an individual that benefits from the administration of an agent according to the present invention. Such an individual in one embodiment suffers from a cancerous condition in the tissue of one or more organs, or is at risk of suffering therefrom.
  • the term "Individual” refers preferably to vertebrates, particular members of the mammalian species, preferably primates including humans. The individual is in one embodiment any human being, male or female, infant, middle-aged or old.
  • the disorder to be treated or prevented in the individual in one embodiment relates to the age of the individual, the general health of the individual, the medications used for treating the individual and whether or not the individual has a prior history of suffering from diseases or disorders that may have or have induced ischemic and/or inflammatory conditions in the individual.
  • treatment refers to the management and care of a patient for the purpose of combating a condition, disease or disorder.
  • the term is intended to include the full spectrum of treatments for a given condition from which the patient is suffering, such as administration of the bioactive agent for the purpose of: alleviating or relieving symptoms or complications; delaying the progression of the condition, partially arresting the clinical manifestations, disease or disorder; curing or eliminating the condition, disease or disorder; and/or preventing or reducing the risk of acquiring the condition, disease or disorder, wherein "preventing” or “prevention” is to be understood to refer to the management and care of a patient for the purpose of hindering the development of the condition, disease or disorder, and includes the administration of the active compounds to prevent or reduce the risk of the onset of symptoms or complications.
  • composition comprising one or more TIMP-1 inhibitors for use in a method of increasing the sensitivity of a cancer cell towards one or more chemotherapeutic agents, in a patient with cancer.
  • the TIMP-1 inhibitor may be administered alone or in combination with said one or more chemotherapeutic agents.
  • the TIMP-1 inhibitor targets TIMP-1 directly; interferes with TIMP-1 binding to cell surface proteins; and/or interferes with TIMP-1 mediated activation of intracellular signalling.
  • a TIMP-1 inhibitor according to the present disclosure in one embodiment is an agent or compound that inhibits TIMP-1 in any way conceivable to the skilled person.
  • a TIMP-1 inhibitor is an agent or compound that inhibits one or more of TIMP-1 expression, TIMP-1 translation, TIMP-1 transcription, TIMP-1 secretion, TIMP- 1 stability, TIMP-1 internalization, TIMP-1 biological activity and TIMP-1 -induced intracellular signalling.
  • the TIMP-1 inhibitor targets the newly identified TIMP-1 binding partner CD74.
  • a TIMP-1 inhibitor is in one embodiment a compound capable of inhibiting the interaction between TIMP-1 and CD74.
  • the TIMP-1 inhibitor decreases intracellular levels of TIMP-1 , such as by inhibiting CD74-mediated TIMP-1 internalization and/or decreasing intracellular CD74-mediated accumulation of TIMP-1 . In one embodiment the TIMP-1 inhibitor is capable of inhibiting internalization of TIMP-1 .
  • the TIMP-1 inhibitor inhibits internalization of TIMP-1 in cells, such as cancer cells, such as cancer cells expressing CD74. In one embodiment the TIMP-1 inhibitor decreases internalization of TIMP-1 in CD74-positive cells.
  • the TIMP-1 inhibitor increases cancer cell sensitivity and/or decreases cancer cell resistance to one or more chemotherapeutics.
  • the present invention in one embodiment discloses a compound capable of inhibiting the interaction between TIMP-1 and CD74.
  • said compound capable of inhibiting the interaction between TIMP-1 and CD74 reduces the
  • said compound capable of inhibiting the interaction between TIMP-1 and CD74 increases sensitivity towards certain chemotherapeutics. In one embodiment said compound capable of inhibiting the interaction between TIMP-1 and CD74 increases sensitivity towards certain
  • chemotherapeutics at least in a specific subset of patients with cancer.
  • said subset of patients with cancer have high or increased TIMP-1 levels and/or CD74-positive cancer cells.
  • a compound capable of inhibiting the interaction between TIMP-1 and CD74 is a binding partner for CD74 other than TIMP-1 , such as MIF (Macrophage migration inhibitory factor) and/or D-dopachrome tautomerase (D-DT/MIF-2).
  • the composition according to the present invention further comprises one or more compounds capable of inhibiting the interaction between TIMP- 1 and one or more binding partners for TIMP other than CD74.
  • the composition further comprises one or more compounds capable of inhibiting the interaction between TIMP-1 and one or more of CD63 (Uniprot entry P08962), CD44 (Uniprot entry P16070), LRP1 (Prolow-density lipoprotein receptor-related protein 1 ; Uniprot entry Q07954), integrin complex and others known to the skilled person.
  • the composition further comprises a CD63 inhibitor.
  • the composition further comprises one or more specific kinase inhibitors, such as kinase inhibitors of intracellular signaling downstream of CD63 and/or CD74. These include Akt and Erk inhibitors.
  • a compound capable of inhibiting the interaction between TIMP-1 and CD74 is a TIMP-1 inhibitor targeting and inhibiting TIMP-1 directly.
  • the TIMP-1 inhibitor is able to reduce the level and/or activity of TIMP-1 .
  • the TIMP-1 inhibitor is able to inhibit TIMP-1 -induced intracellular signaling.
  • the TIMP-1 inhibitor specifically inhibits one or more MMP- independent functions of TIMP-1 , such as inhibits TIMP-1 mediated inhibition of apoptosis
  • the TIMP-1 inhibitor is a TIMP-1 antibody (anti-TIMP-1 antibody).
  • TIMP-1 -binding human antibodies are available, such as those disclosed in WO 2002/086085.
  • a TIMP-1 antibody is in one embodiment selected from the group consisting of a monoclonal antibody; one or more recombinant antibodies; and one or more polyclonal antibodies.
  • Said monoclonal antibody is in one embodiment selected from a chimeric, a humanized or fully human antibody.
  • the TIMP-1 antibody binds to an epitope in the CD74 binding site of TIMP-1 .
  • the TIMP-1 inhibitor interrupts or decreases TIMP-1 binding to the extracellular part of CD74. In one embodiment the TIMP-1 inhibitor is a small molecule TIMP-1 inhibitor.
  • the TIMP-1 inhibitor is a TIMP-1 antisense oligonucleotide providing for inhibition of TIMP-1 gene expression.
  • oligonucleotides are known to the skilled person including RNA, DNA and chemical analogues thereof, including PNA (peptide nucleic acid), PMO
  • the TIMP-1 inhibitor is an agent that down-regulates TIMP-1 , such as an EGF-R inhibitor including EGR-R antibodies such as cetuximab; Statins (HMG- CoA reductase inhibitors) including atorvastatin (Lipitor), fluvastatin (Lescol), lovastatin (Mevacor, Altocor), pitavastatin (Livalo), pravastatin (Pravachol), rosuvastatin (Crestor) and simvastatin (Zocor); and an inhibitor of one or more of HER-1 , HER-1 , HER-3 and HER-4 such as an HER-1 antibody, an HER-1 antibody, an HER-3 antibody and an HER-4 antibody antibody.
  • EGF-R inhibitor including EGR-R antibodies such as cetuximab
  • Statins HMG- CoA reductase inhibitors
  • atorvastatin Lipitor
  • fluvastatin Lescol
  • lovastatin Mevacor
  • the TIMP-1 inhibitor is administered in combination with an agent that down-regulates TIMP-1 .
  • the TIMP-1 inhibitor is a CD74 inhibitor.
  • the compound capable of inhibiting the interaction interaction between TIMP-1 and CD74 is a CD74 inhibitor.
  • said CD74 inhibitor is able to inhibit one or more of CD74 expression, CD74 translation, CD74
  • CD74 secretion CD74 secretion
  • CD74 stability CD74 biological activity
  • CD74 - induced intracellular signalling CD74 - induced intracellular signalling
  • the CD74 inhibitor is a small molecule CD74 inhibitor.
  • the CD74 inhibitor is a CD74 antisense oligonucleotide providing for inhibition of CD74 gene expression.
  • a range of antisense oligonucleotides are known to the skilled person including RNA, DNA and chemical analogues thereof, including PNA (peptide nucleic acid), PMO morpholinophosphordiamidate) and siRNA.
  • the CD74 inhibitor is a CD74 antibody.
  • a "CD74 antibody”, “anti- CD74 antibody”, “CD74 Ab”, “CD74-specific antibody” or “anti-CD74 Ab” is an antibody as described herein, which binds specifically to the antigen CD74.
  • the antibody of the invention is isolated.
  • an "isolated Ab,” as used herein, is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities.
  • An isolated antibody that specifically binds to an epitope, isoform or variant of human CD74 may, however, have cross-reactivity to other related antigens, for instance from other species (such as CD74 species homologs).
  • the CD74 antibody binds to CD74 and interrupts binding of TIMP-1 to CD74 and/or internalization of TIMP-1 .
  • a CD74 antibody is in one embodiment selected from the group consisting of a monoclonal antibody (MAb); one or more recombinant antibodies; and one or more polyclonal antibodies.
  • Said monoclonal antibody is in one embodiment selected from a chimeric, a humanized or fully human antibody.
  • the CD74 antibody is a monoclonal fully human antibody.
  • the CD74 antibody of the invention is selected from the group consisting of multispecific antibodies, bispecific antibodies, diabodies, full-length antibodies, fragments of an antibody e.g., Fab and F(ab')2 fragments, antibody-like polypeptides such as chimeric antibodies and humanized antibodies, antibodies of any isotype, naked antibodies and antibodies conjugated with drug (antibody-drug- conjugate).
  • the CD74 antibody of the invention binds to an epitope of one or more of the four known human isoforms; p43, p41 , p35 and p33.
  • the CD74 antibody is multispecific having specificities for two or more different epitopes such as one or more of the CD74 isoforms p43, p41 , p35 and p33.
  • the CD74 antibody is selected from the group consisting of a CD74 isoform p43 antibody, a CD74 isoform p41 antibody, a CD74 isoform p35 antibody and a CD74 isoform p33 antibody.
  • the CD74 antibody binds to membrane bound CD74.
  • anti-CD74 antibodies are known in the art and any such known antibody or fragment thereof may be utilized.
  • the CD74 antibody is a commercially available CD74 antibody, milatuzumab, a humanized LL1 (hl_L1 ) CD74 antibody, LS-B 1963, LS-B2594, LS-B 1859, LS-B2598, LS-05525, LS-C44929, LN2, PIN.1 , SPM523, LN3, CerCLIP.1 , A.14/19, Bu5, 1 D1 , 5-329 and any other CD74 antibodies known in the art.
  • a humanized LL1 (hl_L1 ) anti-CD74 antibody is disclosed in US 7,312,318.
  • the CD74 antibody binds to the same epitope of CD74 as an LL1 antibody.
  • a patient in need thereof is a patient with cancer.
  • a patient in need thereof is a patient with cancer treated with one or more chemotherapeutic agents.
  • the patient with cancer treated with one or more chemotherapeutic agents has a reduced response rate to said chemotherapeutic agents.
  • a patient in need thereof is a patient with cancer having high or increased TIMP-1 levels.
  • a patient in need thereof is a patient with cancer having a TIMP-1 positive cancer.
  • a patient in need thereof is a patient with cancer having a CD74 positive cancer.
  • a patient with high or increased TIMP-1 levels can refer to one or both of having high or increased TIMP-1 levels inside the cancer cells and outside the cancer cells.
  • the levels may refer to intracellular levels within the cancer cells or extracellular levels. Intracellular levels within the cancer cells may be measured in a tumor sample comprising cancer cells, and extracellular levels may be measured in bodily fluids.
  • high or increased TIMP-1 levels refers to TIMP-1 levels in one or more bodily fluids.
  • Bodily fluids include but are not limited to whole blood, plasma, serum, urine, saliva, sputum and cerebrospinal fluid.
  • high or increased TIMP-1 levels refer to high or increased plasma TIMP-1 levels.
  • a patient with cancer having high or increased TIMP-1 levels in one embodiment means that the patient with cancer has a TIMP-1 plasma level that is above the TIMP-1 plasma level in healthy humans.
  • the patients' TIMP-1 plasma level is above the mean value of healthy humans.
  • the mean value of healthy humans can be identified for example via the TIMP-1 quantitation method described in Holten-Andersen et al 1999 (British Journal of cancer, 80(3/4), 495-503).
  • the patients' TIMP-1 plasma level is above a mean value of 69.2 +/- 13.1 pg 1 (citrate plasma) or above a mean value of 73.5 +/- 14.2 pg 1 (EDTA plasma).
  • the patients' TIMP-1 plasma level is above a reference range (10 th to 90 th centile) of 55.0 - 90.3 pg 1 (citrate plasma) or above a reference range of 58.0 - 91.8 pg ⁇ 1 (EDTA plasma).
  • the patients' TIMP-1 plasma level is above a mean value of 50 pg 1 , such as above a mean value of 60 pg 1 1 ,for example 70 pg 1 , such as 80 pg 1 , for example 90 pg 1 , such as 100 pg 1 , for example 1 10 pg 1 , such as 120 pg 1 , for example 130 pg 1 , such as 140 pg 1 , for example 150 pg 1 , such as 160 pg 1 , for example 170 pg 1 , such as 180 pg 1 , for example 190 pg 1 , such as above a mean value of 200 pg ⁇ 1
  • the patients' TIMP-1 plasma level has a mean value of 50-60 pg 1 , such as 60-70 pg 1 1 ,for example 70-80 pg I "1 , such as 80-90 pg I "1 , for example 90- 100 pg ⁇ 1 , such as 100-1 10 pg ⁇ 1 , for example 1 10-120 pg ⁇ 1 , such as 120-130 pg ⁇ 1 , for example 130-140 pg 1 , such as 140-150 pg 1 , for example 150-160 pg 1 , such as 160-170 pg 1 , for example 170-180 pg 1 , such as 180-190 pg 1 , for example a mean value of 190-200 pg I "1 .
  • High or increased TIMP-1 levels in one embodiment means that the cancer patients' cancer cells have high or increased intracellular levels of TIMP-1 ; or detectable levels. In one embodiment this is defined as high or increased, or detectable, intracellular levels as compared to the intracellular level of TIMP-1 in the surrounding cells; and/or high or increased, or detectable, as compared to the level of TIMP-1 in cancer cells that are responsive to a given chemotherapy; and/or high or increased, or detectable, as compared to the level of TIMP-1 in non-cancer cells.
  • Non-cancer cells may refer to non-cancer cells of the same individual, or one or more different individuals (such as a mean value).
  • Cancer cells that are responsive to a given chemotherapy may refer to cancer cells of the same individual, or one or more different individuals (such as a mean value). In one embodiment responsive to e.g. epirubicin, epirubicin in
  • TIMP-1 positive cancer cells means that levels are detectable, and/or having higher intensity of staining than '-' cells and possibly higher than intermediate staining populations.
  • said patient with cancer has high or increased extracellular TIMP-1 levels and high or increased, or detectable, intracellular TIMP-1 levels.
  • said patient with cancer has high or increased TIMP-1 levels and a TIMP- 1 positive cancer.
  • High or increased, or detectable, intracellular TIMP-1 levels of the cancer patients' cancer cells are in one embodiment meant to refer to TIMP-1 protein, to ensure that exogenous TIMP-1 that has been internalized is measured.
  • the patient with cancer has a CD74 positive cancer. That is, the cancer patients' cancer cells express detectable CD74 and the cancer is characterized as CD74+ and/or CD74 hi .
  • the patient with cancer has high or increased TIMP-1 levels as defined herein and has a CD74 positive cancer.
  • the patient with cancer has a CD63 negative cancer. That is, the cancer patients' cancer cells expression of CD63 is undetectable, absent or low and the cancer is characterized as CD74 -. In one embodiment, the patient with cancer has a CD74 positive and CD63 negative cancer (CD74+/CD63-). Some breast cancers have been characterized as CD74+ and CD63-.
  • the patient with cancer has one or more of high or increased TIMP- 1 levels, a TIMP-1 positive cancer, a CD74 positive and CD63 negative cancer. In one embodiment the patient with cancer has high or increased TIMP-1 levels and a CD74 positive and CD63 negative cancer. In one embodiment, the patient with cancer has a CD74 positive and CD63 positive cancer (CD74+/CD63+). In this embodiment the patient with cancer in one embodiment is co-treated with a CD74 inhibitor to interrupt the CD74-mediated TIMP-1
  • '-' e.g. CD63- or negative
  • '-' means that the marker is undetectable, absent or low, at least lower than an
  • '+' e.g. CD74+ or positive
  • expression is detectable, and/or have higher intensity of staining than '-' cells and possibly higher than intermediate staining populations.
  • 'hi' cells e.g. CD74 hl
  • '+' cells have higher expression and/or intensity of staining than the '+' cells (e.g.
  • CD74 + CD74 + cells.
  • the staining intensity may be determined using conventional FACS methods. Staining intensities may be identified using suffixes +, ++ and +++; all of which are comprised within the term 'CD74 positive' or CD74+ in the present context.
  • the present composition comprising a TIMP-1 inhibitor and optionally one or more chemotherapeutic agents has a greater therapeutic effect that when the one or more chemotherapeutic agents are administered alone.
  • the present composition comprising a TIMP-1 inhibitor and optionally one or more chemotherapeutic agents has a greater therapeutic effect in a patient with cancer having high or increased TIMP-1 levels and/or a CD74-positive cancer, that when the one or more chemotherapeutic agents are administered alone.
  • the present composition comprising a TIMP-1 inhibitor and optionally one or more chemotherapeutic agents has a greater therapeutic effect in a patient with cancer having high or increased TIMP-1 levels and/or a CD74-positive cancer, than in a patient with cancer having low or normal TIMP-1 levels and/or a CD74-negative cancer.
  • Determining if a cancer cell or a tumor cell sample containing cancer cells is CD74 positive, CD63 positive and/or TIMP-1 positive can be done using any conventional methods known to the skilled person. Likewise, the sampling may be done using conventional method known to the skilled person.
  • a sample containing cancer cells wherein said sample is extracted from said patient with cancer, is examined to determine the levels of one or more of TIMP-1 , CD74 and CD63.
  • the levels refer to mRNA levels. In another preferred embodiment the levels refer to protein levels.
  • the TIMP-1 , CD74 and CD63 protein levels may be measured using conventional techniques including immunostaining techniques where antibodies are used to detect proteins in tissues and cells, immunohistochemistry, immunocytochemistry, western blotting, dot blot analysis, and ELISA.
  • the mRNA levels may be measured using conventional techniques including northern blotting, RT-qPCR, SAGE, RNA-Seq, and expression profiling techniques.
  • CD74 may be detected using the methods described in Starlets et al., "Cell-surface CD74 initiates a signaling cascase leading to cell proliferation and survival", Blood, 2006, 107: 4807-4816.
  • CD63 may be detected using the methods described in Caby et al. "Exosomal-like vesicles are present in human blood plasma", International Immunology, vol. 17, No. 7, pp. 879-887.
  • a patient with cancer or a cancer cell refers to a cancer selected from the group consisting of Bone cancer - including Ewing's Sarcoma, Osteosarcoma, Chondrosarcoma; Brain and CNS tumors - including Acoustic Neuroma, Spinal Cord Tumours; Breast cancer - including male breast cancer and Ductal Carcinoma in situ; Colorectal cancer; anal cancer; Endocrine cancers - including Adrenocortical Carcinoma, Pancreatic Cancer, Pituitary Cancer, Thyroid Cancer, Parathyroid Cancer, Thymus Cancer, Multiple Endocrine Neoplasia and Other Endocrine cancers; Gastrointestinal cancers - including Stomach (Gastric) Cancer, Esophageal Cancer, Small Intestine Cancer, Gall Bladder Cancer, Liver Cancer, Extra-Hepatic Bile Duct Cancer and Gastrointestinal Carcinoid Tumour;
  • Genitourinary cancers including Testicular Cancer, Penile Cancer and Prostate Cancer; Gynaecological cancers - including Cervical Cancer, Ovarian Cancer, Vaginal Cancer, Uterus / Endometrium Cancer, Vulva Cancer, Gestational Trophoblastic Cancer, Fallopian Tube cancer and Uterine sarcoma; Head and Neck Cancer - including Oral cavity Cancer, Lip Cancer, Salivary gland Cancer, Larynx Cancer, hypopharynx Cancer, oropharynx Cancer, Nasal Cancer, Paranasal Cancer and Nasopharynx Cancer; Leukaemia - including Childhood Leukaemia, Acute Lymphocytic Leukaemia, Acute Myeloid Leukaemia, Chronic Lymphocytic Leukaemia, Chronic Myeloid Leukaemia, Hairy Cell Leukaemia, BMT for Leukaemia, Patient's Home Pages, Acute Promyelocytic Leukemia and Plasma Cell Leukaemia; Haematological disorders - including Multiple mye
  • Lung cancer including Small Cell Lung Cancer and Non-Small Cell Lung Cancer
  • Lymphoma including Hodgkin's Disease, Non-Hodgkin's Lymphoma and Aids Related Lymphoma
  • Eye cancer including Retinoblastoma and IntraOcular Melanoma
  • Skin cancer including Melanoma, squamous cell carcinoma and basal cell carcinoma
  • Soft tissue Cancer including Soft tissue sarcoma and Kaposi's sarcoma
  • Urinary system cancer including Kidney Cancer, Wilm's Tumour, Bladder Cancer, Urethral Cancer and Transitional Cell Cancer; wherein each of these cancers may or may not be metastatic.
  • composition comprising a TIMP-1 inhibitor according to the present invention for treatment of a cancer and/or for increasing the sensitivity of the cancer to a chemotherapeutic agent, is for use in treating a solid cancer / non- hemotopoietic cancer.
  • present use is not for treating multiple myeloma, non-Hodgkin's lymphoma and chronic lymphocytic leukemia.
  • the one or more chemotherapeutic agents herein may be any chemotherapeutic agent known to the skilled person or a combination thereof.
  • the chemotherapeutic agent of the present invention is a chemotherapeutic agent that has reduced or no effect when TIMP-1 levels are high or increased, or detectable, as defined herein;
  • the chemotherapeutic agents whose effect will be increased when the patient with cancer is treated with a TIMP-1 inhibitor according to the present invention may be identified by evaluating the response rate of said one or more chemotherapeutics and determining the TIMP-1 levels as outlined herein. If the response rate is low, and the TIMP-1 levels are high or increased, or detectable, then the patient is likely to benefit from treatment with a TIMP-1 inhibitor of the present invention.
  • the patient with cancer to be treated with a TIMP-1 inhibitor of the present invention has been and/or is being treated with one or more chemotherapeutic agents.
  • composition of the invention comprises, together or separately, i) a TIMP-1 inhibitor, such as a compound capable of inhibiting the interaction between TIMP-1 and CD74, and ii) one or more chemotherapeutic agents, wherein the TIMP-1 inhibitor and the one or more chemotherapeutic agents are co-administered, which coadministration can be simultaneously, sequentially or separately.
  • a TIMP-1 inhibitor such as a compound capable of inhibiting the interaction between TIMP-1 and CD74
  • chemotherapeutic agents a chemotherapeutic agents
  • the chemotherapeutic agent is selected from the group consisting of alkylating agents, anti-metabolites, anti-microtubule agents, topoisomerase inhibitors, cytotoxic antibiotics and targeted therapies.
  • Alkylating agents are so named because of their ability to alkylate many molecules including DNA.
  • Alkylating agents include the nitrogen mustards, nitrosoureas, tetrazines, aziridines, cisplatins and derivatives, and non-classical alkylating agents.
  • Nitrogen mustards include mechlorethamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide and busulfan.
  • Nitrosoureas include N-Nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU) and semustine (MeCCNU), fotemustine and streptozotocin.
  • Tetrazines include dacarbazine, mitozolomide and temozolomide.
  • Aziridines include thiotepa, mytomycin and diaziquone (AZQ).
  • Cisplatin and derivatives include cisplatin, carboplatin and oxaliplatin.
  • Non-classical alkylating agents include procarbazine and hexamethylmelamine.
  • Anti-metabolites are a group of molecules that impede DNA and RNA synthesis.
  • Subtypes of the anti-metabolites are the anti-folates, fluoropyrimidines,
  • deoxynucleoside analogues and thiopurines include methotrexate and pemetrexed.
  • the fluoropyrimidines include fluorouracil and capecitabine.
  • Fluorouracil is a nucleobase analogue that is metabolised in cells to form at least two active products; 5-fluourouridine monophosphate (FUMP) and 5-fluoro-2'-deoxyuridine 5'-phosphate (fdUMP).
  • Capecitabine is a prodrug of 5-fluorouracil that is broken down in cells to produce the active drug.
  • the deoxynucleoside analogues include cytarabine, gemcitabine, decitabine, Vidaza, fludarabine, nelarabine, cladribine, clofarabine and pentostatin.
  • the thiopurines include thioguanine and mercaptopurine.
  • Anti-microtubule agents prevent microtubule function with two main groups Vinca alkaloids and taxanes.
  • the original vinca alkaloids are natural chemicals that include vincristine and vinblastine.
  • Semi-synthetic vinca alkaloids include: vinorelbine, vindesine, and vinflunine.
  • Taxanes are natural and semi-synthetic drugs and include paclitaxel and docetaxel.
  • Topoisomerase inhibitors are drugs that affect the activity of one of the enzymes: topoisomerase I and topoisomerase II. Topoisomerase I inhibitors include
  • topoisomerase II inhibitors can be divided into two groups; the topoisomerase II poisons and the catalytic inhibitors.
  • Topoisomerase II poisons include etoposide, doxorubicin, mitoxantrone and teniposide.
  • the catalytic inhibitors include novobiocin, merbarone, and aclarubicin as well as ICRF-187.
  • the cytotoxic antibiotics are a varied group of drugs that have different mechanisms of action.
  • the group includes the anthracyclines and other drugs including actinomycin, bleomycin, plicamycin, and mitomycin.
  • the anthracyclines include doxorubicin and daunorubicin and derivatives thereof including epirubicin and idarubicin, as well as pirarubicin, aclarubicin, and mitoxantrone.
  • Actinomycin and Bleomycin intercalates DNA.
  • Some newer anticancer drugs specifically target proteins that are abnormally expressed in cancer cells and that are essential for their growth. Such treatments are often referred to as targeted therapy (as distinct from classic chemotherapy).
  • Antihormone therapy is a form of treatment which suppresses selected hormones. The suppression of certain hormones is beneficial to the cancer patient since certain hormones prompt the growth of a tumor. Antihormone treatment can be specific to gender as well, such as antiestrogen or antitestosterone therapy.
  • Anti-hormone therapies include aromatase inhibitors including irreversible steroidal inhibitors and non-steroidal inhibitors such as Aminoglutethimide, Testolactone (Teslac), Anastrozole (Arimidex), Letrozole (Femara), Exemestane (Aromasin), Vorozole (Rivizor),
  • Formestane (Lentaron) and Fadrozole (Afema); anti-estrogens such as tamoxifen, Fulvestrant (Faslodex), Toremifene, Raloxifene, Selective estrogen receptor modulators (SERM's); anti-androgenic drugs such as Flutamide (Eulexin), nilutamide (Anandron and Nilandron), bicalutamide (Casodex), 5-alpha-reductase inhibitors such as finasteride (Proscar and Propecia), dutasteride (Avodart), bexlosteride, izonsteride, turosteride and epristeride.
  • anti-estrogens such as tamoxifen, Fulvestrant (Faslodex), Toremifene, Raloxifene, Selective estrogen receptor modulators (SERM's)
  • anti-androgenic drugs such as Flutamide (Eulex
  • a range of protein kinases are targets for the kinase inhibitors, including EGFR, ErbB2, VEGFRs, VEGF, BcrAb, SRC, ErbB1 , ALK/Met, Syk, BTK, PDGFR, c-kit, JAK, STAT, RET and BRAF.
  • the kinase inhibitors include afatinib, axitinib, bevacizumab, bosutinib, cetuximab, crizotinib, dasatinib, erlotinib, fostamatinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pagaptanib, ranibizumab, ruxolitinib, sorafenib, SU6656, trastuzumab, tofacitinib, vandetanib and vemurafenib.
  • the chemotherapeutic agent of the invention is a monoclonal antibody that binds to cancer cell-specific antigens and induce an immunological response against the target cancer cell.
  • Therapeutic monoclonal antibodies include Bevacizumab/Avastin (VEGF-A inhibitor), Cetuximab/Erbitux, Panitumumab/Vectibix, Nimotuzumab (EGFR inhibitors), Trastuzumab/Herceptin (HER2/neu receptor inhibitor), Rituximab/Rituxan (CD20) Gemtuzumab ozogamicin/Mylotarg (CD33), Alemtuzumab/Campath (CD52), Brentuximab vedotin/ Adcetris, Denosumab/Prolia, Ibritumomab tiuxetan/Zevalin, Ipilimumab/Yervoy, Ofatumumab/Xolair and
  • said one or more chemotherapeutic agents comprises one or more of epirubicin, 5-Fluorouracil, cyclophosphamide, irinotecan, docetaxel, methotrexate and gemcitabine; such as epirubicin in combination with 5-Fluorouracil and
  • cyclophosphamide such as 5-Flourouracil in combination with irinotecan.
  • GTPase KRas also known as V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog and KRAS, is a protein that in humans is encoded by the KRAS gene.
  • the protein product of the normal KRAS gene performs an essential function in normal tissue signaling, and the mutation of a KRAS gene is an essential step in the development of many cancers.
  • KRas and KRAS may be used interchangeably herein.
  • a single amino acid substitution and in particular a single nucleotide substitution, is responsible for an activating mutation.
  • the transforming protein that results is implicated in various malignancies, including lung
  • adenocarcinoma adenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas, leukemias and colorectal carcinoma.
  • KRAS mutational status of KRAS is indicative for tumor response to treatment with an EGFR inhibitor or antibody, such as cetuximab (Erbitux) and panitumumab (Vectibix).
  • an EGFR inhibitor or antibody such as cetuximab (Erbitux) and panitumumab (Vectibix).
  • KRAS wild-type tumors are generally responsive to treatment with EGFR inhibitors, while tumors with mutated (at one or more positions) KRAS is generally less or not responsive to treatment.
  • KRAS mutational analysis is commercially available from a number of laboratories.
  • a new treatment regimen is disclosed for those cancer patients whose tumors harbors a KRas mutation, which are today excluded from treatment with an EGFR inhibitor, such as cetuximab, alone.
  • a composition comprising a compound capable of inhibiting the interaction interaction between TIMP-1 and CD74 for use in the treatment of a patient with cancer having one or more tumor KRas mutations and having high or increased TIMP-1 levels.
  • Treatment may in one embodiment mean treatment of the cancer, or it may be treatment of the cancer by increasing the sensitivity towards anticancer agents.
  • ⁇ patient with cancer having one or more tumor KRas mutations' means that the cancer patients tumor cells harbors one or more mutations in the KRAS gene.
  • composition comprising a compound capable of inhibiting the interaction interaction between TIMP-1 and CD74 and an EGFR inhibitor for use in the treatment of a patient with cancer having one or more tumor KRas mutations and having high or increased TIMP-1 levels.
  • a method of treating a patient with cancer with one or more KRas mutations and having high or increased TIMP-1 levels comprising administering to said patient a compound capable of inhibiting the interaction interaction between TIMP-1 and CD74, alone or in combination with an EGFR inhibitor.
  • said EGFR inhibitor is an EGFR antibody (anti-EGFR antibody).
  • An EGFR antibody is in one embodiment selected from the group consisting of a monoclonal antibody; one or more recombinant antibodies; and a polyclonal antibody.
  • Said monoclonal antibody is in one embodiment selected from a chimeric, a humanized or fully human antibody.
  • said EGFR antibody is selected from the group consisting of cetuximab (Erbitux), panitumumab (Vectibix), gefitinib (Iressa), erlotinib hydrochloride (Tarceva), and others known to the skilled person. Selecting a cancer patient
  • Responsive to treatment in the present context refers to treatment with one or more TIMP-1 inhibitors as defined herein; alone or in combination with one or more chemotherapeutics.
  • a sample obtained from said patient is a bodily fluid, such as whole blood, plasma, serum, urine, saliva, sputum and cerebrospinal fluid. It is also an aspect to provide a method for increasing the response rate of cancer patients to chemotherapeutic agents, said method comprising the steps of examining one or more of: i) the cancer cell expression of CD74, and ii) the level of TIMP-1 in a sample obtained from said cancer patient, wherein the patient is selected for treatment when having high or increased expression of TIMP-1 and a CD74-positive cancer.
  • the method further comprises the steps of examining the cancer cell expression of CD63, wherein a CD63 negative cancer is indicative of said patient with cancer being responsive to treatment.
  • a TIMP-1 inhibitor such as a compound capable of inhibiting the interaction interaction between TIMP-1 and CD74, such as a CD74 inhibitor; alone or in combination with a chemotherapeutic agent, such as an EGFR inhibitor, such as an EGFR antibody, said method comprising examining i) the cancer cell expression of CD74 and/or CD63 and ii) the level of TIMP-1 in a sample obtained from said cancer patient, wherein expression of TIMP-1 and CD74 and/or CD63 is indicative of said patient with cancer being responsive to treatment.
  • a method for selecting a patient with cancer for treatment with a TIMP-1 inhibitor such as a compound capable of inhibiting the interaction interaction between TIMP-1 and CD74, such as a CD74 inhibitor; alone or in combination with a chemotherapeutic agent, such as an EGFR inhibitor, such as an EGFR antibody; said method comprising examining i) the mutational status of KRas and ii) the level of TIMP-1 in a sample obtained from said cancer patient, wherein the identification of one or more KRas mutations and concomitant high or increased levels of TIMP-1 is indicative of said patient with cancer being responsive to treatment with said compound.
  • a method for screening for compounds being an agonist or antagonist for CD74 comprising: contacting a CD74 polypeptide with TIMP-1 in the presence and absence of a candidate compound, and comparing the interaction of the TIMP-1 and said CD74 polypeptide in the presence of said candidate compound with their interaction in the absence of said candidate compound, whereby a candidate compound that enhances the interaction of said TIMP-1 with said CD74 polypeptide is identified as an agonist of CD74, and a candidate compound that inhibits the interaction of said TIMP-1 with said CD74 polypeptide is identified as an antagonist of CD74.
  • the TIMP-1 inhibitor or a composition comprising the same, is to be administered to an individual in need of treatment in
  • the dosage requirements will vary with the particular drug composition employed, the route of administration and the particular subject being treated, which depend on the severity and the sort of the disorder as well as on the weight and general state of the subject. It will also be recognized by one skilled in the art that the optimal quantity and spacing of individual dosages will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and that such optima can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment, i.e., the number of doses of a compound given per day for a defined number of days, can be ascertained using conventional course of treatment determination tests.
  • a therapeutically effective amount is in one embodiment an amount sufficient to cure, prevent, reduce the risk of, alleviate or partially arrest the clinical manifestations of a given disease or disorder and its complications.
  • the amount that is effective for a particular therapeutic purpose will depend on the severity and the sort of the disorder as well as on the weight and general state of the subject. An amount adequate to accomplish this is defined as a "therapeutically effective amount”. Examples
  • the aim of this study was to confirm the binding of CD74 and TIMP-1 in mammalian cells.
  • the high CD74 expressing breast cancer cell line MDA-MB-231 was incubated with rTIMP-1 for 1 hr and subjected to Co-immunoprecipitation.
  • the high CD74 expressing breast cancer cell line, MDA-MB-231 , used in this study was purchased from ATCC (Rockville, MD). The cells were cultured in DMEM supplemented with 10 % FCS in the presence of 50 ⁇ g/ml rTIMP-1 , 5 % C0 2 at 37 ⁇ C. Co-lmmunoprecipitation (co-IP). The Dynabeads Co-lmmunoprecipitation Kit (Life Technologies, San Diego, Ca) was used for co-IP experiments. The co-IP was carried out according to the manufacture's instruction with minor adjustments.
  • MDA- MB-231 cells were incubated with rTIMP-1 (2.5 ⁇ g/ml) for 30 min and subsequently lysed in modified Extraction Buffer (1 x Extraction Buffer, 100 mM NaCI, 2 mM MgCI 2 , 1 mM DTT and PMSF).
  • modified Extraction Buffer 1 x Extraction Buffer, 100 mM NaCI, 2 mM MgCI 2 , 1 mM DTT and PMSF.
  • Supernatants were incubated with antibody coupled beads (C- 16, Santa Cruz, VT4 clone and goat IgG control) for immunprecipitation of CD74, TIMP-1 and negative control, respectively, for 1 hr, 4 °C. After several wash steps the immunoprecipitated proteins were eluted and subjected to Western Blot analysis. Antibodies. For Western Blot; TIMP-1 (clone VT7), CD74 (LN-2, Santa Cruz
  • the 293T lysate was included as a positive control for CD74 whereas the IgG control was included as a negative control where we don't expect to see any bound proteins. From figure 1 we can see that we don't have any CD74 in the IgG control sample which indicates that there is no unspecific binding of CD74 to the beads. However, we do see CD74 in the TIMP-1 IP sample which matches the size of the bands in the 293T positive control.
  • the aim of this study was to investigate the role of CD74 in TIMP-1 uptake by the cells. In order to do that we down -regulated CD74 by siRNA and compared the uptake of TIMP-1 to wild type cells.
  • the high CD74 expressing breast cancer cell line, MDA-MB-231 , used in this study was purchased from ATCC (Rockville, MD). The cells were cultured in DMEM supplemented with 10 % FCS in the absence or presence of 50 ⁇ g/ml rTIMP-1 , 5 % C0 2 at 37 °C.
  • CD74 was transiently down-regulated by siRNA (Sigma, SASI_Hs01_00142175). Cells were plated 24 hour prior to transfection and medium was changed 1 hour before start of transfection. According to manufacturer's instruction CD74 siRNA was transfected into MDA-MB-231 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA).
  • TIMP-1 (clone VT7)
  • CD74 (LN-2, Santa Cruz Biotechnology)
  • HSP70 (AB-2 (W27), Fremont, CA)
  • the membranes were incubated with the appropriate secondary antibodies.
  • the antigen was detected using the Amersham ECL-Select Western Blotting detection reagent (GE Healthcare Life Sciences, Piscataway, NJ) according to manufacturer's instruction and visualized by UVP BioSpectrum Imaging system.
  • CD74 is an evolutionarily conserved type II membrane protein initially described to be involved in several key processes of the immune system.
  • TIMP-1 internalization in human cancer as we have shown in human cancer cell lines.
  • a screening assay to find a TIMP-1 inhibitor capable of increasing the sensitivity towards one or more chemotherapeutic agents is performed in vitro by adding to a cell culture a TIMP-1 inhibitor with or without TIMP-1 protein (or in TIMP-1 expressing and TIMP-1 non-expressing cells) and see if the effect of certain anti-cancer treatments are increased (increased chemosensitivity) by adding a TIMP-1 inhibitor (alone or in combination) to the cells.
  • a TIMP-1 inhibitor may be a compound capable of inhibiting the interaction between TIMP-1 and CD74 and/or between TIMP-1 and CD63.
  • Kinase inhibitors of intracellular signaling downstream of CD63 and/or CD74 can also be added.
  • TIMP-1 :CD74 interaction When one or more inhibitors of the TIMP-1 :CD74 interaction have been identified and validated in preclinical in vitro and in vivo models, the most promising inhibitors will be taken to preclinical toxicity and pharmacology studies in two species, e.g. rats and dogs, and different doses of the inhibitor in question will be tested for toxicity, following international guidelines for such studies. Moreover, the pharmacokinetics (half-life, T1/2 etc.) will be determined from blood from the treated animals. Based on all these preclinical studies a protocol for a clinical phase 1 study will be developed. A clinical phase 1 study has as its primary objective to test toxicity of the TIMP-1 :CD74 inhibitor in human beings.
  • Suitable individuals/patients are identified and after having signed an informed consent form they can be enrolled in the clinical study. Patients will be exposed in groups of three to increasing concentrations of the inhibitor in question. When toxicity is observed, 3 more patients will be exposed to the inhibitor but at the former dose level. If no toxicity is observed here, this dose is the recommended dose for subsequent clinical phase 2 study in which one will look for efficacy of the drug. It will be possible to include measurements of so-called surrogate biomarkers in the phase 1 study. Moreover, it will be possible to restrict the enrollment of patients in the phase 1 study to patients with specific diseases and specific tumor cell characteristics, e.g. positive for TIMP-1 , CD74 and/or CD63. ITEMS
  • a composition comprising a TIMP-1 inhibitor for use in increasing the sensitivity of a cancer cell towards one or more chemotherapeutic agents,
  • a method of increasing the sensitivity of a cancer cell towards one or more chemotherapeutic agents comprising administering a composition comprising a TIMP-1 inhibitor to a patient in need thereof, alone or in combination with said one or more chemotherapeutic agents).
  • a composition comprising i) a TIMP-1 inhibitor and ii) a chemotherapeutic agent for use in the treatment of cancer and/or for use in increasing the sensitivity of a cancer cell towards one or more chemotherapeutic agents.
  • composition for use according to any of the preceding items wherein said TIMP-1 inhibitor is able to decrease intracellular tumor levels of TIMP-1 and/or able to decrease tumor cell internalization of TIMP-1 .
  • composition for use according to any of the preceding items, wherein said compound capable of inhibiting the interaction between TIMP-1 and CD74 is a CD74 inhibitor.
  • CD74 inhibitor is a CD74 antibody.
  • composition for use according to any of the preceding items wherein said composition comprises a CD74 inhibitor such as a CD74 antibody and a CD63 inhibitor such as a CD63 antibody.
  • MIF Macrophage migration inhibitory factor
  • D-DT/MIF-2 D-dopachrome tautomerase
  • chemotherapeutic agent is an EGFR inhibitor.
  • composition for use according to any of the preceding items wherein said EGFR antibody is selected from the group consisting of cetuximab (Erbitux), panitumumab (Vectibix), gefitinib (Iressa) and erlotinib hydrochloride (Tarceva).
  • said one or more chemotherapeutic agent is epirubicin, epirubicin in combination with 5-Fluorouracil and cyclophosphamide, or 5-Flourouracil and irinotecan.
  • composition for use according to the preceding items wherein said composition is to be administered to a cancer patient.
  • composition for use according to any of the preceding items, wherein said patient with cancer has high or increased tumor TIMP-1 levels.
  • composition for use according to any of the preceding items wherein said patient with cancer has one or more tumor KRas mutations and high or increased TIMP-1 levels.
  • composition for use according to any of the preceding items wherein said patient with cancer has high or increased tumor TIMP-1 levels and also expresses CD74 and/or CD63.
  • composition for use according to any of the preceding items, wherein said TIMP-1 inhibitor is to be administered in combination with a chemotherapeutic agent wherein said TIMP-1 inhibitor is to be administered in combination with a chemotherapeutic agent.
  • composition for use according to any of the preceding items wherein said TIMP-1 inhibitor and said chemotherapeutic agent are administered separately, sequentially or simultaneously.
  • composition for use according to any of the preceding items wherein said cancer is selected from the group consisting of colorectal cancer, metastatic colorectal cancer (mCRC), lung cancer including small cell lung cancer and non-small cell lung cancer, breast cancer, head and neck cancer, squamous cell carcinoma of the head and neck, hematological malignancies,
  • mCRC metastatic colorectal cancer
  • lung cancer including small cell lung cancer and non-small cell lung cancer
  • breast cancer breast cancer
  • head and neck cancer squamous cell carcinoma of the head and neck
  • hematological malignancies hematological malignancies
  • compositions for use according to any of the preceding items wherein said composition further comprises one or more specific kinase inhibitors, such as kinase inhibitors of intracellular signaling downstream of CD63 and/or CD74.
  • a method for selecting a patient with cancer for treatment with a TIMP-1 inhibitor such as a compound capable of inhibiting the interaction interaction between TIMP-1 and CD74, such as a CD74 inhibitor; alone or in combination with a chemotherapeutic agent, such as an EGFR inhibitor, such as an EGFR antibody, said method comprising examining i) the mutational status of KRas and ii) the level of TIMP-1 in a sample obtained from said cancer patient, wherein the identification of one or more KRas mutations and concomitant high levels of TIMP-1 is indicative of said patient with cancer being responsive to treatment.
  • a method for selecting a patient with cancer for treatment with a TIMP-1 inhibitor such as a compound capable of inhibiting the interaction interaction between TIMP-1 and CD74, such as a CD74 inhibitor; alone or in combination with a chemotherapeutic agent, such as an EGFR inhibitor, such as an EGFR antibody, said method comprising examining i) the cancer cell expression of CD74 and/or CD63 and ii) the level of TIMP-1 in a sample obtained from said cancer patient, wherein expression of TIMP-1 and CD74 and/or CD63 is indicative of said patient with cancer being responsive to treatment.
  • a TIMP-1 inhibitor such as a compound capable of inhibiting the interaction interaction interaction between TIMP-1 and CD74, such as a CD74 inhibitor
  • a chemotherapeutic agent such as an EGFR inhibitor, such as an EGFR antibody
  • a method for screening for compounds being an agonist or antagonist for CD74 comprising: contacting a CD74 polypeptide with TIMP-1 in the presence and absence of a candidate compound, and comparing the interaction of the TIMP-1 and said CD74 polypeptide in the presence of said candidate compound with their interaction in the absence of said candidate compound, whereby a candidate compound that enhances the interaction of said TIMP-1 with said
  • CD74 polypeptide is identified as an agonist of CD74, and a candidate compound that inhibits the interaction of said TIMP-1 with said CD74 polypeptide is identified as an antagonist of CD74.

Abstract

The present invention relates to a method of targeting cancer cells having an intracellular and/or extracellular environment of increased TIMP-1 levels by inhibition of TIMP-1 to increase the response rate of chemotherapeutic compounds. The TIMP-1 inhibitor in one embodiment is a compound capable of interfering with the bona fide interaction between TIMP-1 and CD74.

Description

TIMP-1 binding partner Field of invention
The present invention relates to a method of targeting cancer cells having an intracellular and/or extracellular environment of increased TIMP-1 levels by inhibition of TIMP-1 in order to increase the effect (increase sensitivity or reduce resistance) of a chemotherapeutic compound administered to said cancer cells.
Background of invention
The TIMP-1 (tissue inhibitor of metalloproteases-1 ) glycoprotein is a natural inhibitor of the matrix metalloproteinases (MMP), a group of peptidases involved in degradation of the extracellular matrix. TIMP-1 blood levels have been proposed as a diagnostic and prognostic marker for certain cancers, based on a correlation between high levels and occurrence of disease, disease stage and outcome.
The cancer and/or blood levels of TIMP-1 have also been shown to correlate inversely with the response rate to some chemotherapeutics. One MMP-independent function of TIMP-1 is inhibition of apoptosis, which may be involved in resistance to
chemotherapeutics.
Some cancer cells have high intracellular levels of TIMP-1 protein but no
corresponding TIMP-1 mRNA suggesting that TIMP-1 is taken up by cancer cells such as by internalization. Known cell surface binding partners for TIMP-1 include CD63 (Jung et al. EMBO J. 2006 Sep 6;25(17):3934-42). However, cells that do not express CD63 also internalizes TIMP-1 suggesting that additional cell surface binding partners for TIMP-1 exist.
Summary of invention
The response rate to chemotherapeutics may be increased if the high levels of TIMP associated with poor response to chemotherapeutics are inhibited. Specifically the response rate to chemotherapeutics may be increased if the internalization of TIMP-1 into cancer cells is reduced or inhibited, such as by inhibiting the newly identified interaction between TIMP-1 and the cell surface protein CD74. Disclosed herein is a method of treating a patient with cancer, such as by improving the response rate of one or more chemotherapeutic agents, said method comprising administering a TIMP-1 inhibitor as defined herein, whereby the sensitivity towards said one or more chemotherapeutic agents is increased and the response rate improved.
The TIMP-1 inhibitor may be administered alone or in combination with said one or more chemotherapeutic agents.
In one embodiment the TIMP-1 inhibitor targets TIMP-1 directly; interferes with TIMP-1 binding to cell surface proteins; interferes with TIMP-1 mediated activation of intracellular signalling.
In one embodiment the TIMP-1 inhibitor is a compound capable of inhibiting the interaction between TIMP-1 and CD74. In one embodiment the TIMP-1 inhibitor is capable of decreasing cellular internalization of TIMP-1 in a cell. In one embodiment said cell is a cancer cell, such as a CD74-positive cancer cell.
In one embodiment said patient with cancer has high or increased TIMP-1 levels, such as in bodily fluids including plasma, and/or in the cancer cells of said patient, and/or CD74-positive cancer cells.
Figures
Figure 1 : Western Blot. Protein complex formation of TIMP-1 and CD74 as revealed by Co-lmmunoprecipitation.
Figure 2; western blot. Uptake of TIMP-1 is visualized in MDA-MB-231 cells +/- CD74 expression at 4 and 24 hours.
Detailed description of the invention
Chemotherapy is a category of cancer treatment that uses chemical substances, especially one or more anti-cancer drugs (chemotherapeutic agents). Chemotherapy may be given with a curative intent or it may aim to prolong life or to palliate symptoms. It is often used in conjunction with other cancer treatments, such as radiation therapy or surgery. Traditional chemotherapeutic agents act by killing cells that divide rapidly. Some newer anti-cancer agents (for example, various monoclonal antibodies) are not indiscriminately cytotoxic, but rather target proteins that are abnormally expressed in cancer cells and that are essential for their growth. Such treatments are often referred to as targeted therapy (as distinct from classic chemotherapy) and are often used alongside traditional chemotherapeutic agents in anti-neoplastic treatment regimens.
The present inventors have previously shown that cells that do not express the known TIMP-1 binding partner CD63 can still internalize TIMP-1 , suggesting that other cell surface binding partners for TIMP-1 exist. The present inventors have now identified CD74 as a bona fide TIMP-1 binding partner, and have shown that CD74 expression correlates with the extent of TIMP-1 internalization. Thus, CD74 has a role in TIMP-1 internalization, causing an increased intracellular level of TIMP-1 in CD74 expressing cells. Increased intracellular levels of TIMP-1 may subsequently decrease the sensitivity towards certain anti-cancer agents, such as by affecting anti-apoptotic intracellular signaling.
The invention relates to improving the effect of anti-cancer agents such as anti- hormones, classic chemotherapeutic drugs and biologicals, such as antibodies and kinase inhibitors. More specifically, the invention relates to inhibition of the biological activities/functions of TIMP-1 in patients suffering from a cancer disease and being treated with said anti-cancer agent, in order to target the cancer and/or increase the sensitivity towards said anti-cancer agent. The inhibition of TIMP-1 activities/functions can be accomplished by directly targeting the TIMP-1 protein or by inhibiting TIMP-1 protein interaction with its binding partners, e.g. cell surface binding proteins. The inventors have identified herein CD74 as a new cell surface binding partner for TIMP-1 , which is shown to be involved in TIMP-1 internalization into cancer cells.
Examples of TIMP-1 inhibition according to the present invention include use of a neutralizing drug, e.g. an anti-TIMP-1 antibody; inhibition of the mRNA expression of TIMP-1 ; inhibition of the interaction between TIMP-1 protein and one or more binding partners located at cancer cell surfaces, such as by targeting at least CD74 or targeting CD74 and CD63; inhibiting TIMP-1 cellular internalization through prevention of its binding to one or more specific cell surface interaction partners, preferably comprising a compound capable of inhibiting the interaction between TIMP-1 and CD74; inhibiting TIMP-1 -induced intracellular signaling through its binding to cell surface proteins;
abolishing TIMP-1 mediated reduction in cellular sensitivity to anti-cancer drugs through inhibition of one or more of TIMP-1 cell surface binding, TIMP-1 internalization, TIMP-1 intracellular signaling or any way by which TIMP-1 mediates resistance to chemotherapeutics. TIMP-1
TIMP-1 (UniProt entry P01033; Metalloproteinase inhibitor 1 ) is one among four known endogenous inhibitors of the matrix metalloproteinases (MMPs). It is a 24 kD highly glycosylated protein produced and secreted by many different cell types. The main function of TIMP-1 is believed to be extracellular inhibition of the MMPs. However, more recent research has pointed to MMP-independent biological functions of TIMP-1 , including inhibition of apoptosis. TIMP-1 functions as a growth factor that regulates cell differentiation, migration and cell death and activates cellular signaling cascades via CD63 and ITGB1 . With respect to treatment of cancer, an association between high tumor cell and/or high patient with cancer blood levels of TIMP-1 protein and a reduced effect of certain types of chemotherapy, including biological anticancer therapy and endocrine therapy, has been observed and validated in the clinical setting. For example, it is shown that while patients with primary breast cancer and TIMP-1 positive cancer cells do not gain additional benefit from exchanging methotrexate with epirubicin when given in combination with 5-Fluorouracil and cyclophosphamide, in patients with no or little TIMP-1 tumor cell protein, the epirubicin based treatment resulted in a significant benefit compared to the non-epirubicin treatment. This suggests that TIMP-1 is involved in determining the sensitivity (high levels conferring resistance) to epirubicin in clinical adjuvant breast cancer treatment. Results from the present inventors show that patients with metastatic breast cancer, receiving a combination of docetaxel and gemcitabin versus docetaxel alone, and having no or little TIMP-1 protein expression in their cancer cells had a doubling of their survival while this difference was not observed among patients with TIMP-1 protein expressing cancer cells.
In colorectal cancer the inventors have previously shown that patients with metastatic colorectal cancer receiving treatment with 5-Flourouracil and irinotecan and having high pre-treatment plasma TIMP-1 levels have a significantly lower objective response rate and a significantly shorter overall survival than patients with low pre-treatment plasma TIMP-1 levels, suggesting that TIMP-1 is involved in determining sensitivity to this drug combination. Interesting and surprisingly, a similar association was not observed when metastatic colorectal cancer patients were treated with 5-Flourouracil and oxaliplatin. The precise biological mechanisms for this effect of TIMP-1 (decreased anti-tumor activity of certain anti-cancer drugs) are not clearly understood. Binding of TIMP-1 to the transmembrane protein CD63 with subsequent activation of the Akt survival pathway has been described. Moreover, the literature indicates that cell surface binding of TIMP-1 can also induce signaling though Erk (reviewed in Sci Signal. 2008 Jul 8;1 (27):re6. doi: 10.1 126/scisignal.127re6).
It has been shown that TIMP-1 protein produced by a mesenchymal stem cell culture can be internalized by human breast cancer cells in vitro when co-cultured (Kuvaja et al., Exp Cell Res. 2012 Jun 10;318(10):1094-103. doi: 10.1016/j.yexcr.2012.03.009)
The present inventors have performed extensive immunohistochemistry and in situ hybridization for TIMP-1 in formalin fixed paraffin embedded breast cancer tissue and observed that some patients have TIMP-1 positive tumor cells by
immunohistochemistry while these tumors are negative for TIMP-1 mRNA. These observations lead to the hypothesis that cancer cells can bind and internalize TIMP-1 . Thus, cancer cells may internalize or take up TIMP-1 from the surroundings, rather than producing TIMP-1 by intracellular protein synthesis (transcription/translation).
Known cell surface binding partners for TIMP-1 include CD63 (Jung et al. EMBO J. 2006 Sep 6;25(17):3934-42). The inventors have stained human breast cancer tissue biopsies for the known TIMP-1 binding partner CD63, and surprisingly have observed that some of the tumors that are TIMP-1 protein positive but TIMP-1 mRNA negative did not express CD63. This lead the inventors to propose that other cell surface binding partners for TIMP-1 than CD63 could be in play. The inventors therefore initiated research with the aim of identifying new TIMP-1 cell surface binding partners.
CD74
CD74 (also known as HLA-DR antigens-associated invariant chain; la antigen- associated invariant chain; li, p33) is a transmembrane protein with a short cytoplasmic tail. It participates in several key processes of the immune system, including antigen presentation, B-cell differentiation and inflammatory signaling. CD74 is a protein trafficking regulator and a cell membrane receptor for the pro-inflammatory cytokine MIF (Macrophage migration inhibitory factor), D-dopachrome tautomerase (D-DT/MIF- 2) and bacterial proteins. Cell surface CD74 is very rapidly internalized both with and without CD74 antibodies bound. CD74 is highly expressed in inflammatory disorders and some cancer types including hematopoietic cancers and some solid tumors (see e.g. Berkova et al.: "Milatuzumab - a promising new immunotherapeutic agent". Expert Opin Investig Drugs. 2010 Jan;19(1 ):141 -9 and WO12104344A1 ). Various formats of a humanized CD74-specific monoclonal antibody, hLLI, have been proposed for treatment of CD74- positive tumors. CD74-specific antibodies are currently studied for the treatment of multiple myeloma, non-Hodgkin's lymphoma and chronic lymphocytic leukemia.
The terms "CD74" and "CD74 antigen" are used interchangeably herein. Unless specified otherwise, the terms include any variants, isoforms and species homologs of human CD74 which are naturally expressed by cells or are expressed on cells transfected with the CD74 gene. At least four human isoforms are known to exist; p43, p41 , p35 and p33 (Borghese F et al., Expert Opin Ther Targets 201 1 ; 15(3) : 237-251 ). These result from alternative transcript splicing and two translation start sites. p43 (also known as CD74 isoform 1 , isoform a, or "long"; see UniProt entry P04233-1 ) contains 296 amino acids, with residues 73-296 forming the extracellular portion. p35 (also known as CD74 isoform 2, isoform b or "short"; see Uniprot entry P04233-2) lacks residues 209-272 from the extracellular part due to alternative splicing. p41 and p33 arise from an alternative translation start site (48 bp downstream; 16 amino acids shorter protein) leading to variants lacking the endoplasmic reticulum (ER) retention signal that is present within these 16 amino acids, but having an identical extracellular part as p43 and p35, respectively.
As used herein, "internalization", when used in the context of TIMP-1 includes any mechanism by which TIMP-1 is internalized from the cell-surface into a CD74- expressing cell. Antibodies
A TIMP-1 inhibitor in the present context in one embodiment refers to an antibody, preferably an antibody that selectively targets TIMP-1 or CD74. An antibody targeting TIMP-1 or CD74 in one embodiment prevents cellular internalization of TIMP-1 .
Immunoglobulins are a class of structurally related glycoproteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight chains and one pair of heavy (H) chains, all four inter-connected by disulfide bonds. The structure of immunoglobulins has been well characterized. Briefly, each heavy chain typically is comprised of a heavy chain variable region (VH) and a heavy chain constant region (CH) typically comprised of three domains, CH1 , CH2, and CH3. Each light chain typically is comprised of a light chain variable region (VL) and a light chain constant region, typically comprised of one domain (CL). The VH and VL regions may be further subdivided into regions of hypervariability also termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FRs).
The term "antibody" or "Ab" in the context of the present invention refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative of either thereof, which has the ability to specifically bind to an antigen under typical physiological conditions. The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with an antigen. An antibody may also be multispecific, having specificities for two or more different epitopes, typically non-overlapping. Examples of multispecific antibodies include bispecific antibodies, diabodies, and similar antibody molecules. As indicated above, the term antibody herein, unless otherwise stated, includes fragments of an antibody that retain the ability to specifically bind to the antigen. The antigen-binding function of an antibody may be performed by fragments of a full-length antibody, e.g., Fab and F(ab')2 fragments. In the context of the present invention the term antibody, unless specified otherwise, includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides such as chimeric antibodies and humanized antibodies. An antibody as generated can possess any isotype. The term "epitope" means a protein determinant capable of specific binding to an antibody. The terms "human antibody" include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline
immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody", as used herein, is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences.
The terms "monoclonal antibody" (mAb), refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Accordingly, the term "human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. The human monoclonal antibodies may be produced by a hybridoma which includes a B cell obtained from a transgenic or transchromosomal non-human animal, such as a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene, fused to an immortalized cell.
As used herein, "isotype" refers to the immunoglobulin class (for instance IgGI, lgG2, lgG3, lgG4, IgD, IgA, IgE, or IgM) that is encoded by heavy chain constant region genes. The term "full-length antibody" when used herein, refers to an antibody which contains all heavy and light chain constant and variable domains that are normally found in an antibody of that isotype. Treatment
In the present context, the term chemotherapeutic agent comprises all conceivable anti-cancer agents including the traditional chemotherapeutics as well as targeted therapy. In the broadest aspect of the invention there is provided a composition comprising a TIMP-1 inhibitor for use in a method of increasing the sensitivity of a cancer cell towards one or more chemotherapeutic agents, in a patient with cancer. The patient with cancer may be defined as specified elsewhere herein. It is understood that increasing the sensitivity towards a chemotherapeutic agent is synonymous to increasing the response rate, reducing resistance and enhancing the effect to said chemotherapeutic agents. Increasing the sensitivity of a cancer cell means that the cancer cell will become responsive to the chemotherapeutic agent where it was more or less non-responsive; in one embodiment effectively enabling use of a lower dosage of the chemotherapeutic agents to achieve a sufficient response rate.
It is an aspect to provide a method for increasing the sensitivity of a cancer cell towards one or more chemotherapeutic agents, said method comprising administering to a patient with cancer in need thereof a composition comprising a TIMP-1 inhibitor.
It is an aspect to provide a composition comprising a TIMP-1 inhibitor for the manufacture of a medicament for use in a method of increasing the sensitivity of a cancer cell towards one or more chemotherapeutic agents in a patient with cancer.
Also disclosed is a method of reducing cellular TIMP-1 internalization, said method comprising administering to a patient in need thereof a composition comprising a TIMP- 1 inhibitor. In one embodiment the method reduces the cellular TIMP-1 internalization in a cancer cell, such as a CD74-positive cancer cell.
Reducing TIMP-1 internalization in cancer cells in one embodiment increases TIMP-1 concentration in the tumor microenvironment, which may reduce the propensity of the tumor to metastasize.
Also disclosed is a composition comprising, separately or together, i) a TIMP-1 inhibitor and ii) one or more chemotherapeutic agents for use in a method of treating cancer.
Also disclosed is a composition comprising, separately or together, i) a TIMP-1 inhibitor and ii) one or more chemotherapeutic agents for use in a method of increasing the sensitivity of a cancer cell towards said one or more chemotherapeutic agents.
Disclosed herein is a composition comprising i) a compound capable of inhibiting the interaction between TIMP-1 and CD74 alone or in combination with ii) one or more chemotherapeutic agents for use in the treatment of cancer. Disclosed herein is a composition comprising i) a compound capable of inhibiting the interaction between TIMP-1 and CD74 alone or in combination with ii) one or more chemotherapeutic agents for use in a method of increasing the sensitivity of a cancer cell towards said one or more chemotherapeutic agents.
In one embodiment, where the composition comprises two (or more)
compounds/agents, said two or more compounds/agents may be administered simultaneous, separately or sequentially.
Also disclosed herein is a method of inhibiting the interaction between TIMP-1 and CD74, said method comprising administration of a TIMP-1 inhibitor to a patient in need thereof, such as to a cancer patient. Disclosed herein is a method of inhibiting the interaction between TIMP-1 and CD74, said method comprising administration of a compound capable of inhibiting the interaction between TIMP-1 and CD74 to a patient in need thereof, such as to a cancer patient. In one embodiment said patient with cancer has high or increased TIMP-1 levels, such as in in bodily fluids including plasma, and/or in the cancer cells of said patient, and/or has CD74-positive cancer cells.
In one embodiment a composition comprising a TIMP-1 inhibitor comprises two or more TIMP-1 inhibitors as defined herein. In one embodiment the two TIMP-1 inhibitors comprises a compound that inhibits TIMP-1 directly, and a compound that inhibits CD74 directly.
An individual in need as referred to herein is in one embodiment an individual that benefits from the administration of an agent according to the present invention. Such an individual in one embodiment suffers from a cancerous condition in the tissue of one or more organs, or is at risk of suffering therefrom. The term "Individual" refers preferably to vertebrates, particular members of the mammalian species, preferably primates including humans. The individual is in one embodiment any human being, male or female, infant, middle-aged or old. The disorder to be treated or prevented in the individual in one embodiment relates to the age of the individual, the general health of the individual, the medications used for treating the individual and whether or not the individual has a prior history of suffering from diseases or disorders that may have or have induced ischemic and/or inflammatory conditions in the individual.
The terms "treatment" and "treating" as used herein refer to the management and care of a patient for the purpose of combating a condition, disease or disorder. The term is intended to include the full spectrum of treatments for a given condition from which the patient is suffering, such as administration of the bioactive agent for the purpose of: alleviating or relieving symptoms or complications; delaying the progression of the condition, partially arresting the clinical manifestations, disease or disorder; curing or eliminating the condition, disease or disorder; and/or preventing or reducing the risk of acquiring the condition, disease or disorder, wherein "preventing" or "prevention" is to be understood to refer to the management and care of a patient for the purpose of hindering the development of the condition, disease or disorder, and includes the administration of the active compounds to prevent or reduce the risk of the onset of symptoms or complications. The term "palliation", and variations thereof, as used herein, means that the extent and/or undesirable manifestations of a physiological condition or symptom are lessened and/or time course of the progression is slowed or lengthened, as compared to not administering compositions of the present invention.
TIMP-1 inhibitor
In one embodiment there is provided a composition comprising one or more TIMP-1 inhibitors for use in a method of increasing the sensitivity of a cancer cell towards one or more chemotherapeutic agents, in a patient with cancer. The TIMP-1 inhibitor may be administered alone or in combination with said one or more chemotherapeutic agents.
In one embodiment the TIMP-1 inhibitor targets TIMP-1 directly; interferes with TIMP-1 binding to cell surface proteins; and/or interferes with TIMP-1 mediated activation of intracellular signalling.
A TIMP-1 inhibitor according to the present disclosure in one embodiment is an agent or compound that inhibits TIMP-1 in any way conceivable to the skilled person. In one embodiment a TIMP-1 inhibitor is an agent or compound that inhibits one or more of TIMP-1 expression, TIMP-1 translation, TIMP-1 transcription, TIMP-1 secretion, TIMP- 1 stability, TIMP-1 internalization, TIMP-1 biological activity and TIMP-1 -induced intracellular signalling. In one embodiment the TIMP-1 inhibitor targets the newly identified TIMP-1 binding partner CD74. A TIMP-1 inhibitor is in one embodiment a compound capable of inhibiting the interaction between TIMP-1 and CD74.
In one embodiment the TIMP-1 inhibitor decreases intracellular levels of TIMP-1 , such as by inhibiting CD74-mediated TIMP-1 internalization and/or decreasing intracellular CD74-mediated accumulation of TIMP-1 . In one embodiment the TIMP-1 inhibitor is capable of inhibiting internalization of TIMP-1 .
In one embodiment the TIMP-1 inhibitor inhibits internalization of TIMP-1 in cells, such as cancer cells, such as cancer cells expressing CD74. In one embodiment the TIMP-1 inhibitor decreases internalization of TIMP-1 in CD74-positive cells.
In one embodiment the TIMP-1 inhibitor increases cancer cell sensitivity and/or decreases cancer cell resistance to one or more chemotherapeutics.
The present invention in one embodiment discloses a compound capable of inhibiting the interaction between TIMP-1 and CD74. In one embodiment, said compound capable of inhibiting the interaction between TIMP-1 and CD74 reduces the
intracellular level of TIMP-1 . In one embodiment said compound capable of inhibiting the interaction between TIMP-1 and CD74 increases sensitivity towards certain chemotherapeutics. In one embodiment said compound capable of inhibiting the interaction between TIMP-1 and CD74 increases sensitivity towards certain
chemotherapeutics at least in a specific subset of patients with cancer. In one embodiment said subset of patients with cancer have high or increased TIMP-1 levels and/or CD74-positive cancer cells.
In one embodiment, a compound capable of inhibiting the interaction between TIMP-1 and CD74 is a binding partner for CD74 other than TIMP-1 , such as MIF (Macrophage migration inhibitory factor) and/or D-dopachrome tautomerase (D-DT/MIF-2). In one embodiment, the composition according to the present invention further comprises one or more compounds capable of inhibiting the interaction between TIMP- 1 and one or more binding partners for TIMP other than CD74. In one embodiment the composition further comprises one or more compounds capable of inhibiting the interaction between TIMP-1 and one or more of CD63 (Uniprot entry P08962), CD44 (Uniprot entry P16070), LRP1 (Prolow-density lipoprotein receptor-related protein 1 ; Uniprot entry Q07954), integrin complex and others known to the skilled person. In one embodiment the composition further comprises a CD63 inhibitor. In one embodiment the composition further comprises one or more specific kinase inhibitors, such as kinase inhibitors of intracellular signaling downstream of CD63 and/or CD74. These include Akt and Erk inhibitors.
Direct TIMP-1 inhibitor
In one embodiment, a compound capable of inhibiting the interaction between TIMP-1 and CD74 is a TIMP-1 inhibitor targeting and inhibiting TIMP-1 directly. In one embodiment the TIMP-1 inhibitor is able to reduce the level and/or activity of TIMP-1 . In one embodiment the TIMP-1 inhibitor is able to inhibit TIMP-1 -induced intracellular signaling.
In one embodiment the TIMP-1 inhibitor specifically inhibits one or more MMP- independent functions of TIMP-1 , such as inhibits TIMP-1 mediated inhibition of apoptosis In one embodiment the TIMP-1 inhibitor is a TIMP-1 antibody (anti-TIMP-1 antibody). TIMP-1 -binding human antibodies are available, such as those disclosed in WO 2002/086085. A TIMP-1 antibody is in one embodiment selected from the group consisting of a monoclonal antibody; one or more recombinant antibodies; and one or more polyclonal antibodies. Said monoclonal antibody is in one embodiment selected from a chimeric, a humanized or fully human antibody. In one embodiment the TIMP-1 antibody binds to an epitope in the CD74 binding site of TIMP-1 .
In one embodiment the TIMP-1 inhibitor interrupts or decreases TIMP-1 binding to the extracellular part of CD74. In one embodiment the TIMP-1 inhibitor is a small molecule TIMP-1 inhibitor.
In one embodiment the TIMP-1 inhibitor is a TIMP-1 antisense oligonucleotide providing for inhibition of TIMP-1 gene expression. A range of antisense
oligonucleotides are known to the skilled person including RNA, DNA and chemical analogues thereof, including PNA (peptide nucleic acid), PMO
morpholinophosphordiamidate) and siRNA.
In one embodiment the TIMP-1 inhibitor is an agent that down-regulates TIMP-1 , such as an EGF-R inhibitor including EGR-R antibodies such as cetuximab; Statins (HMG- CoA reductase inhibitors) including atorvastatin (Lipitor), fluvastatin (Lescol), lovastatin (Mevacor, Altocor), pitavastatin (Livalo), pravastatin (Pravachol), rosuvastatin (Crestor) and simvastatin (Zocor); and an inhibitor of one or more of HER-1 , HER-1 , HER-3 and HER-4 such as an HER-1 antibody, an HER-1 antibody, an HER-3 antibody and an HER-4 antibody antibody.
In one embodiment the TIMP-1 inhibitor is administered in combination with an agent that down-regulates TIMP-1 . CD74 inhibitor
In one embodiment the TIMP-1 inhibitor is a CD74 inhibitor. In one embodiment of the present invention, the compound capable of inhibiting the interaction interaction between TIMP-1 and CD74 is a CD74 inhibitor. In one embodiment said CD74 inhibitor is able to inhibit one or more of CD74 expression, CD74 translation, CD74
transcription, CD74 secretion, CD74 stability, CD74 biological activity and CD74 - induced intracellular signalling.
In one embodiment the CD74 inhibitor is a small molecule CD74 inhibitor. In one embodiment the CD74 inhibitor is a CD74 antisense oligonucleotide providing for inhibition of CD74 gene expression. A range of antisense oligonucleotides are known to the skilled person including RNA, DNA and chemical analogues thereof, including PNA (peptide nucleic acid), PMO morpholinophosphordiamidate) and siRNA. In one embodiment the CD74 inhibitor is a CD74 antibody. A "CD74 antibody", "anti- CD74 antibody", "CD74 Ab", "CD74-specific antibody" or "anti-CD74 Ab" is an antibody as described herein, which binds specifically to the antigen CD74. In a particular embodiment, the antibody of the invention is isolated. An "isolated Ab," as used herein, is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities. An isolated antibody that specifically binds to an epitope, isoform or variant of human CD74 may, however, have cross-reactivity to other related antigens, for instance from other species (such as CD74 species homologs). Preferably the CD74 antibody binds to CD74 and interrupts binding of TIMP-1 to CD74 and/or internalization of TIMP-1 .
A CD74 antibody is in one embodiment selected from the group consisting of a monoclonal antibody (MAb); one or more recombinant antibodies; and one or more polyclonal antibodies. Said monoclonal antibody is in one embodiment selected from a chimeric, a humanized or fully human antibody. In a particular embodiment the CD74 antibody is a monoclonal fully human antibody.
In another embodiment the CD74 antibody of the invention is selected from the group consisting of multispecific antibodies, bispecific antibodies, diabodies, full-length antibodies, fragments of an antibody e.g., Fab and F(ab')2 fragments, antibody-like polypeptides such as chimeric antibodies and humanized antibodies, antibodies of any isotype, naked antibodies and antibodies conjugated with drug (antibody-drug- conjugate). In one embodiment the CD74 antibody of the invention binds to an epitope of one or more of the four known human isoforms; p43, p41 , p35 and p33. In one embodiment the CD74 antibody is multispecific having specificities for two or more different epitopes such as one or more of the CD74 isoforms p43, p41 , p35 and p33. In one embodiment the CD74 antibody is selected from the group consisting of a CD74 isoform p43 antibody, a CD74 isoform p41 antibody, a CD74 isoform p35 antibody and a CD74 isoform p33 antibody. Preferably the CD74 antibody binds to membrane bound CD74.
Many examples of anti-CD74 antibodies are known in the art and any such known antibody or fragment thereof may be utilized. In one embodiment the CD74 antibody is a commercially available CD74 antibody, milatuzumab, a humanized LL1 (hl_L1 ) CD74 antibody, LS-B 1963, LS-B2594, LS-B 1859, LS-B2598, LS-05525, LS-C44929, LN2, PIN.1 , SPM523, LN3, CerCLIP.1 , A.14/19, Bu5, 1 D1 , 5-329 and any other CD74 antibodies known in the art. A humanized LL1 (hl_L1 ) anti-CD74 antibody is disclosed in US 7,312,318. In one embodiment the CD74 antibody binds to the same epitope of CD74 as an LL1 antibody.
Patient
In one embodiment, a patient in need thereof according to the present invention is a patient with cancer. In one embodiment, a patient in need thereof is a patient with cancer treated with one or more chemotherapeutic agents. In one embodiment the patient with cancer treated with one or more chemotherapeutic agents has a reduced response rate to said chemotherapeutic agents. In one embodiment, a patient in need thereof is a patient with cancer having high or increased TIMP-1 levels. In one embodiment, a patient in need thereof is a patient with cancer having a TIMP-1 positive cancer. In one embodiment, a patient in need thereof is a patient with cancer having a CD74 positive cancer.
A patient with high or increased TIMP-1 levels can refer to one or both of having high or increased TIMP-1 levels inside the cancer cells and outside the cancer cells. Thus the levels may refer to intracellular levels within the cancer cells or extracellular levels. Intracellular levels within the cancer cells may be measured in a tumor sample comprising cancer cells, and extracellular levels may be measured in bodily fluids.
In one embodiment high or increased TIMP-1 levels refers to TIMP-1 levels in one or more bodily fluids. Bodily fluids include but are not limited to whole blood, plasma, serum, urine, saliva, sputum and cerebrospinal fluid.
In one embodiment high or increased TIMP-1 levels refer to high or increased plasma TIMP-1 levels. A patient with cancer having high or increased TIMP-1 levels in one embodiment means that the patient with cancer has a TIMP-1 plasma level that is above the TIMP-1 plasma level in healthy humans.
In one embodiment the patients' TIMP-1 plasma level is above the mean value of healthy humans. The mean value of healthy humans can be identified for example via the TIMP-1 quantitation method described in Holten-Andersen et al 1999 (British Journal of cancer, 80(3/4), 495-503).
In one embodiment the patients' TIMP-1 plasma level is above a mean value of 69.2 +/- 13.1 pg 1 (citrate plasma) or above a mean value of 73.5 +/- 14.2 pg 1 (EDTA plasma).
In one embodiment the patients' TIMP-1 plasma level is above a reference range (10th to 90th centile) of 55.0 - 90.3 pg 1 (citrate plasma) or above a reference range of 58.0 - 91.8 pg Γ1 (EDTA plasma).
In one embodiment the patients' TIMP-1 plasma level is above a mean value of 50 pg 1 , such as above a mean value of 60 pg 1 1 ,for example 70 pg 1 , such as 80 pg 1 , for example 90 pg 1 , such as 100 pg 1 , for example 1 10 pg 1 , such as 120 pg 1 , for example 130 pg 1 , such as 140 pg 1 , for example 150 pg 1 , such as 160 pg 1 , for example 170 pg 1 , such as 180 pg 1 , for example 190 pg 1 , such as above a mean value of 200 pg Γ1
In one embodiment the patients' TIMP-1 plasma level has a mean value of 50-60 pg 1 , such as 60-70 pg 1 1 ,for example 70-80 pg I"1 , such as 80-90 pg I"1 , for example 90- 100 pg Γ1 , such as 100-1 10 pg Γ1 , for example 1 10-120 pg Γ1 , such as 120-130 pg Γ1 , for example 130-140 pg 1 , such as 140-150 pg 1 , for example 150-160 pg 1 , such as 160-170 pg 1 , for example 170-180 pg 1 , such as 180-190 pg 1 , for example a mean value of 190-200 pg I"1.
High or increased TIMP-1 levels in one embodiment means that the cancer patients' cancer cells have high or increased intracellular levels of TIMP-1 ; or detectable levels. In one embodiment this is defined as high or increased, or detectable, intracellular levels as compared to the intracellular level of TIMP-1 in the surrounding cells; and/or high or increased, or detectable, as compared to the level of TIMP-1 in cancer cells that are responsive to a given chemotherapy; and/or high or increased, or detectable, as compared to the level of TIMP-1 in non-cancer cells. Non-cancer cells may refer to non-cancer cells of the same individual, or one or more different individuals (such as a mean value). Cancer cells that are responsive to a given chemotherapy may refer to cancer cells of the same individual, or one or more different individuals (such as a mean value). In one embodiment responsive to e.g. epirubicin, epirubicin in
combination with 5-Fluorouracil and cyclophosphamide, or 5-Flourouracil and irinotecan. In one embodiment said patient with cancer has a TIMP-1 positive (TIMP-1 + or TIMP- 1 hl) cancer. TIMP-1 positive cancer cells means that levels are detectable, and/or having higher intensity of staining than '-' cells and possibly higher than intermediate staining populations. In one embodiment said patient with cancer has high or increased extracellular TIMP-1 levels and high or increased, or detectable, intracellular TIMP-1 levels. In one embodiment said patient with cancer has high or increased TIMP-1 levels and a TIMP- 1 positive cancer. High or increased, or detectable, intracellular TIMP-1 levels of the cancer patients' cancer cells are in one embodiment meant to refer to TIMP-1 protein, to ensure that exogenous TIMP-1 that has been internalized is measured.
In one embodiment, the patient with cancer has a CD74 positive cancer. That is, the cancer patients' cancer cells express detectable CD74 and the cancer is characterized as CD74+ and/or CD74hi.
In one embodiment the patient with cancer has high or increased TIMP-1 levels as defined herein and has a CD74 positive cancer.
In one embodiment, the patient with cancer has a CD63 negative cancer. That is, the cancer patients' cancer cells expression of CD63 is undetectable, absent or low and the cancer is characterized as CD74 -. In one embodiment, the patient with cancer has a CD74 positive and CD63 negative cancer (CD74+/CD63-). Some breast cancers have been characterized as CD74+ and CD63-.
In one embodiment the patient with cancer has one or more of high or increased TIMP- 1 levels, a TIMP-1 positive cancer, a CD74 positive and CD63 negative cancer. In one embodiment the patient with cancer has high or increased TIMP-1 levels and a CD74 positive and CD63 negative cancer. In one embodiment, the patient with cancer has a CD74 positive and CD63 positive cancer (CD74+/CD63+). In this embodiment the patient with cancer in one embodiment is co-treated with a CD74 inhibitor to interrupt the CD74-mediated TIMP-1
internalization, and is also treated with a CD63 inhibitor to interrupt the CD63-mediated TIMP-1 internalization.
In this respect, it is understood that for a given marker the attributed suffix '+' or 'hi' has clear meaning to the skilled person. For instance, '-' (e.g. CD63- or negative) means that the marker is undetectable, absent or low, at least lower than an
intermediate expressing population. Likewise, '+' (e.g. CD74+ or positive) means that expression is detectable, and/or have higher intensity of staining than '-' cells and possibly higher than intermediate staining populations. Furthermore, 'hi' cells (e.g. CD74hl) have higher expression and/or intensity of staining than the '+' cells (e.g.
CD74+) cells. The staining intensity may be determined using conventional FACS methods. Staining intensities may be identified using suffixes +, ++ and +++; all of which are comprised within the term 'CD74 positive' or CD74+ in the present context.
In one embodiment the present composition comprising a TIMP-1 inhibitor and optionally one or more chemotherapeutic agents has a greater therapeutic effect that when the one or more chemotherapeutic agents are administered alone.
In one embodiment the present composition comprising a TIMP-1 inhibitor and optionally one or more chemotherapeutic agents has a greater therapeutic effect in a patient with cancer having high or increased TIMP-1 levels and/or a CD74-positive cancer, that when the one or more chemotherapeutic agents are administered alone.
In one embodiment the present composition comprising a TIMP-1 inhibitor and optionally one or more chemotherapeutic agents has a greater therapeutic effect in a patient with cancer having high or increased TIMP-1 levels and/or a CD74-positive cancer, than in a patient with cancer having low or normal TIMP-1 levels and/or a CD74-negative cancer. Determining if a cancer cell or a tumor cell sample containing cancer cells is CD74 positive, CD63 positive and/or TIMP-1 positive can be done using any conventional methods known to the skilled person. Likewise, the sampling may be done using conventional method known to the skilled person. In one embodiment a sample containing cancer cells, wherein said sample is extracted from said patient with cancer, is examined to determine the levels of one or more of TIMP-1 , CD74 and CD63. In one embodiment the levels refer to mRNA levels. In another preferred embodiment the levels refer to protein levels.
The TIMP-1 , CD74 and CD63 protein levels may be measured using conventional techniques including immunostaining techniques where antibodies are used to detect proteins in tissues and cells, immunohistochemistry, immunocytochemistry, western blotting, dot blot analysis, and ELISA.
The mRNA levels may be measured using conventional techniques including northern blotting, RT-qPCR, SAGE, RNA-Seq, and expression profiling techniques.
CD74 may be detected using the methods described in Starlets et al., "Cell-surface CD74 initiates a signaling cascase leading to cell proliferation and survival", Blood, 2006, 107: 4807-4816.
CD63 may be detected using the methods described in Caby et al. "Exosomal-like vesicles are present in human blood plasma", International Immunology, vol. 17, No. 7, pp. 879-887.
In one embodiment, a patient with cancer or a cancer cell according to the invention refers to a cancer selected from the group consisting of Bone cancer - including Ewing's Sarcoma, Osteosarcoma, Chondrosarcoma; Brain and CNS tumors - including Acoustic Neuroma, Spinal Cord Tumours; Breast cancer - including male breast cancer and Ductal Carcinoma in situ; Colorectal cancer; anal cancer; Endocrine cancers - including Adrenocortical Carcinoma, Pancreatic Cancer, Pituitary Cancer, Thyroid Cancer, Parathyroid Cancer, Thymus Cancer, Multiple Endocrine Neoplasia and Other Endocrine cancers; Gastrointestinal cancers - including Stomach (Gastric) Cancer, Esophageal Cancer, Small Intestine Cancer, Gall Bladder Cancer, Liver Cancer, Extra-Hepatic Bile Duct Cancer and Gastrointestinal Carcinoid Tumour;
Genitourinary cancers - including Testicular Cancer, Penile Cancer and Prostate Cancer; Gynaecological cancers - including Cervical Cancer, Ovarian Cancer, Vaginal Cancer, Uterus / Endometrium Cancer, Vulva Cancer, Gestational Trophoblastic Cancer, Fallopian Tube cancer and Uterine sarcoma; Head and Neck Cancer - including Oral cavity Cancer, Lip Cancer, Salivary gland Cancer, Larynx Cancer, hypopharynx Cancer, oropharynx Cancer, Nasal Cancer, Paranasal Cancer and Nasopharynx Cancer; Leukaemia - including Childhood Leukaemia, Acute Lymphocytic Leukaemia, Acute Myeloid Leukaemia, Chronic Lymphocytic Leukaemia, Chronic Myeloid Leukaemia, Hairy Cell Leukaemia, BMT for Leukaemia, Patient's Home Pages, Acute Promyelocytic Leukemia and Plasma Cell Leukaemia; Haematological disorders - including Multiple myeoloma, Myelodysplastic Syndromes, Myeloproliferative
Disorders, Aplastic Anaemia, Fanconi Anaemia and Waldenstrom's Macroglobulinemia; Lung cancer - including Small Cell Lung Cancer and Non-Small Cell Lung Cancer; Lymphoma - including Hodgkin's Disease, Non-Hodgkin's Lymphoma and Aids Related Lymphoma; Eye cancer - including Retinoblastoma and IntraOcular Melanoma; Skin cancer - including Melanoma, squamous cell carcinoma and basal cell carcinoma; and Soft tissue Cancer - including Soft tissue sarcoma and Kaposi's sarcoma; Urinary system cancer - including Kidney Cancer, Wilm's Tumour, Bladder Cancer, Urethral Cancer and Transitional Cell Cancer; wherein each of these cancers may or may not be metastatic.
In one embodiment the composition comprising a TIMP-1 inhibitor according to the present invention, for treatment of a cancer and/or for increasing the sensitivity of the cancer to a chemotherapeutic agent, is for use in treating a solid cancer / non- hemotopoietic cancer. In one embodiment the present use is not for treating multiple myeloma, non-Hodgkin's lymphoma and chronic lymphocytic leukemia.
Chemotherapeutic agent
The one or more chemotherapeutic agents herein may be any chemotherapeutic agent known to the skilled person or a combination thereof. Preferably the chemotherapeutic agent of the present invention is a chemotherapeutic agent that has reduced or no effect when TIMP-1 levels are high or increased, or detectable, as defined herein;
and/or when TIMP-1 is internalized via CD74 and/or CD63. The chemotherapeutic agents whose effect will be increased when the patient with cancer is treated with a TIMP-1 inhibitor according to the present invention may be identified by evaluating the response rate of said one or more chemotherapeutics and determining the TIMP-1 levels as outlined herein. If the response rate is low, and the TIMP-1 levels are high or increased, or detectable, then the patient is likely to benefit from treatment with a TIMP-1 inhibitor of the present invention.
In one embodiment the patient with cancer to be treated with a TIMP-1 inhibitor of the present invention has been and/or is being treated with one or more chemotherapeutic agents.
In one embodiment the composition of the invention comprises, together or separately, i) a TIMP-1 inhibitor, such as a compound capable of inhibiting the interaction between TIMP-1 and CD74, and ii) one or more chemotherapeutic agents, wherein the TIMP-1 inhibitor and the one or more chemotherapeutic agents are co-administered, which coadministration can be simultaneously, sequentially or separately.
In one embodiment the chemotherapeutic agent is selected from the group consisting of alkylating agents, anti-metabolites, anti-microtubule agents, topoisomerase inhibitors, cytotoxic antibiotics and targeted therapies.
Alkylating agents are so named because of their ability to alkylate many molecules including DNA. Alkylating agents include the nitrogen mustards, nitrosoureas, tetrazines, aziridines, cisplatins and derivatives, and non-classical alkylating agents. Nitrogen mustards include mechlorethamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide and busulfan. Nitrosoureas include N-Nitroso-N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU) and semustine (MeCCNU), fotemustine and streptozotocin. Tetrazines include dacarbazine, mitozolomide and temozolomide. Aziridines include thiotepa, mytomycin and diaziquone (AZQ). Cisplatin and derivatives include cisplatin, carboplatin and oxaliplatin. Non-classical alkylating agents include procarbazine and hexamethylmelamine.
Anti-metabolites are a group of molecules that impede DNA and RNA synthesis.
Subtypes of the anti-metabolites are the anti-folates, fluoropyrimidines,
deoxynucleoside analogues and thiopurines. The anti-folates include methotrexate and pemetrexed. The fluoropyrimidines include fluorouracil and capecitabine. Fluorouracil is a nucleobase analogue that is metabolised in cells to form at least two active products; 5-fluourouridine monophosphate (FUMP) and 5-fluoro-2'-deoxyuridine 5'-phosphate (fdUMP). Capecitabine is a prodrug of 5-fluorouracil that is broken down in cells to produce the active drug. The deoxynucleoside analogues include cytarabine, gemcitabine, decitabine, Vidaza, fludarabine, nelarabine, cladribine, clofarabine and pentostatin. The thiopurines include thioguanine and mercaptopurine.
Anti-microtubule agents prevent microtubule function with two main groups Vinca alkaloids and taxanes. The original vinca alkaloids are natural chemicals that include vincristine and vinblastine. Semi-synthetic vinca alkaloids include: vinorelbine, vindesine, and vinflunine. Taxanes are natural and semi-synthetic drugs and include paclitaxel and docetaxel. Topoisomerase inhibitors are drugs that affect the activity of one of the enzymes: topoisomerase I and topoisomerase II. Topoisomerase I inhibitors include
camptothecin (CPT) and the CPT-analogues irinotecan and topotecan, topoisomerase II inhibitors can be divided into two groups; the topoisomerase II poisons and the catalytic inhibitors. Topoisomerase II poisons include etoposide, doxorubicin, mitoxantrone and teniposide. The catalytic inhibitors include novobiocin, merbarone, and aclarubicin as well as ICRF-187.
The cytotoxic antibiotics are a varied group of drugs that have different mechanisms of action. The group includes the anthracyclines and other drugs including actinomycin, bleomycin, plicamycin, and mitomycin. The anthracyclines include doxorubicin and daunorubicin and derivatives thereof including epirubicin and idarubicin, as well as pirarubicin, aclarubicin, and mitoxantrone. Actinomycin and Bleomycin intercalates DNA. Some newer anticancer drugs specifically target proteins that are abnormally expressed in cancer cells and that are essential for their growth. Such treatments are often referred to as targeted therapy (as distinct from classic chemotherapy). These include various immunotherapeutic agents, monoclonal antibodies, biologicals, anti- hormone therapies and kinase inhibitors. Antihormone therapy is a form of treatment which suppresses selected hormones. The suppression of certain hormones is beneficial to the cancer patient since certain hormones prompt the growth of a tumor. Antihormone treatment can be specific to gender as well, such as antiestrogen or antitestosterone therapy. Anti-hormone therapies include aromatase inhibitors including irreversible steroidal inhibitors and non-steroidal inhibitors such as Aminoglutethimide, Testolactone (Teslac), Anastrozole (Arimidex), Letrozole (Femara), Exemestane (Aromasin), Vorozole (Rivizor),
Formestane (Lentaron) and Fadrozole (Afema); anti-estrogens such as tamoxifen, Fulvestrant (Faslodex), Toremifene, Raloxifene, Selective estrogen receptor modulators (SERM's); anti-androgenic drugs such as Flutamide (Eulexin), nilutamide (Anandron and Nilandron), bicalutamide (Casodex), 5-alpha-reductase inhibitors such as finasteride (Proscar and Propecia), dutasteride (Avodart), bexlosteride, izonsteride, turosteride and epristeride. A range of protein kinases are targets for the kinase inhibitors, including EGFR, ErbB2, VEGFRs, VEGF, BcrAb, SRC, ErbB1 , ALK/Met, Syk, BTK, PDGFR, c-kit, JAK, STAT, RET and BRAF.
The kinase inhibitors include afatinib, axitinib, bevacizumab, bosutinib, cetuximab, crizotinib, dasatinib, erlotinib, fostamatinib, gefitinib, ibrutinib, imatinib, lapatinib, lenvatinib, mubritinib, nilotinib, panitumumab, pazopanib, pagaptanib, ranibizumab, ruxolitinib, sorafenib, SU6656, trastuzumab, tofacitinib, vandetanib and vemurafenib.
In one embodiment the chemotherapeutic agent of the invention is a monoclonal antibody that binds to cancer cell-specific antigens and induce an immunological response against the target cancer cell. Therapeutic monoclonal antibodies include Bevacizumab/Avastin (VEGF-A inhibitor), Cetuximab/Erbitux, Panitumumab/Vectibix, Nimotuzumab (EGFR inhibitors), Trastuzumab/Herceptin (HER2/neu receptor inhibitor), Rituximab/Rituxan (CD20) Gemtuzumab ozogamicin/Mylotarg (CD33), Alemtuzumab/Campath (CD52), Brentuximab vedotin/ Adcetris, Denosumab/Prolia, Ibritumomab tiuxetan/Zevalin, Ipilimumab/Yervoy, Ofatumumab/Xolair and
Tositumomab/Bexxar.
In one embodiment, said one or more chemotherapeutic agents comprises one or more of epirubicin, 5-Fluorouracil, cyclophosphamide, irinotecan, docetaxel, methotrexate and gemcitabine; such as epirubicin in combination with 5-Fluorouracil and
cyclophosphamide, such as 5-Flourouracil in combination with irinotecan.
KRas mutation
GTPase KRas also known as V-Ki-ras2 Kirsten rat sarcoma viral oncogene homolog and KRAS, is a protein that in humans is encoded by the KRAS gene. The protein product of the normal KRAS gene performs an essential function in normal tissue signaling, and the mutation of a KRAS gene is an essential step in the development of many cancers. KRas and KRAS may be used interchangeably herein.
In some embodiments, a single amino acid substitution, and in particular a single nucleotide substitution, is responsible for an activating mutation. The transforming protein that results is implicated in various malignancies, including lung
adenocarcinoma, mucinous adenoma, ductal carcinoma of the pancreas, leukemias and colorectal carcinoma.
The mutational status of KRAS is indicative for tumor response to treatment with an EGFR inhibitor or antibody, such as cetuximab (Erbitux) and panitumumab (Vectibix). Thus, KRAS wild-type tumors are generally responsive to treatment with EGFR inhibitors, while tumors with mutated (at one or more positions) KRAS is generally less or not responsive to treatment. KRAS mutational analysis is commercially available from a number of laboratories.
In one aspect of the invention a new treatment regimen is disclosed for those cancer patients whose tumors harbors a KRas mutation, which are today excluded from treatment with an EGFR inhibitor, such as cetuximab, alone.
In one embodiment there is provided a composition comprising a compound capable of inhibiting the interaction interaction between TIMP-1 and CD74 for use in the treatment of a patient with cancer having one or more tumor KRas mutations and having high or increased TIMP-1 levels. Treatment may in one embodiment mean treatment of the cancer, or it may be treatment of the cancer by increasing the sensitivity towards anticancer agents. Ά patient with cancer having one or more tumor KRas mutations' means that the cancer patients tumor cells harbors one or more mutations in the KRAS gene. In one embodiment there is provided a composition comprising a compound capable of inhibiting the interaction interaction between TIMP-1 and CD74 and an EGFR inhibitor for use in the treatment of a patient with cancer having one or more tumor KRas mutations and having high or increased TIMP-1 levels. Also provided is a method of treating a patient with cancer with one or more KRas mutations and having high or increased TIMP-1 levels, said method comprising administering to said patient a compound capable of inhibiting the interaction interaction between TIMP-1 and CD74, alone or in combination with an EGFR inhibitor. In one embodiment, said EGFR inhibitor is an EGFR antibody (anti-EGFR antibody). An EGFR antibody is in one embodiment selected from the group consisting of a monoclonal antibody; one or more recombinant antibodies; and a polyclonal antibody. Said monoclonal antibody is in one embodiment selected from a chimeric, a humanized or fully human antibody.
In one embodiment, said EGFR antibody is selected from the group consisting of cetuximab (Erbitux), panitumumab (Vectibix), gefitinib (Iressa), erlotinib hydrochloride (Tarceva), and others known to the skilled person. Selecting a cancer patient
It is also an aspect to provide a method for selecting a patient with cancer for treatment with a TIMP-1 inhibitor according to the present invention, said method comprising examining i) the level of TIMP-1 in a sample obtained from said patient with cancer, and/or ii) the cancer cell expression of CD74; wherein high or increased, or detectable, expression of TIMP-1 and/or a CD74-positive cancer is indicative of said patient with cancer being responsive to treatment. Responsive to treatment in the present context refers to treatment with one or more TIMP-1 inhibitors as defined herein; alone or in combination with one or more chemotherapeutics. In one embodiment a sample obtained from said patient is a bodily fluid, such as whole blood, plasma, serum, urine, saliva, sputum and cerebrospinal fluid. It is also an aspect to provide a method for increasing the response rate of cancer patients to chemotherapeutic agents, said method comprising the steps of examining one or more of: i) the cancer cell expression of CD74, and ii) the level of TIMP-1 in a sample obtained from said cancer patient, wherein the patient is selected for treatment when having high or increased expression of TIMP-1 and a CD74-positive cancer.
In one embodiment the method further comprises the steps of examining the cancer cell expression of CD63, wherein a CD63 negative cancer is indicative of said patient with cancer being responsive to treatment. In one embodiment, there is provided a method for selecting a patient with cancer for treatment with a TIMP-1 inhibitor, such as a compound capable of inhibiting the interaction interaction between TIMP-1 and CD74, such as a CD74 inhibitor; alone or in combination with a chemotherapeutic agent, such as an EGFR inhibitor, such as an EGFR antibody, said method comprising examining i) the cancer cell expression of CD74 and/or CD63 and ii) the level of TIMP-1 in a sample obtained from said cancer patient, wherein expression of TIMP-1 and CD74 and/or CD63 is indicative of said patient with cancer being responsive to treatment.
In one embodiment, there is provided a method for selecting a patient with cancer for treatment with a TIMP-1 inhibitor, such as a compound capable of inhibiting the interaction interaction between TIMP-1 and CD74, such as a CD74 inhibitor; alone or in combination with a chemotherapeutic agent, such as an EGFR inhibitor, such as an EGFR antibody; said method comprising examining i) the mutational status of KRas and ii) the level of TIMP-1 in a sample obtained from said cancer patient, wherein the identification of one or more KRas mutations and concomitant high or increased levels of TIMP-1 is indicative of said patient with cancer being responsive to treatment with said compound. Screening
In one embodiment, there is provided a method for screening for compounds being an agonist or antagonist for CD74 comprising: contacting a CD74 polypeptide with TIMP-1 in the presence and absence of a candidate compound, and comparing the interaction of the TIMP-1 and said CD74 polypeptide in the presence of said candidate compound with their interaction in the absence of said candidate compound, whereby a candidate compound that enhances the interaction of said TIMP-1 with said CD74 polypeptide is identified as an agonist of CD74, and a candidate compound that inhibits the interaction of said TIMP-1 with said CD74 polypeptide is identified as an antagonist of CD74.
Administration and dosage
According to the present invention the TIMP-1 inhibitor, or a composition comprising the same, is to be administered to an individual in need of treatment in
pharmaceutically effective doses or a therapeutically effective amount. The dosage requirements will vary with the particular drug composition employed, the route of administration and the particular subject being treated, which depend on the severity and the sort of the disorder as well as on the weight and general state of the subject. It will also be recognized by one skilled in the art that the optimal quantity and spacing of individual dosages will be determined by the nature and extent of the condition being treated, the form, route and site of administration, and the particular patient being treated, and that such optima can be determined by conventional techniques. It will also be appreciated by one of skill in the art that the optimal course of treatment, i.e., the number of doses of a compound given per day for a defined number of days, can be ascertained using conventional course of treatment determination tests.
A therapeutically effective amount is in one embodiment an amount sufficient to cure, prevent, reduce the risk of, alleviate or partially arrest the clinical manifestations of a given disease or disorder and its complications. The amount that is effective for a particular therapeutic purpose will depend on the severity and the sort of the disorder as well as on the weight and general state of the subject. An amount adequate to accomplish this is defined as a "therapeutically effective amount". Examples
Example 1 : Co-lmmunoprecipitation studies
The aim of this study was to confirm the binding of CD74 and TIMP-1 in mammalian cells. In short, the high CD74 expressing breast cancer cell line MDA-MB-231 was incubated with rTIMP-1 for 1 hr and subjected to Co-immunoprecipitation.
Material and Methods
Cells. The high CD74 expressing breast cancer cell line, MDA-MB-231 , used in this study was purchased from ATCC (Rockville, MD). The cells were cultured in DMEM supplemented with 10 % FCS in the presence of 50 μg/ml rTIMP-1 , 5 % C02 at 37 <C. Co-lmmunoprecipitation (co-IP). The Dynabeads Co-lmmunoprecipitation Kit (Life Technologies, San Diego, Ca) was used for co-IP experiments. The co-IP was carried out according to the manufacture's instruction with minor adjustments. In short, MDA- MB-231 cells were incubated with rTIMP-1 (2.5 μg/ml) for 30 min and subsequently lysed in modified Extraction Buffer (1 x Extraction Buffer, 100 mM NaCI, 2 mM MgCI2, 1 mM DTT and PMSF). Supernatants were incubated with antibody coupled beads (C- 16, Santa Cruz, VT4 clone and goat IgG control) for immunprecipitation of CD74, TIMP-1 and negative control, respectively, for 1 hr, 4 °C. After several wash steps the immunoprecipitated proteins were eluted and subjected to Western Blot analysis. Antibodies. For Western Blot; TIMP-1 (clone VT7), CD74 (LN-2, Santa Cruz
Biotechnology). For Co-IP; TIMP-1 (clone VT4), CD74 (C-16, Santa Cruz
Biotechnology) and normal goat IgG (Control IgG, Santa Cruz Biotechnology)
Reagents. 293T lysate (sc-175300, Santa Cruz Biotechnology).
Immunoblot analysis. Equal volumens of protein samples in Laemmli Sample Buffer, 10 % mecaptoethanol (Bio-Rad, Hercules, CA) were heated for 10 min at 70 °C and subsequently subjected to reducing SDS-PAGE. After electrophoresis, the proteins were blottet on to PVDF membranes which were subsequently blocked with 5 % milk in 1 x PBS + 0.1 % Tween 20 (PBS-T) for 1 hr at room temperature. The membranes were then incubated overnight with primary antibodies in PBS-T containing 2.5 % milk. After 3 washes in PBS-T, the membranes were incubated with the appropriate secondary antibodies. The antigen was detected using the Amersham ECL-Select Western Blotting detection reagent (GE Healthcare Life Sciences, Piscataway, NJ) according to manufacturer's instruction and visualized by UVP BioSpectrum Imaging system. Results
The 293T lysate was included as a positive control for CD74 whereas the IgG control was included as a negative control where we don't expect to see any bound proteins. From figure 1 we can see that we don't have any CD74 in the IgG control sample which indicates that there is no unspecific binding of CD74 to the beads. However, we do see CD74 in the TIMP-1 IP sample which matches the size of the bands in the 293T positive control.
Conclusion
In the Co-immunoprecipitation studies we have successfully confirmed the interaction between CD74 and TIMP-1 in a mammalian cell system.
Example 2: Uptake studies
The aim of this study was to investigate the role of CD74 in TIMP-1 uptake by the cells. In order to do that we down -regulated CD74 by siRNA and compared the uptake of TIMP-1 to wild type cells.
Material and Methods
Cells. The high CD74 expressing breast cancer cell line, MDA-MB-231 , used in this study was purchased from ATCC (Rockville, MD). The cells were cultured in DMEM supplemented with 10 % FCS in the absence or presence of 50 μg/ml rTIMP-1 , 5 % C02 at 37 °C.
Transient knock down of CD74. CD74 was transiently down-regulated by siRNA (Sigma, SASI_Hs01_00142175). Cells were plated 24 hour prior to transfection and medium was changed 1 hour before start of transfection. According to manufacturer's instruction CD74 siRNA was transfected into MDA-MB-231 cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA).
Uptake studies. Medium was changed 96 hours after transfection and replaced with medium containing 50 μg/ml rTIMP. Before cell lysis, cells were washed 3 times in ice cold PBS. Whole cell lysates were obtained by lysing the cell monolayer with M-PER Mammalian Protein Extraction Reagent supplemented with protease and phosphatase inhibitors (Thermo Scientific, Rockford, IL).
Antibodies. TIMP-1 (clone VT7), CD74 (LN-2, Santa Cruz Biotechnology) and HSP70 (AB-2 (W27), Fremont, CA)
Immunoblot analysis. Protein concentration of collected samples was measured using the BCA protein Assay Kit (Novagen, San Diego, CA). Equal amounts of protein samples in Laemmli Sample Buffer, 10 % mecaptoethanol (Bio-Rad, Hercules, CA) were heated for 10 min at 70 °C and subsequently subjected to reducing SDS-PAGE. After electrophoresis, the proteins were blottet on to PVDF membranes which were subsequently blocked with 5 % milk in 1 x PBS + 0.1 % Tween 20 (PBS-T) for 1 hr at room temperature. The membranes were then incubated overnight with primary antibodies in PBS-T containing 2.5 % milk. After 3 washes in PBS-T, the membranes were incubated with the appropriate secondary antibodies. The antigen was detected using the Amersham ECL-Select Western Blotting detection reagent (GE Healthcare Life Sciences, Piscataway, NJ) according to manufacturer's instruction and visualized by UVP BioSpectrum Imaging system.
Results
Cells +/- CD74 expression were incubated in medium containing 50 μg/ml rTIMP-1 for 4- and 24 hours. Cells without CD74 expression displayed an impaired ability to take up rTIMP-1 from the medium. This was seen for both of the selected time points fig. 2. Conclusion
As can be seen from fig. 2 we successfully down -regulated the expression of CD74 by siRNA. Furthermore, lower levels of CD74 in the cells correlate with a reduced uptake of rTIMP-1 from the medium. These results show that CD74 is involved in the uptake of TIMP-1 in cancer cells.
Example 3
We first incubated human breast cancer cells grown in culture with recombinant TIMP- 1 . Already following 10 minutes of incubation with TIMP-1 , the cancer cells have internalized TIMP-1 . At 6 hours post incubation, even more TIMP-1 protein is to be seen within the cancer cells. We then next incubated the cancer cells with recombinant TIMP-1 and after 24 hours of incubation the cancer cells were exposed to
chemotherapy (epirubicin). As control we included the parent cell line without TIMP-1 incubation. Incubation and subsequent internalization of TIMP-1 resulted in a significant reduction in epirubicin-induced cytotoxicity. In a third experiment we down- regulated CD63 in the cancer cells and (... ) suggesting that other internalization proteins may be involved in TIMP-1 internalization.
We therefore performed yeast two hybrid experiments where TIMP-1 was the bait and a library derived from normal breast epithelial served as the prey. When searching for potential cell surface proteins among the obtained hits, we identified CD74 as a potential TIMP-1 binding partner. CD74 is an evolutionarily conserved type II membrane protein initially described to be involved in several key processes of the immune system.
A Western blot on which immunoprecipated TIMP-1 :CD74 protein complexes were loaded clearly shown that by using a TIMP-1 antibody for immunoprecipitation, CD74 can be identified on the gel and vice versa supporting that TIMP-1 and CD74 indeed are binding partners.
Specific (shRNA) down regulation of CD74 resulted in a notable decrease in TIMP-1 internalization suggesting that CD74 plays an important role in TIMP-1 cellular internalization.
In order to obtain information on the expression of CD63 and CD74 in human cancer tissue samples, we performed CD63 and CD74 immunohistochemistry on breast cancer samples. We identified cancers that had TIMP-1 protein in the cancer cells, and no CD63 but CD74 on the cell surface suggesting that TIMP-1 can be internalized without the presence of CD63 and these data also points to CD74 being involved in
TIMP-1 internalization in human cancer as we have shown in human cancer cell lines.
Example 4
A screening assay to find a TIMP-1 inhibitor capable of increasing the sensitivity towards one or more chemotherapeutic agents is performed in vitro by adding to a cell culture a TIMP-1 inhibitor with or without TIMP-1 protein (or in TIMP-1 expressing and TIMP-1 non-expressing cells) and see if the effect of certain anti-cancer treatments are increased (increased chemosensitivity) by adding a TIMP-1 inhibitor (alone or in combination) to the cells. A TIMP-1 inhibitor may be a compound capable of inhibiting the interaction between TIMP-1 and CD74 and/or between TIMP-1 and CD63. Kinase inhibitors of intracellular signaling downstream of CD63 and/or CD74 can also be added.
Example 5
When one or more inhibitors of the TIMP-1 :CD74 interaction have been identified and validated in preclinical in vitro and in vivo models, the most promising inhibitors will be taken to preclinical toxicity and pharmacology studies in two species, e.g. rats and dogs, and different doses of the inhibitor in question will be tested for toxicity, following international guidelines for such studies. Moreover, the pharmacokinetics (half-life, T1/2 etc.) will be determined from blood from the treated animals. Based on all these preclinical studies a protocol for a clinical phase 1 study will be developed. A clinical phase 1 study has as its primary objective to test toxicity of the TIMP-1 :CD74 inhibitor in human beings. Suitable individuals/patients are identified and after having signed an informed consent form they can be enrolled in the clinical study. Patients will be exposed in groups of three to increasing concentrations of the inhibitor in question. When toxicity is observed, 3 more patients will be exposed to the inhibitor but at the former dose level. If no toxicity is observed here, this dose is the recommended dose for subsequent clinical phase 2 study in which one will look for efficacy of the drug. It will be possible to include measurements of so-called surrogate biomarkers in the phase 1 study. Moreover, it will be possible to restrict the enrollment of patients in the phase 1 study to patients with specific diseases and specific tumor cell characteristics, e.g. positive for TIMP-1 , CD74 and/or CD63. ITEMS
A composition comprising a TIMP-1 inhibitor for use in increasing the sensitivity of a cancer cell towards one or more chemotherapeutic agents,
(or; A method of increasing the sensitivity of a cancer cell towards one or more chemotherapeutic agents comprising administering a composition comprising a TIMP-1 inhibitor to a patient in need thereof, alone or in combination with said one or more chemotherapeutic agents).
A composition comprising i) a TIMP-1 inhibitor and ii) a chemotherapeutic agent for use in the treatment of cancer and/or for use in increasing the sensitivity of a cancer cell towards one or more chemotherapeutic agents.
The composition for use according to any of the preceding items, wherein said TIMP-1 inhibitor is able to decrease intracellular tumor levels of TIMP-1 and/or able to decrease tumor cell internalization of TIMP-1 .
The composition for use according to any of the preceding items, wherein said TIMP-1 inhibitor is a compound capable of inhibiting the interaction between TIMP-1 and CD74.
The composition for use according to any of the preceding items, wherein said compound capable of inhibiting the interaction between TIMP-1 and CD74 is a CD74 inhibitor. The composition for use according to any of the preceding items, wherein said CD74 inhibitor is a CD74 antibody.
The composition for use according to any of the preceding items, wherein said CD74 antibody is milatuzumab.
The composition for use according to any of the preceding items, wherein said composition comprises a CD74 inhibitor such as a CD74 antibody and a CD63 inhibitor such as a CD63 antibody.
The composition for use according to any of the preceding items, wherein said CD74 inhibitor is able to reduce the level and/or activity of CD74.
The composition for use according to any of the preceding items, wherein said TIMP-1 inhibitor is able to reduce the level and/or activity of TIMP-1 .
The composition for use according to any of the preceding items, wherein said TIMP-1 inhibitor is able to inhibit TIMP-1 -induced intracellular signaling.
The composition for use according to any of the preceding items, wherein said TIMP-1 inhibitor is a TIMP-1 antibody.
The composition for use according to any of the preceding items, wherein said TIMP-1 inhibitor decreases the mRNA expression of CD74 and/or TIMP-1 . The composition for use according to any of the preceding items, wherein said compound capable of inhibiting the interaction between TIMP-1 and CD74 is a binding partner for CD74 other than TIMP-1 , such as MIF (Macrophage migration inhibitory factor) and/or D-dopachrome tautomerase (D-DT/MIF-2). The composition for use according to any of the preceding items, wherein said chemotherapeutic agent is an EGFR inhibitor.
The composition for use according to any of the preceding items, wherein said EGFR inhibitor is an EGFR antibody.
The composition for use according to any of the preceding items, wherein said EGFR antibody is selected from the group consisting of cetuximab (Erbitux), panitumumab (Vectibix), gefitinib (Iressa) and erlotinib hydrochloride (Tarceva). The composition for use according to any of the preceding items, wherein said one or more chemotherapeutic agent is epirubicin, epirubicin in combination with 5-Fluorouracil and cyclophosphamide, or 5-Flourouracil and irinotecan. The composition for use according to the preceding items, wherein said composition is to be administered to a cancer patient.
The composition for use according to any of the preceding items, wherein said patient with cancer has high or increased tumor TIMP-1 levels. The composition for use according to any of the preceding items, wherein said patient with cancer has one or more tumor KRAS mutation(s).
The composition for use according to any of the preceding items, wherein said patient with cancer has one or more tumor KRas mutations and high or increased TIMP-1 levels.
The composition for use according to any of the preceding items, wherein said patient with cancer has high or increased tumor TIMP-1 levels and also expresses CD74 and/or CD63.
The composition for use according to any of the preceding items, wherein said TIMP-1 inhibitor is to be administered in combination with a chemotherapeutic agent.
The composition for use according to any of the preceding items, wherein said TIMP-1 inhibitor and said chemotherapeutic agent are administered separately, sequentially or simultaneously.
The composition for use according to any of the preceding items, wherein said cancer is selected from the group consisting of colorectal cancer, metastatic colorectal cancer (mCRC), lung cancer including small cell lung cancer and non-small cell lung cancer, breast cancer, head and neck cancer, squamous cell carcinoma of the head and neck, hematological malignancies,
The composition for use according to any of the preceding items, wherein said composition further comprises one or more specific kinase inhibitors, such as kinase inhibitors of intracellular signaling downstream of CD63 and/or CD74. A method for selecting a patient with cancer for treatment with a TIMP-1 inhibitor, such as a compound capable of inhibiting the interaction interaction between TIMP-1 and CD74, such as a CD74 inhibitor; alone or in combination with a chemotherapeutic agent, such as an EGFR inhibitor, such as an EGFR antibody, said method comprising examining i) the mutational status of KRas and ii) the level of TIMP-1 in a sample obtained from said cancer patient, wherein the identification of one or more KRas mutations and concomitant high levels of TIMP-1 is indicative of said patient with cancer being responsive to treatment.
A method for selecting a patient with cancer for treatment with a TIMP-1 inhibitor, such as a compound capable of inhibiting the interaction interaction between TIMP-1 and CD74, such as a CD74 inhibitor; alone or in combination with a chemotherapeutic agent, such as an EGFR inhibitor, such as an EGFR antibody, said method comprising examining i) the cancer cell expression of CD74 and/or CD63 and ii) the level of TIMP-1 in a sample obtained from said cancer patient, wherein expression of TIMP-1 and CD74 and/or CD63 is indicative of said patient with cancer being responsive to treatment.
30. A method for screening for compounds being an agonist or antagonist for CD74 comprising: contacting a CD74 polypeptide with TIMP-1 in the presence and absence of a candidate compound, and comparing the interaction of the TIMP-1 and said CD74 polypeptide in the presence of said candidate compound with their interaction in the absence of said candidate compound, whereby a candidate compound that enhances the interaction of said TIMP-1 with said
CD74 polypeptide is identified as an agonist of CD74, and a candidate compound that inhibits the interaction of said TIMP-1 with said CD74 polypeptide is identified as an antagonist of CD74.

Claims

Claims
A composition comprising a TIMP-1 inhibitor for use in a method of increasing the sensitivity of a cancer cell towards one or more chemotherapeutic agents in a patient with cancer.
The composition according to claim 1 comprising, together or separately, i) a TIMP-1 inhibitor and ii) one or more chemotherapeutic agents, wherein said composition is for use in a method of increasing the sensitivity of a cancer cell towards said one or more chemotherapeutic agents in a patient with cancer.
The composition for use according to any of the preceding claims, wherein said sensitivity is increased by inhibiting the interaction between TIMP-1 and CD74; and/or inhibiting CD74-mediated cancer cell TIMP-1 internalization.
The composition for use according to any of the preceding claims, wherein said patient with cancer has high or increased TIMP-1 levels.
The composition for use according to any of the preceding claims, wherein said patient with cancer has high or increased extracellular TIMP-1 levels and/or high or increased, or detectable, intracellular TIMP-1 levels.
The composition for use according to any of the preceding claims, wherein said patient with cancer has high or increased TIMP-1 levels in one or more bodily fluids, such as one or more bodily fluids selected from the group consisting of whole blood, plasma, serum, urine, saliva, sputum and cerebrospinal fluid.
The composition for use according to any of the preceding claims, wherein the TIMP-1 plasma level of said patient with cancer is above a mean value of 50 1 , such as 60 1 1 ,for example 70 1 , such as 80 1 , for example 90 μς 1 , such as 100 μς 1 , for example 1 10 μς 1 , such as 120 μς 1 , for example 130 μς 1 , such as 140 μς 1 , for example 150 μς 1 , such as 160 μς 1 , for example 170 μς 1 , such as 180 μς 1 , for example 190 μς 1 , such as above a mean value of 200 Γ1
8. The composition for use according to any of the preceding claims, wherein said patient with cancer has high or increased, or detectable, TIMP-1 levels in a sample comprising cancer cells.
9. The composition for use according to any of the preceding claims, wherein said patient with cancer has a TIMP-1 positive cancer.
10. The composition for use according to any of the preceding claims, wherein said patient with cancer has a CD74 positive cancer.
1 1 . The composition for use according to any of the preceding claims, wherein said patient with cancer has a CD63 negative cancer.
12. The composition for use according to any of the preceding claims, wherein said patient with cancer has one or more of high or increased TIMP-1 levels, a TIMP-1 positive cancer, a CD74 positive and CD63 negative cancer.
13. The composition for use according to any of the preceding claims, wherein said patient with cancer has high or increased TIMP-1 levels and a CD74 positive cancer.
14. The composition for use according to any of the preceding claims, wherein said patient with cancer has high or increased TIMP-1 levels, a CD74 positive and a CD63 negative cancer.
15. The composition for use according to any of the preceding claims, wherein said patient with cancer has one or more of high or increased TIMP-1 levels, a TIMP-1 positive cancer, a CD74 positive and CD63 positive cancer.
16. The composition for use according to any of the preceding claims, wherein said composition comprising a TIMP-1 inhibitor and optionally one or more chemotherapeutic agents has a greater therapeutic effect in a patient with cancer having high or increased TIMP-1 levels and/or a CD74-positive cancer, than in a patient with cancer having low or normal TIMP-1 levels and/or a CD74-negative cancer.
17. The composition for use according to any of the preceding claims, wherein said composition comprises two or more TIMP-1 inhibitors.
18. The composition for use according to any of the preceding claims, wherein said TIMP-1 inhibitor is a compound capable of inhibiting the interaction between TIMP-1 and CD74.
19. The composition for use according to any of the preceding claims, wherein said TIMP-1 inhibitor is a compound capable of inhibiting CD-74 mediated cancer cell TIMP-1 internalization.
20. The composition for use according to any of the preceding claims, wherein said
TIMP-1 inhibitor is a CD74 inhibitor.
21 . The composition for use according to any of the preceding claims, wherein said CD74 inhibitor is a CD74 antibody.
22. The composition for use according to any of the preceding claims, wherein said CD74 antibody is selected from the group consisting of a monoclonal antibody (MAb); one or more recombinant antibodies; and one or more polyclonal antibodies, multispecific antibodies, bispecific antibodies, diabodies, full-length antibodies, fragments of an antibody such as Fab and F(ab')2 fragments.
23. The composition for use according to any of the preceding claims, wherein said monoclonal CD74 antibody is selected from the group consisting of a chimeric, a humanized or fully human antibody.
24. The composition for use according to any of the preceding claims, wherein said CD74 antibody is milatuzumab.
25. The composition for use according to any of the preceding claims, wherein said CD74 antibody binds to an epitope of one or more of the human isoforms of
CD74: p43, p41 , p35 and p33.
26. The composition for use according to any of the preceding claims, wherein said CD74 inhibitor is able to inhibit one or more of CD74 expression, CD74 translation, CD74 transcription, CD74 secretion, CD74 stability, CD74 biological activity and CD74 -induced intracellular signalling.
27. The composition for use according to any of the preceding claims, wherein said TIMP-1 inhibitor is able to inhibit one or more of TIMP-1 expression, TIMP-1 translation, TIMP-1 transcription, TIMP-1 secretion, TIMP-1 stability, TIMP-1 internalization, TIMP-1 biological activity, TIMP-1 binding to cell surface proteins, TIMP-1 internalization and TIMP-1 -induced intracellular signalling.
28. The composition for use according to any of the preceding claims, wherein said TIMP-1 inhibitor is a TIMP-1 antibody.
29. The composition for use according to any of the preceding claims, wherein said two or more TIMP-1 inhibitors comprises a compound that inhibits TIMP-1 directly and a compound that inhibits CD74 directly.
30. The composition for use according to any of the preceding claims, wherein said composition comprises a CD74 inhibitor and a CD63 inhibitor.
31 . The composition for use according to any of the preceding claims, wherein said composition further comprises one or more specific kinase inhibitors, such as kinase inhibitors of intracellular signaling downstream of CD63 and/or CD74.
32. The composition for use according to any of the preceding claims, wherein said TIMP-1 inhibitor and said one or more chemotherapeutic agents are
administered separately, sequentially or simultaneously.
33. The composition for use according to any of the preceding claims, wherein said chemotherapeutic agents are selected from the group consisting of alkylating agents, anti-metabolites, anti-microtubule agents, topoisomerase inhibitors, cytotoxic antibiotics and targeted therapies including immunotherapeutic agents, monoclonal antibodies, biologicals, anti-hormone therapies and kinase inhibitors.
34. The composition for use according to any of the preceding claims, wherein said cancer is selected from the group consisting of Bone cancer - including Ewing's Sarcoma, Osteosarcoma, Chondrosarcoma; Brain and CNS tumors - including Acoustic Neuroma, Spinal Cord Tumours; Breast cancer - including male breast cancer and Ductal Carcinoma in situ; Colorectal cancer; anal cancer; Endocrine cancers - including Adrenocortical Carcinoma, Pancreatic Cancer, Pituitary Cancer, Thyroid Cancer, Parathyroid Cancer, Thymus Cancer, Multiple Endocrine Neoplasia and Other Endocrine cancers; Gastrointestinal cancers - including Stomach (Gastric) Cancer, Esophageal Cancer, Small Intestine Cancer, Gall Bladder Cancer, Liver Cancer, Extra-Hepatic Bile Duct Cancer and Gastrointestinal Carcinoid Tumour; Genitourinary cancers - including Testicular Cancer, Penile Cancer and Prostate Cancer; Gynaecological cancers - including Cervical Cancer, Ovarian Cancer, Vaginal Cancer, Uterus /
Endometrium Cancer, Vulva Cancer, Gestational Trophoblastic Cancer, Fallopian Tube cancer and Uterine sarcoma; Head and Neck Cancer - including Oral cavity Cancer, Lip Cancer, Salivary gland Cancer, Larynx Cancer, hypopharynx Cancer, oropharynx Cancer, Nasal Cancer, Paranasal Cancer and Nasopharynx Cancer; Leukaemia - including Childhood Leukaemia, Acute Lymphocytic Leukaemia, Acute Myeloid Leukaemia, Chronic Lymphocytic Leukaemia, Chronic Myeloid Leukaemia, Hairy Cell Leukaemia, BMT for Leukaemia, Patient's Home Pages, Acute Promyelocytic Leukemia and Plasma Cell Leukaemia; Haematological disorders - including Multiple myeoloma, Myelodysplastic Syndromes, Myeloproliferative Disorders, Aplastic Anaemia, Fanconi Anaemia and Waldenstrom's Macroglobulinemia; Lung cancer - including Small Cell Lung Cancer and Non-Small Cell Lung Cancer; Lymphoma - including Hodgkin's Disease, Non-Hodgkin's Lymphoma and Aids Related Lymphoma; Eye cancer - including Retinoblastoma and IntraOcular Melanoma; Skin cancer - including Melanoma, squamous cell carcinoma and basal cell carcinoma; and Soft tissue Cancer - including Soft tissue sarcoma and
Kaposi's sarcoma; Urinary system cancer - including Kidney Cancer, Wilm's Tumour, Bladder Cancer, Urethral Cancer and Transitional Cell Cancer;
wherein each of these cancers may or may not be metastatic.
35. A method for selecting a patient with cancer for treatment with a TIMP-1
inhibitor, said method comprising examining i) the cancer cell expression of CD74 and/or CD63 and ii) the level of TIMP-1 in a sample obtained from said cancer patient, wherein high or increased, or detectable, expression of TIMP-1 and detectable CD74 and/or CD63 is indicative of said patient with cancer being responsive to treatment.
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