WO2007014744A2 - Promedicament ctl - Google Patents

Promedicament ctl Download PDF

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Publication number
WO2007014744A2
WO2007014744A2 PCT/EP2006/007581 EP2006007581W WO2007014744A2 WO 2007014744 A2 WO2007014744 A2 WO 2007014744A2 EP 2006007581 W EP2006007581 W EP 2006007581W WO 2007014744 A2 WO2007014744 A2 WO 2007014744A2
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WO
WIPO (PCT)
Prior art keywords
region
polypeptide according
cd95l
polypeptide
cells
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PCT/EP2006/007581
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English (en)
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WO2007014744A3 (fr
Inventor
Klaus Pfizenmaier
Harald Wajant
Iris Watermann
Jeannette Gerspach
Dafne MÜLLER
Original Assignee
Universität Stuttgart
Julius Maximilians-Universität Würzburg
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Publication of WO2007014744A2 publication Critical patent/WO2007014744A2/fr
Publication of WO2007014744A3 publication Critical patent/WO2007014744A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70575—NGF/TNF-superfamily, e.g. CD70, CD95L, CD153, CD154
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6835—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site
    • A61K47/6849—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment the modifying agent being an antibody or an immunoglobulin bearing at least one antigen-binding site the antibody targeting a receptor, a cell surface antigen or a cell surface determinant
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to a polypeptide with preferably antitumoral and/or immune modulating cytokine properties which can be activated via in vivo processing comprising a C-terminal region having specific biological activity, wherein at the N-terminus of said C-terminal region an inhibitory region, a region having a processing site, and a region which is capable of selectively recognising a macromolecule on a cell surface or a component of the extracellular matrix is arranged.
  • TNF-family e.g. TNF or FasL (CD95L)
  • TNF recombinant ligands of the TNF-family
  • FasL CD95L
  • a treatment with TNF can only be carried out successfully using specific laborious treatment protocols (e.g. using the so-called "Isolated Limb Perfusion") for very limited indications (melanoma/sarcoma-metastases of the extremities). From these clinical data it can be estimated that about a 10-fold to 100-fold higher TNF dose as the MTD (Maximum Tolerated Dose) which is set up by massive systemic side effects would have to be used for achieving an antitumoral effect.
  • MTD Maximum Tolerated Dose
  • the problem underlying the present invention is to avoid or to minimise the unwanted effects of a treatment with therapeutically active polypeptide agents, like for example substances containing FasL or TNF while at the same time conserve or even enhance the therapeutically effective, e.g. anti-tu moral, properties of the active substance like e.g. FasL or TNF.
  • the present invention provides a polypeptide having an amino acid sequence comprising at least one region (1 ) having at least one processing site and further comprising, for example from N- to C-terminal,
  • region inhibiting the biological activity of region (4) by intramolecular or intermolecular binding and/or interaction
  • targeting-module selectively recognising a specific macromolecule on a cell surface and/or component of the extracellular matrix
  • region (4) wherein the biological activity of region (4) can be reconstituted by in vivo processing of the at least one processing site in at least one region (1 ). If region (1 ) is not processed the activity of region (4) stays inhibited and therefore the polypeptide is biologically, e.g. in the sense of the effect of the cytokine (for example the effect of FasL or TNF) inactive.
  • the position of region (1 ) in the polypeptide can be any position within the claimed polypeptide which does not have a significant negative effect on the functional properties of one of regions (2) to (4), and which is located N-terminal of region (3). This includes any position between regions (2) and (3), i.e. interregional, as well as any position within region (2), i.e. intraregional.
  • region and “module” are used as synonyms in the following.
  • fragment describes a shortened version of a molecule.
  • derivative and “mutant” refer to any derivatives and mutants, respectively, of a molecule which has essentially the same functional and/or biological property of said molecule.
  • nucleic acids, derivatives and mutants, respectively may contain deletions, additions and/or substitutions of bases the presence or absence or substitution of which has no essential impact on the activity of the respective transcription products, e.g. bases which are located in a transcriptionally inactive part of the nucleotide sequence. This also includes derivatives and mutants having a different nucleotide sequence but encoding the same amino acid sequence.
  • Deletions, additions and/or substitutions which lead to a minor enhancement or decrease of the transcription rate are also included.
  • amino acid sequences, derivatives and mutants, respectively may contain deletions, additions and/or substitutions of amino acids the absence, presence or substitution of which does not have a substantial effect on the activity of the polypeptide, e.g. conservative substitutions of amino acids, e.g. the substitution of amino acids by amino acids having similar chemical characteristics.
  • derivatives also includes any post-translation modifications, e.g. molecules having a different glycosylation pattern than the wild-type.
  • Polypeptides according to the present invention will be further also called "C- terminal TNF-family Ligand (CTL)-Prodrug". If the following refers to polypeptides in which region (4) is derived from a certain ligand of the TNF-family, e.g. FasL or TNF, they will be called after said region. If region (4) for example comprises a FasL derivative the polypeptide according to the present invention will be called FasL-Prodrug.
  • CTL C- terminal TNF-family Ligand
  • the CTL-Prodrugs according to the present invention are modular structure agents and according to an especially preferred embodiment homotrimer or homohexamer fusion proteins having for example one cytokine as an anti- tumoral substance in region (4) which selectively releases its biological effect in a pathologically altered tissue, e.g. a tumor area, through its linkage to the three further functional modules.
  • This is for example obtained by linking a therapeutically selective substance, e.g. the FasL molecule, to the C-terminus of a targeting module (3), e.g.
  • tumor specific antibodies or derivatives thereof like scFv-antibodies being specific for the target tissue, wherein the targeting module is N-terminally preceded through a processable region (1 ) by an inhibitor (2) against the therapeutically effective substance (4).
  • a processable region (1 ) may as well be contained in the inhibitor region (2).
  • the inhibitor is thus selectively - A - inactivated in the target tissue, e.g. the tumor area, preferably by removing it from the fusion protein by a selective proteolytic cleavage of the processing region (1 ). Accordingly, a bioactive agent, e.g. FasL, being bound to a selective targeting module, is generated.
  • a cytokine which may for example be used as a therapeutically selective substance is preferably a ligand of the TNF-family, more preferably FasL, TNF, TRAIL, CD40L, LIGHT or APRIL or a biologically effective derivative or a biologically effective mutant of said molecules.
  • the inhibitor (2) is a peptide inhibitor, preferably an extracellular domain of the receptor corresponding to region (4) or an antibody fragment (e.g. a scFV) with specifity of region (4).
  • the C-terminus of ligands of the TNF-family can be very important for the optimal activity of the molecules.
  • the activity of said cyotkines is very often reduced. This particularly applies to FasL.
  • the principle of the CTL-Prodrug has inter alia the advantage that in the construction of the prodrug the C-terminus of the component being responsible for the biological effect of the fusion protein is free. Therefore, after processing a cytokine derivative having a maximum of activity can be obtained.
  • the preparation of antibody-cytokine fusion proteins according to the prior art often leads to an aggregation of the fusion proteins.
  • FasL in the cytokine module the aggregation effects a constitutive activation of FasL resulting in a nonspecific, e.g. potentially systemic, activity of the respective fusion proteins and therefore may result in severe side effects.
  • the principle of the CTL- Prodrug inter alia has the advantage that also aggregating domains of antibodies can be used for the construction of prodrugs having a free C-terminus ligand.
  • the prodrug construction of the present invention high local effective concentration of the therapeutically effective substance, e.g. FasL, can be obtained without resulting in an elevated systemic level of the therapeutic agent (e.g. FasL in serum) and thus without resulting in side effects which limit the therapy.
  • the therapeutic agent e.g. FasL in serum
  • an effect corresponding to naturally occurring membrane- FasL is obtained by the presentation of FasL on cells being mediated by the targeting module (e.g. an antibody) after processing when using FasL as therapeutically effective region which means that an optimal activation of Fas is obtained.
  • soluble ligands and ligands being located on membranes of several members of the TNF-family, in particular FasL, TNF, TRAIL, CD40, is well known and may be dependent on the affinity of the different types of ligands to their receptors, the differences in the binding of receptor subtypes and/or the differences in the formation of receptor clusters in the membrane and therefore the activation of signal cascades.
  • a therapeutic agent being specifically adjusted and/or optimised for the respective tumor entity is obtained.
  • a local activation of a respective ligand and accordingly of the corresponding receptor in vivo is made available by providing a CTL-Prodrug having a free C-terminus which can be accumulated at the desired target by its antibody portion (region (3), e.g. an scFv) and subsequently activated via proteolysis.
  • the activation is obtained by cell associated proteases which are particularly found at the desired target.
  • the local activity is determined by the specificity of the antibodies used as well as by the restricted expression of the respective proteases at the target.
  • the polypeptide according to the present invention is based on a new prodrug technology and is a construct which is as a preferred embodiment a recombinant fusion protein having in principle a defined sequel of the following structural elements (as a monomer) (from N-terminal to C-terminal): (i) a specific region binding and inactivating protein or peptide, respectively, (ii) a variable linker peptide having a specific protease cleavage site, (iii) a murine, humanised or human single chain antibody fragment (scFv) having a defined antigen specificity, comprising a V H -linker-V L or a peptide having comparable characteristics, and (iv) a ligand of the TNF-family which e.g. corresponds to the wild-type ligand or the extracellular domain thereof or biologically active variants derived thereof.
  • the inhibitor module (2) is a receptor or, respectively, the ligand binding domain thereof for the cytokine part in module (4).
  • the inhibitor module preferably has at least one binding site for the therapeutically effective region (4).
  • the inhibitor module comprises the complete or partial extracellular domain of the human Fas receptor when using FasL in region (4).
  • Other proteins which specifically bind FasL e.g. fragments of Fas or Fas derived from other species or protein having a viral origin as well as synthetic peptides to derived thereof which has FasL-binding properties and interfere with the FasL-binding to the Fas-receptors are also suitable.
  • the cytokine receptor is the complete or partial extracellular domain of a receptor of the TNF-receptor family and/or a TNF ligand binding virus protein or a biologically active mutant of said domain or a synthetic TNF ligand binding compound.
  • the inhibitor module (2) comprises an antibody or a fragment thereof having a defined antigen specificity for the therapeutically effective region (4).
  • the inhibitor module comprises a single chain antibody fragment (scFv), more preferably a human scFv, having a defined antigen specificity for the therapeutically effective region (4) and the capability to block the biological activity of said region (4). Therefore, in an especially preferred embodiment of the present invention, the scFv possesses the capacity to neutralize and/or block the bioactivity of region (4), e.g. the scFv blocks FasL activity.
  • the fusion protein according to the present invention is biologically inactive in this state, i.e. being in its proform (prodrug).
  • the processing module (1 ) is for example protease sensitive (i.e. the processing site corresponds to a recognition site of a protease).
  • the processing site corresponds to a recognition site of a protease.
  • the processing module is further preferably made to contain at least one preferably more selective cleavage site(s) for extracellular or cell associated proteases which are preferably expressed in tumor tissue. Suitable cleavage sites are for example those of urokinase type plasminogen activator (uPA), tissue plasminogen activator (tPA), activated clotting factor Vila, matrix metalloproteases, like MMP-2 and MMP-9 and/or membrane bound FAP-protease which is selectively expressed in the stroma of tumors.
  • uPA urokinase type plasminogen activator
  • tPA tissue plasminogen activator
  • activated clotting factor Vila matrix metalloproteases, like MMP-2 and MMP-9 and/or membrane bound FAP-protease which is selectively expressed in the stroma of tumors.
  • protease sensitive cleavage sites are those of matrix metalloproteases which are associated with the process of metastasis and angiogenesis (e.g. MMP-9-recognition site Gly-Pro- Leu-Gly-Val-Arg-Gly-Lys), of heparanase, of enzymes which are preferably found in necrotic lesions, as well as of enzymes which are associated with prostate cancer (e.g. PSMA, PSA cleavable processing module Glutaryl-4-(hydrocypropyl)- Ala-Ser-cyclohexaglycyl-Gln-Ser-Leu-COOH).
  • matrix metalloproteases which are associated with the process of metastasis and angiogenesis
  • MMP-9-recognition site Gly-Pro- Leu-Gly-Val-Arg-Gly-Lys heparanase
  • enzymes which are preferably found in necrotic lesions as well as of enzymes which are associated
  • the structure of the linker is chosen so that the protease recognition sequence is freely accessible, i.e. effective processing by the specific protease is possible, and remaining amino acids of the linker(s) which eventually remain at the split off part which contains the therapeutically effective module after the cleavage of the fusion protein do not have any negative influence on the bioactivity of the therapeutically effective region.
  • the polypeptide according to the present invention additionally comprises a trimerization module.
  • the position of the trimerization module in the polypeptide can be any position within the claimed polypeptide which does not have a significant negative effect on the functional properties of one of regions (1 ) to (4). This also includes, for example, any position between the inhibitor module (2) and the processing module (1 ) and/or between the processing module (1) and the targeting module (3) and/or between the inhibitor module (2) and the targeting module (3).
  • the trimerization module is located directly N-terminal or C-terminal of the inhibitor module (2) of the fusion polypeptide of the present invention.
  • the trimerization module comprises a naturally occurring or synthetic peptide having intrinsic trimerization properties.
  • a particularly suitable example of such a peptide is a domain of the tenascin molecule (AA 110-139, Swissprot #P10039, (chicken), or Swissprot #P24821 (human)). It guarantees the covalent homotrimer linking of the fusion proteins during the biogenesis and may improve the binding of the inhibitor module (2) to the therapeutically effective module (4).
  • the ligands of the TNF- family and their cognate receptors have intrinsic trimerization properties.
  • An optional trimerization region accordingly provides a further stabilisation of the inactive state of the fusion protein. The particular importance of the stabilisation for the maintenance of the trimeric processed, i.e. activated form of the prodrug depends on the particular case.
  • the targeting module (3) preferably binds to a molecule on the cell surface which is expressed in tumor lesions and/or proliferating endothelial cells which are associated with the process of angiogenesis, and/or in reactive fibroblasts of the tumor stroma, and/or immune cells.
  • the targeting module (3) is specific for a component of the extracellular matrix which is present in tumor lesions and/or area of angiogenesis of pathological lesions.
  • the targeting module (3) is specific for a component of the malignant tumor cell itself.
  • region or module, respectively, (3) comprises an antibody (e.g. murine, humanised or human) or a fragment thereof, e.g.
  • a Fab-fragment or a typical single chain antibody fragment being prepared by methods known in the art which is of murine origin, humanised by using CDR-grafting origin or completely human origin having a specificity for e.g. an antigen which is selectively or dominantly, respectively, expressed in tumor tissue, wherein it can be expressed by the malignant cells itself or, preferably, in the non-malignant part of the tumor, the stroma cells or the tumor endothelium.
  • an antigen which is selectively or dominantly, respectively, expressed in tumor tissue wherein it can be expressed by the malignant cells itself or, preferably, in the non-malignant part of the tumor, the stroma cells or the tumor endothelium.
  • These antigens of non-malignant tissue parts of a solid tumor are on the one hand genetically invariant and on the other hand are present in various tumor entities and therefore are universal tumor markers.
  • antigens examples include the VEGFR-complex and the VEGFR/VEGF-complex, respectively, (being examples of receptor-ligand- complexes) as well as integrin ⁇ v ⁇ 3, endosialine and the fibronectin-isoform bFn as selective target structures of the tumor endothelium and the so-called Fibroblast Activation Protein (FAP) as a selective marker of a component of the extracellular matrix which serves as a target and which is present in the tumor stroma.
  • FAP Fibroblast Activation Protein
  • these antigens can for example be determined using a specific, highly affine scFv. Therefore, in a preferred embodiment of the present invention, the scFv comprising region (3) is SC40 or SC36.
  • Suitable targeting modules are peptides and protein domains, respectively, which e.g. naturally bind to a structure being typical for a tumor, tumor stroma, or tumor endothelium, respectively, but also artificial antibodies, and peptide aptamers.
  • the therapeutically effective module (4) preferably comprises an amino acid sequence of a cytokine or of a therapeutically effective fragment thereof.
  • region (4) comprises the amino acid sequence of a ligand of the TNF-family, preferably of FasL, particularly preferred of a protein being identical to a processed mature wild-type TNF-family ligand molecule or a derivative or a mutant derived therefrom having selective receptor binding properties or mutants and derivatives, respectively, which have been optimised regarding their specific bioactivity or other properties (stability, resistance to the proteases etc.), and most preferred of a protein being identical to a processed mature wild-type FasL molecule or a derivative or mutant derived therefrom having selective receptor binding properties or mutants or derivatives, respectively, which have been optimised regarding the specific bioactivity or other properties (stability, resistance to the proteases etc.).
  • the specific target molecule of region (4) is a cytokine receptor corresponding to the target molecule being expressed at a cell membrane.
  • the polypeptide according to the present invention may contain further regions. For example, marker sequences which allow an improved purification of recombinantly obtained proteins and the in vitro analysis may be added. By doing so, the polypeptide according to the present invention may for example have at the N-terminus and/or between two adjacent regions at least one detectable label.
  • a detectable label may be for example a tag, e.g. for purification, e.g. a flag tag or a myc tag.
  • the preferred FasL-Prodrug of the present invention is a non-covalently linked homotrimer molecule or if a respective trimerization region has been inserted, a covalently linked homotrimer molecule or in case of intermolecular interaction a homohexamer molecule consisting of monomers of the fusion of four functional regions, the tumor specific targeting module (3) (scFv or other binder), the FasL- module (4) and the blocking FasL binding protein (extracellular receptor domain or a peptide derived thereof or a FasL specific scFv)-module (2) as well as a specific protease cleavage site module (1 ) preferably between module (2) and module (3) which is in its complete state inactive concerning a FasL effect, as described above.
  • the tumor specific targeting module (3) scFv or other binder
  • FasL- module (4) the blocking FasL binding protein (extracellular receptor domain or a peptide derived thereof or a FasL specific scFv
  • the FasL-Prodrug is specifically enriched in the tumor area due to its targeting module (region (3)) and is further processed by the tumor itself or by the proteases formed by the reactive tumor stroma / tumor vascular system (e.g. FAP, uPA, tPA, MMP2, factor Vila), i.e. the inhibiting module/peptide (2) is cleaved off.
  • the FasL receptor fragment/inhibitor peptide dissociates from the FasL-module (4) the latter thus becomes bioactive (i.e. the biological activity of the region is released by the processing of the processing site in region (1 )).
  • Selectivity of the FasL effect is therefore achieved with the FasL prodrug according to the present invention by two factors: On the one hand by the selective enrichment of the inactive product in the tumor being mediated by region (3) and the retention of the biologically active fragment consisting of region (3) and region (4) also after the proteolytic activation and on the other hand by the locally specific conversion of the prodrugs by proteases which exclusively or preferably are detectable with a significant activity in the tumor area.
  • the CTL-Prodrug has the amino acid sequence SEQ ID No. 1 which shows an especially preferred amino acid sequence of CD95-TNC-PL-SC40-F-CD95L.
  • the CTL-Prodrug has the amino acid sequence SEQ ID No. 3 which shows an especially preferred amino acid sequence of L-CD95-TNC-PL-SC36-F-CD95L.
  • nucleic acid comprising a nucleotide sequence which encodes the polypeptide according to the present invention.
  • the term , nucleic acid means native, half-synthetic, synthetic or modified nucleic acid molecule made of deoxyribonucleotides and/or ribonucleotides and/or modified nucleotides.
  • the nucleic acid has the nucleotide sequence SEQ ID No.
  • CD95-TNC- PL-SC40-F-CD95L comprising a signalling peptide sequence: NT 1-57, a sequence of the human extracellular domain of CD95 (AA 15-173): NT 64-540, a trimerization domain of chicken tenascin: NT 553-660, a sequence of the MMP2- specific cleavage-sites PL2: NT 679-768, a sequence of the scFv-antibody- fragment SC40: NT 781-1524, a sequence of the Flag-tag: NT 1540-1563, and a sequence of the human extracellular domain of CD95L: NT 1576-2004.
  • the nucleic acid has the nucleotide sequence SEQ ID No. 4 which shows an especially preferred nucleotide sequence coding for CD95-TNC-PL-SC36-F-CD95L comprising a signalling peptide sequence: NT 1-57, a sequence of the human extracellular domain of CD95 (AA 15-173): NT 64-540, a trimerization domain of chicken tenascin: NT 553-660, a sequence of the MMP2-specific cleavage-sites PL2: NT 679-768, a sequence of the scFv-antibody-fragment SC36: NT 781-1560, a sequence of the Flag-tag: NT 1570-1593 and a sequence of the human extracellular domain of CD95L: NT 1606-2034.
  • SEQ ID No. 4 shows an especially preferred nucleotide sequence coding for CD95-TNC-PL-SC36-F-CD95L comprising a signalling peptide sequence: NT 1-57,
  • the present invention provides a vector comprising the above defined nucleic acid.
  • the vector according to the present invention is not limited and suitable vectors are known in the prior art.
  • the vector can be expressed and/or amplified in any prokaryotic or eukaryotic cell. Additionally, the vector preferably comprises suitable regulatory elements like promoters, enhancers, terminators etc. The vector further may be used for stable integration of the nucleic acid according to the present invention into genetic material of a host cell.
  • the present invention further provides a host cell containing the above-mentioned nucleic acid and/or the above-mentioned vector.
  • Suitable host cells are for example all mammalian cells, particularly cultivated cells like e.g. COS- or CHO- cells.
  • the present invention further provides a method for the production of the polypeptide according to the present invention comprising the steps of
  • the polypeptide according to the present invention is preferably obtained by expression using suitable expression systems, preferably as a secreted product of stable transfectants of the cell line CHO DG44 or after transient expression in COS7-cells.
  • suitable expression systems e.g. Pichia pastoris, insect cells or mammalian cells as well as the expression vectors suitable for the secretion in the respective cell system, are described e.g. in Brocks et al. (Immunotechnology 1 :173-184. 1997) for mammalian cells and insect cells.
  • pPICZalpha-vectors Immunotechnology 1 :173-184. 1997) for mammalian cells and insect cells.
  • the polypeptide according to the present invention, the nucleic acid and/or the vector can be used advantageously for the production of pharmaceutical compositions for treating disorders like for example tumor disorders in an individual/patient.
  • a further embodiment of the present invention is a pharmaceutical composition comprising in an pharmaceutically effective amount the polypeptide according to the present invention and/or the nucleic acid according to the present invention and/or the vector according to the present invention, eventually along with one or more pharmaceutically acceptable adjuvants, diluants and/or carriers.
  • the pharmaceutical composition can preferably be used for therapeutic treatment of cancer disorders and/or infection disorders and/or metabolic diseases.
  • Particularly preferred applications of the pharmaceutical composition according to the present invention are the treatment of solid tumors as well as of angiogenesis in pathological lesions.
  • the pharmaceutical composition according to the present invention can be in any form known in the prior art. In a preferred embodiment it is solid, liquid or an aerosol.
  • the present invention comprises a method of treatment which contains the administration of a therapeutically effective amount of the pharmaceutical composition according to the present invention to a patient in need of a respective treatment.
  • Suitable ways of administration of the pharmaceutical composition are known to the persons skilled in the art and comprise for example oral, intravenous, intraarterial, intramuscular, nasal, subcutaneous, intratumoral, rectal and topic applications.
  • An intravenous application may be for example carried out in form of a bolus injection with subsequent intersection intervals and/or in form of an infusion.
  • human as well as animals patients may be treated.
  • the method of treatment is preferably applied to patients with one of the above disorders.
  • the figures show:
  • Fig. 1 shows a scheme of the CD95L fusion proteins used in this study.
  • L leader peptide
  • SC40 human FAP-specific single chain antibody fragment "40”
  • SC36 human and mouse FAP-specific single chain antibody fragment "36”
  • F Flag tag
  • CD95L human CD95L (aa 139-281)
  • CD95 human CD95 (aa 15-173)
  • TNC chicken tenascin aa (110-139)
  • PL MMP2-sensitive linker
  • scFv ⁇ CD95L CD95L specific scFv antibody fragment.
  • Fig. 2 shows the cytotoxic activity of anti-Flag M2 mAb crosslinked CD95L fusion proteins.
  • HT1080 squares
  • HT1080FAP cells circles
  • cells were treated with 1 ⁇ g/ml cycloheximide and challenged in duplicates or triplicates with the indicated concentrations of the various CD95L fusion proteins in the presence (filled symbols) or absence (open symbols) of 1 ⁇ g/ml of the crosslinking anti-Flag mAb M2. After additional 16 h cell viability was determined by crystal violet staining.
  • Fig. 3 shows the FAP binding of CD95L fusion proteins.
  • HT1080 and HT1080FAP cells were incubated with the indicated fusion proteins on ice and after repeated washing, bound proteins were detected by FACS using anti-Flag mAb M2 and FITC-labelled anti mouse IgG.
  • Fig. 4 shows the FAP-dependent stimulation of non-apoptotic CD95L signalling by SC40-CD95L-PD.
  • HT1080 and HT1080FAP cells were seeded in 96-well plates. Next day, medium was replaced by culture medium containing zVAD (20 ⁇ M) and cells were stimulated in triplicates with the indicated concentrations of the prodrug, M2-crosslinked prodrug or M2-crosslinked sCD95L. After 4 h supernatants were removed and their relative IL8 content was determined by ELISA.
  • Fig. 5 shows that SC40-CD95L-PD induced apoptosis occurs via CD95 and is dependent on FAP expression and MMP activity.
  • A left panel, HT1080 and HT1080FAP cells were incubated for 16 h with SC40-CD95L-PD in the presence and absence of the MMP specific inhibitor llomastat (25 ⁇ M) and supernatants were analysed by anti-Flag Western Blot; right panel, SC40- CD95L-PD (12 ⁇ g/ml) was incubated with rec.
  • HT1080FAP cells were seeded and next day challenged with CD95L-PD (15 ng/ml) in duplicates. Where indicated cells were pretreated with the CD95L neutralizing mAb NOK-1 (1 ⁇ g/ml, left panel), with the FAP blocking MB36 (10 ⁇ g/ml, middle panel) or with the inhibitor llomastat (ILO, 25 ⁇ M, right panel).
  • Fig. 6 shows the characterization of SC40-CD95L-PD.
  • A SC40-CD95L-PD was separated under reducing and non-reducing conditions by SDS-PAGE and visualized by Western Blot with anti-Flag antibody.
  • B SC40-CD95L-PD was treated with the indicated concentrations of the chemical crosslinker BS 3 and analyzed under reducing conditions by Western Blot.
  • C SC40- CD95L-PD and the High Molecular Weight Marker for Native Electrophoresis (GE Healthcare, Uppsala, Sweden) were resolved by native polyacrylamide gel electrophoresis. Proteins were finally visualized by Western Blot analysis.
  • D Proposed organization of SC40-CD95L-PD hexamers.
  • Fig. 7 shows that SC36-CD95L-PD induced apoptosis occurs via CD95 and is dependent on FAP expression and MMP activity.
  • A Triplicates of HT1080 and HT1080FAP cells were incubated for 16 h in the presence of CHX (1 ⁇ g/ml) with the indicated concentrations of SC36-CD95L and SC36-CD95L- PD. Cell viability finally was determined by crystal violet staining.
  • B HT1080 and HT1080FAP cells were incubated for 16 h with SC36-CD95L- PD in the presence and absence of the MMP specific inhibitor llomastat (25 ⁇ M) and supernatants then were analysed by anti-Flag Western Blot.
  • HT1080FAP cells were seeded and challenged the next day with SC36- CD95L-PD (5 ng/ml) in presence or absence of the CD95L neutralizing mAb NOK-1 (1 ⁇ g/ml, left panel), the FAP neutralizing scFv SC36 (50 ⁇ g/ml, middle panel) or the MMP inhibitor llomastat (25 ⁇ M, right panel). After additional 16 h cell viability was determined by crystal violet staining.
  • Fig. 8 shows the analyses of systemic toxicity and antitumoral activity of the CD95L prodrug.
  • C57BL/6xCBA/J mice received a single i.v. injection of 50 ⁇ g SC36-CD95L or 80 ⁇ g SC36-CD95L-PD or as positive control 10 ⁇ g sCD95L followed by 10 ⁇ g M2 for secondary crosslinking. Liver tissues were removed either directly after dead or 6 h after injection and analysed for caspase 3 activity using the fluorigenic substrate Ac-DEVD-AMC.
  • B 1 ,5x10 6 parental and FAP expressing HT1080 cells were injected s.c. into the back of NMRI nu/nu mice and treatment was started 1 day later.
  • mice received 4 consecutive s.c. injections with 30 ⁇ g SC36-CD95L-PD close to the site of tumor cell transplantation. On day 1 after tumor cell injection mice were treated twice, followed by daily single treatment on day 2 and 3. Tumor growth was monitored at the time points indicated. Target antigen expressing tumor cells showed reduced tumor growth as compared to contrail HT1080 cells.
  • Fig. 9 shows a model of FAP-dependent and MMP-mediated activation of the FAP specific CD95L prodrug.
  • the FAP specific CD95L prodrug stays inactive on antigen negative cells.
  • the scFv module of the prodrug traps this molecule on the cell surface.
  • the still inactive CD95L prodrug now can be processed by cell surface associated MMPs leading to the release of the trimeric inhibitory CD95 domain.
  • the unmasked, membrane bound CD95L now mimicks membrane CD95L exerting high signal capacity towards CD95 expressing cells.
  • Fig. 10 shows a scheme of the native structure of soluble FasL (1) and FasL- Prodrug fusion proteins having a free FasL-C-terminus in a trimer arrangement (2) (intra-molecular interaction of Fas and the FasL module).
  • Fiq. 11 shows the encoding DNA-sequence (upper) and amino acid sequence (one letter code, lower) of the polypeptide having the module structure L- CD95-TNC-PL-SC40-F-CD95L according to the present invention.
  • Fig. 12 shows the amino acid sequence of the polypeptide having the module structure L-CD95-TNC-PL-SC40-F-CD95L (SEQ ID No. 1) according to the present invention.
  • Fig. 13 shows the DNA-sequence of the polypeptide having the module structure L-CD95-TNC-PL-SC40-F-CD95L (SEQ ID No. 2) according to the present invention.
  • Fig. 14 shows the amino acid sequence of the polypeptide having the module structure L-CD95-TNC-PL-SC36-F-CD95L (SEQ ID No. 3) according to the present invention.
  • Fig. 15 shows the DNA-sequence of the polypeptide having the module structure L-CD95-TNC-PL-SC36-F-CD95L (SEQ ID No. 4) according to the present invention.
  • Example 1 Material and Methods
  • CD95 is used as a synonym of "Fas” and “CD95L” is used as a synonym of "FasL”.
  • HEK293 cells were obtained from the American Type Culture Collection (Manassas, VA). Cells were cultured in RPMI 1640 medium (Biochrom, Berlin, Germany) supplemented with 5% fetal calf serum (FCS). HT1080FAP cells were grown in the presence of 250 ⁇ g/ml G418. Stably transfected cells expressing the fusion proteins were cultivated in the presence of 5 ⁇ g/ml puromycin.
  • the various CD95L fusion proteins were produced in HEK293 cells after transient transfection of the corresponding expression plasmids using lipofectamine 2000 (Invitrogen, Düsseldorf, Germany). After 3 days of cell culture in serum free medium (Optimem, Invitrogen, Düsseldorf, Germany) supernatants were collected.
  • HEK293 were stably transfected with the plRESpuro3 derived expression constructs by selection with puromycin. For protein production stable clones were expanded and grown to approximately 75 % confluence in FCS supplemented medium. FCS-containing medium was then removed and serum free Optimem medium was added.
  • HT1080 and HT1080FAP cells were incubated for 2 h at 4 0 C with 5 ug/ml of the indicated CD95L fusion protein. After washing three times with PBS, 0,2 % FCS, 0,02% sodium azide, bound fusion proteins were detected by anti-Flag mAb M2 (1 ⁇ g/ml Sigma, Steinheim, Germany) and fluoresceine isothiocyanate labelled rabbit anti-mouse IgG Ab (1 ⁇ g/ml Sigma, Steinheim, Germany). Analyses were performed using EPICS-XL (Coulter, Krefeld, Germany) according to standard procedures.
  • the indicated CD95L fusion protein (200 ng) was incubated with varying concentrations of the chemical crosslinker BS 3 (Pierce, Rockford, USA) for 30 min on ice in a total volume of 18 ⁇ l.
  • the crosslinking reaction was quenched by adding 2 ⁇ l 1 M Tris-HCI pH 7.5 and proteins were finally analysed by SDS-PAGE and Western Blot.
  • the purified proteins (12 ug/ml) were incubated with 2 ⁇ g/ml recombinant MMP-2 (Merck Bioscience GmbH, Schwalbach, Germany) in reaction buffer (20 ⁇ M ZnSO 4 , 1 mM CaCI 2 , 0,05 % Brij-35, 50 mM Tris-HCI, pH 7.5) for 6 h at 37°C.
  • Prodrug processing and activity were finally analyzed by SDS-PAGE, Western Blot and viability assay.
  • HT1080 and HT1080FAP cells (2x10 4 /well) were grown over night in 100 ⁇ l culture medium in 96-well plates.
  • HT1080 and HT1080FAP cells (2x10 4 /well) were grown over night in 100 ⁇ l culture medium in 96-well plates. The following day, cells were challenged in duplicates or triplicates with the indicated concentrations of the various CD95L fusion proteins in the presence or absence of 1 ⁇ g/ml Flag-specific mAb M2 and after 16 h cell viability was determined by crystal violet staining. Assays were performed in presence of 1 ⁇ g/ml cycloheximide to sensitize cells for apoptosis induction.
  • CD95L fusion proteins were incubated with 1 ⁇ g/ml anti-CD95L NOK-1 (BD Biosciences, Heidelberg, Germany) prior stimulation. FAP-dependency of prodrug activation was shown by preincubation of the cells with 10 ⁇ g/ml of the recombinant anti- FAP minibody MB36 or 50 ⁇ g/ml of the corresponding recombinant scFv SC36.
  • HT1080 and HT1080FAP cells (1.5 x 10 4 /well) were seeded in 96-well tissue culture plates and grown overnight. Next day, the medium was replaced by medium containing 20 ⁇ M z-VAD to avoid apoptosis induction and cells were stimulated in triplicates with the indicated concentrations of the CD95L prodrug. After 4 h supematants were removed and analyzed using an IL8 ELISA kit (BD Biosciences, Heidelberg, Germany) according to the manufacturer's instructions.
  • Subcutaneous tumor growth of parental HT1080 and FAP transfectants was assessed by subcutaneous injection of 1 ,5x10 6 cells suspended in 100 ⁇ l of 0,9 % NaCI solution into the back of narcotized (Isofluran) NMRI nu/nu mice. Mice were treated on day 1 after tumor cell injection twice with 30 ⁇ g SC36-CD95L-PD, followed by a daily single treatment on day 2 and 3 in the area of tumor cell application. Tumor growth was monitored. Tumor bearing mice were sacrificed when tumor size exceeded 250 mm 2 .
  • mice were treated with the indicated doses of the reagents by intravenous application. Survival and phenotypical signs of sickness were monitored for 6 h before mice were sacrificed for determination of caspase-3 activity in liver tissues.
  • CD95L fusion protein lacking the inhibitory CD95 modul and possessing only the protease sensitive linker.
  • This fusion protein again showed no significant apoptosis induction upon secondary crosslinking on HT1080 cells (Fig. 2D).
  • CD95L prodrug in which the CD95L carboxy-terminus was preserved.
  • CD95-PL- SC40-CD95L the inhibitory extracellular domain of CD95 preceded the single chain-CD95L domain.
  • CD95-PL-SC40-CD95L was comparatively active as SC40- CD95L on FAP-expressing HT1080 cells, but unexpectedly was also highly active on FAP-negative parental HT1080 cells after secondary crosslinking (Fig. 2E). The latter observation indicated that the CD95L module of the CD95-PL-SC40-CD95L fusion protein was not or only inefficiently blocked by the inhibitory CD95 domain of the molecule.
  • trimerization domain of tenascin-C (TNC 1 ref. 11 ) between the inhibitory CD95 domain and the SC40-CD95L modules.
  • the trimerization domain of TNC was immediately placed after the CD95 domain (CD95-TNC-PL-SC40-CD95L) and in a second variant the TNC trimerization domain was placed between the protease sensitive linker and the scFv domain (CD95-PL-TNC-SC40-CD95L), (Fig. 1 ).
  • Both CD95L prodrugs were almost as active as SC40-CD95L or secondarily crosslinked CD95L on FAP- expressing cells (Fig.
  • CD95-TNC-PL-SC40-CD95L prodrug showed less background activity after crosslinking (Fig. 2) and better specific binding to the FAP antigen (Fig. 3) than the CD95-PL-TNC-SC40-CD95L variant, we decided to concentrate subsequent analyses on the CD95-TNC-PL-SC40-CD95L prodrug.
  • this prodrug will be designated in the following SC40-CD95L-PD.
  • SC40-CD95L- PD also induced, in a FAP-dependent manner, non-apoptotic CD95L signaling (IL8 induction) when apoptosis induction was blocked by caspase inhibitors (Fig. 4).
  • SC40-CD95L-PD was detectable in the supernatant of HT1080FAP cells, but not in the supernatant of HT1080 cells (Fig. 5A, left panel).
  • MMPs in FAP-dependent processing of SC40-CD95L-PD
  • complete processing was achieved by prodrug incubation with recombinant MMP2 (Fig. 5A, right panel).
  • Example 7 A SC40-CD95L-PD variant in which the MMP2-sensitive linker has been replaced by an uPA-sensitive linker
  • HT1080 cells also express the tumor associated protease urokinase-type plasminogen activator (uPA).
  • uPA tumor associated protease urokinase-type plasminogen activator
  • trimeric scFv-CD95L fusion proteins stimulate CD95 up to 1000 fold less efficient than their corresponding secondarily aggregated counterparts, but the fusion proteins can be converted to membrane CD95L-like, highly active CD95 stimulating entities by binding to a cell surface antigen. In principle, this allows antigen restricted and therefore local activation of CD95 in vivo.
  • this approach does not work with scFv molecules displaying an intrinsic aggregation tendency as the latter leads to secondary aggregation thereby nonspecifically activating the CD95L module within the corresponding fusion protein.
  • the CD95L prodrug concept outlined above could therefore provide a mean also allowing the use of such scFv molecules for safe targeting of CD95.
  • a CD95L prodrug in which the scFv SC40, displaying no tendency to autoaggregate, has been replaced by a scFv (SC36) of the same specificity (FAP), but with known autoaggregation.
  • SC36-CD95L fusion protein displayed a high activity to stimulate CD95 also on FAP-negative cells (Fig. 7A, left panel).
  • the corresponding SC36-derived CD95L prodrug showed no activity on FAP-negative cells, but strongly activated CD95 in a FAP dependent manner on HT1080FAP cells (Fig. 7A, right panel).
  • processed SC36-CD95L prodrug could only be detected in supernatants of FAP expressing, but not parental, antigen negative HT1080 cells (Fig. 7B).
  • the MMP inhibitor llomastat blocked cleavage of the SC36-CD95L of HT1080FAP cells, confirming specific action of MMPs (Fig. 7B).
  • the SC36-CD95L prodrug induced apoptosis on FAP expressing HT1080 cells via CD95 in a FAP and MMP dependent manner, evident from blocking of cell death by CD95L neutralizing NOK-1 Ab, FAP-neutralizing scFv SC36, and MMP inhibitor llomastat, respectively (Fig. 7C).
  • the prodrug design described above allows to use scFvs as targeting module which exert an intrinsic tendency to autoaggregate.
  • SC36- CD95L and the corresponding prodrug were applied in equivalent molarities.
  • a second intravenous injection was given within 30 min after the injection of CD95L containing a FLAG epitope allowing secondary cross-linking with the FLAG-specific mAb M2. +Mice died within 1 h; #mice died within 3.5 h.
  • the unmasked CD95L module stays membrane bound and now mimicks membrane CD95L, exerting a high signal capacity towards CD95 expressing cells in an autotropic and juxtatropic signaling mode.
  • tumor selective activation of the prodrug is encompassed by tumor associated proteases, we envisage that this principle can be applied to any cell surface molecule expressed by the tumor cells themselves, the tumor vasculature or stroma, as exemplified here using FAP as a model antigen.
  • the animal studies provide first evidence for a target dependent processing and functional, tumor restricted activation of the CD95L prodrug in vivo, warranting further studies on the systemic applicability, safety and antitumor efficacy of CD95L prodrugs.

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Abstract

La présente invention concerne un polypeptide présentant de préférence des propriétés de cytokines antitumorales et/ou immunomodulatrices et pouvant être activé par maturation in vivo, ce polypeptide comprenant une région C-terminale possédant une activité biologique spécifique. Au niveau de la terminaison N de cette région C-terminale se trouvent une région inhibitrice, une région pourvue d'un site de maturation et une région capable de reconnaître sélectivement une macromolécule sur une surface cellulaire ou un constituant de la matrice extracellulaire.
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