DE102005036542A1 - CTL prodrug - Google Patents

Abstract

The The present invention relates to a polypeptide having preferably antitumoral and / or immunomodulatory cytokine properties by Processing in vivo is activatable comprising a C-terminal Region of specific biological activity at the N-terminal end an inhibitory region, a region with a processing site and a region containing a macromolecule on a cell surface or a component of the extracellular Matrix selectively recognizes, is arranged.

Description

The The present invention relates to a polypeptide having preferably antitumoral and / or immunomodulatory cytokine properties by Processing in vivo is activatable comprising a C-terminal Region of specific biological activity at the N-terminal end an inhibitory region, a region with a processing site and a region containing a macromolecule on a cell surface or a component of the extracellular Matrix selectively recognizes, is arranged.

Of the Use of recombinant ligands of the TNF family, e.g. TNF or FasL (CD95L), for the treatment of, for example, tumors So far, due to strong systemic side effects that as Therapy limiting are to be regarded, strongly limited. So is e.g. a treatment with TNF only under special, elaborate Treatment protocols (e.g., by so-called "Isolated Limb Perfusion") in very limited Indications (melanoma / sarcoma metastases of the extremities) with success feasible. From these clinical data one can estimate that about a 10 to 100-fold higher TNF dose as the MTD (Engl Tolerated can ") would be required for anti-tumoral efficacy, as it is the massive systemic Side effects would allow. Treatment concepts based on the activation of Fas, the receptor FasL, are so far no attempts have been made since a systemic Activation of this receptor very rapidly induces apoptosis in hepatocytes and thus to acute, in the experimental animal fatal liver failure leads.

Of the The present invention is therefore based on the object, the undesirable Consequences of treatment with therapeutically active polypeptide active ingredients, such as. FasL or TNF-containing substances to avoid or to diminish while at the same time the therapeutically effective, for example antitumoral properties the active substance, such as FasL or TNF, or even enhanced.

These The object is achieved by the embodiments characterized in the claims solved the present invention.

• (1) eine Region („Inhibitor"), welche die biologische Aktivität der Region (4) durch intramolekulare oder intermolekulare Bindung und/oder Wechselwirkung hemmt,
• (2) eine Region, die mindestens eine Prozessierungsstelle aufweist,
• (3) eine Region(„Targeting-Modul"), die ein spezifisches Makromolekül auf einer Zelloberfläche und/oder eine Komponente der extrazellulären Matrix selektiv erkennt, und
• (4) eine Region („therapeutisch wirksame Region oder Substanz") mit einer biologischen Aktivität für ein spezifisches Zielmolekül,

wobei die biologische Aktivität der Region (4) durch Prozessierung in vivo der mindestens einen Prozessierungsstelle in Region (2) freisetzbar ist. Ohne Prozessierung der Region (2) bleibt die Aktivität der Region (4) inhibiert und das Polypeptid daher biologisch, z.B. im Sinne der Zytokinwirkung (beispielsweise der FasL oder TNF-Wirkung), inaktiv.In particular, the invention provides a polypeptide having an amino acid sequence comprising N- to C-terminal

• (1) a region ("inhibitor") which inhibits the biological activity of the region (4) by intramolecular or intermolecular bonding and / or interaction,
• (2) a region having at least one processing station,
• (3) a region ("targeting module") that selectively recognizes a specific macromolecule on a cell surface and / or a component of the extracellular matrix, and
• (4) a region ("therapeutically effective region or substance") having a biological activity for a specific target molecule,

wherein the biological activity of the region (4) is releasable by processing in vivo the at least one processing site in region (2). Without processing of the region (2), the activity of the region (4) remains inhibited and the polypeptide is therefore biologically inactive, eg in the sense of cytokine action (for example FasL or TNF action).

The Terms "region" and "module" are hereafter used synonymously. The term "fragment" refers to a truncated form of a molecule. The terms "derivative" and "mutant" refer to each Derivative, or each mutant of a molecule, which is essentially the have the same functional and / or biological properties of the molecule. In Terms of nucleic acids can Derivatives or mutants deletions, additions and / or substitutions of bases, their absence, presence or substitution no significant influence the activity of the gene product has, for example, bases that are transcriptional inactive part of a nucleotide sequence. This also includes Derivatives and mutants, which at different nucleotide sequence the same amino acid sequence encode. Deletions, additions and / or substitutions that belong to a slight increase or reduction in transcription rate are also included. In terms of amino acid sequences can Derivatives or mutants deletions, additions and / or substitutions of amino acids whose absence, presence or substitution does not exist significant influence the activity of the polypeptide, for example conservative substitutions of amino acids, i.e. Replacing amino acids by amino acids with similar ones chemical properties. The term "derivative" also includes post-translational Modifications, such as different from the wild type Glycosylation pattern, a.

Polypeptides of the invention will also be referred to hereinafter generally as the "C-terminal TNF family ligand (CTL) prodrug". If in the following polypeptides according to the invention are meant in which the region (4) is derived from a particular ligand of the TNF family, for example the FasL or TNF, these are named corresponding to this region. Is the region (4) eg from a FasL derivative so the polypeptide of the invention is referred to as FasL prodrug. The CTL prodrugs according to the invention are modular active compounds and, according to a particularly preferred embodiment, a preferably homotrimeric or homohexameric fusion protein with, for example, a cytokine as antitumoral active substance in region (4), which specifically targets its biological activity in the diseased tissue by linking with three further functional modules, eg a tumor area, releases. This is achieved by genetically linking the therapeutically active substance, for example the FasL molecule, to the C terminus of a targeting tissue-specific targeting module (3), eg tumor-specific antibodies or derivatives thereof, such as scFv antibodies, N-terminally preceded by a processable region (2) an inhibitor (1) against the therapeutically active substance (4) which is selectively inactivated in the target tissue, such as the tumor area, by processing the region (2), preferably by targeted proteolytic cleavage from the Fusion protein is removed, and so bound to the selective targeting module, bioactive agent, such as FasL arises.

The For example, as a therapeutically effective substance usable cytokine is preferably a ligand of the TNF family, more preferably FasL, TNF, TRAIL, CD40L, LIGHT or APRIL or a biologically active Derivative or a biologically active mutant of these molecules. In a preferred embodiment In the present invention, the inhibitor (1) is a peptide inhibitor, preferably an extracellular domain of the receptor corresponding to the region (4). The C-terminus of ligands the TNF family can be used for the optimal activity of the molecule be very important. After the fusion of peptides to the C-terminus is the activity Therefore, these cytokines are often very severely restricted. This is especially true for the FasL. The CTL prodrug principle has therefore u.a. the advantage that in the construction the prodrug the C-terminus of the actual biological active component remains free of the fusion protein and so after processing a cytokine derivative with maximum activity can arise.

Lots Aggregate antibody domains spontaneous. The preparation of antibody-cytokine fusion proteins according to the state the technology leads therefore typically common also for the aggregation of the fusion protein. When using FasL In the cytokine module, this aggregation causes constitutive activation FasL, which is an undirected, i. potentially systemic activity of the subject Fusion protein and thus serious side effects. The CTL prodrug principle therefore has u.a. the advantage that to Construction of prodrugs with free ligand C-terminus also used aggregating antibody domains can be.

With the prodrug structure according to the invention is it possible locally high active concentrations of the therapeutically active substance, e.g. FasL, without causing systemically elevated therapeutic levels (for example FasL in serum) and thus therapy-limiting side effects comes. In particular, in the case of FasL, for example, it is considered therapeutic effective region after being processed by the targeting module (e.g., antibody) mediated cellular presentation the FasL achieves an effect similar to that of the natural membrane FasL, i.e. Optimal activation of Fas occurs. The differential biological effect more soluble and membrane-bound Ligands of some members of the TNF family, v.a. FasL, TNF, TRAIL, CD40, is known and hangs with the affinity of different forms of ligands to their receptors, the different ones Binding to receptor subtypes and / or different ability for receptor clustering in the membrane and thus activation the signal processes together.

By Selection of specificity of the targeting module (3) and the processing region (2) can be to the respective tumor entity specifically tailored / optimized therapeutic agent are produced. By preparing a CTL prodrug with free C-terminus, the in the desired Destination by an antibody portion enriched and there is activated by proteolysis, becomes a local activation the corresponding ligand and thus the corresponding to this Receptor in vivo allows. The activation takes place by cell-associated proteases, the especially at the desired Destination occur. The local activity is then on the one hand the specificity of the antibody used conditionally, but also by the preferred expression corresponding Proteases at the destination.

The (Amino acid sequence) regions or modules of the polypeptide according to the invention are described in detail below with respect to preferred embodiments.

The polypeptide of the invention is based on a novel prodrug technology and is a construct which, according to a preferred embodiment, is a recombinant fusion protein which comprises in principle a defined sequence of the following structural elements (in the monomer) (N-terminal to C-terminal): (1 (2) a variable linker peptide having specific protease cleavage sites, (3) a murine, humanized or human single chain antibody fragment (scFv) of defined anti-gageneity, consisting of: (3) a protein or peptide specifically binding to and inactivating the region (4) V _H -linker V _L or a peptide with comparable property, and (4) a ligand of the TNF family which corresponds, for example, to the wild-type ligand or its extracellular domain or biologically active variants derived therefrom.

The Inhibitor module (1) is in accordance with a preferred embodiment of the polypeptide of the invention a receptor or its ligand binding domain for the cytokine part in the module (4). The inhibitor module preferably has at least one binding site for the therapeutically effective region (4). In a preferred embodiment of the present invention the inhibitor module in case of using FasL in region (1) the complete or partial extracellular domain a human Fas receptor. Other, specifically FasL binding proteins, for example Fas fragments or Fas of other species origin or Proteins of viral origin and synthetic derived therefrom Peptides, the FasL binding property and interfere with FasL binding to Fas receptors, are also suitable. In a further preferred embodiment In the present invention, the cytokine receptor is the full or partial extracellular domain a receptor of the TNF receptor family and / or a TNF ligand-binding Virus protein or a biologically active mutant of this domain or a synthetic TNF ligand-binding compound. Because of the bond or interaction of the inhibitor with the therapeutically effective Module is the fusion protein according to the invention biologically inactive in this state, i. it is in the proform (Prodrug).

The Processing module (2) is, for example, protease-sensitive (i.e. the processing station corresponds to the recognition sequence of a Protease) and preferably in amino acid composition and total length so get that one high-affinity, stable binding of the N-terminal module in the molecule (1), e.g. a FasL inhibitor (e.g., the extracellular Fas receptor domain) to the Region (4), e.g. FasL allows, so that thereby the binding of the Ligand portion of the module (4) to the corresponding to this ligand, cell-expressed receptors in vivo is prevented. The processing module ("Linker") is still preferably before in such a way that he at least one, preferably a plurality of selective cleavage sites for such extracellular or cell-associated proteases, which are preferably selective be detected in tumor tissue. Examples of suitable cleavage sites are the one for Urokinase-type plasminogen activator (uPA), tissue plasminogen activator (tPA), the activated coagulation factor VIIa, matrix metalloproteases, like MMP-2 and MMP-9, and for the highly selectively in the stroma of tumors membrane-bound expressed FAP protease. Particularly preferred protease-sensitive cleavage sites are those of the process of metastasis and angiogenesis standing matrix metalloproteases (e.g., MMP-9 recognition sequence Gly-Pro-Leu-Gly-Val-Arg-Gly-Lys), of heparanase, of enzymes that preferentially occur in necrotic lesions, and enzymes associated with prostate cancer (e.g., PSMA, PSA, fissionable processing module glutaryl- (4-hydroxypropyl) -Ala-Ser-cyclohexaglycyl-Gln-Ser-Leu-COOH). The structure of the linker is chosen such that the protease recognition sequence freely accessible is, i. effective processing by specific proteases possible and after cleavage of the fusion protein, optionally at the cleaved Part containing the therapeutically effective module, remaining amino acids of the Linker's bioactivity therapeutically effective region.

In a preferred embodiment has the polypeptide of the invention additionally a trimerization module between the inhibitor module (1) and the Processing module (2) or between the processing module (2) and the targeting module (3). According to a particularly preferred embodiment comprises the trimerization module is natural occurring or synthetic peptide having intrinsic trimerization properties. A particularly suitable example of such a peptide is one domain of the tenascin molecule (AA 110-139, Swissprot # P10039, (chicken) or Swissprot # P24821 (human)). It guarantees the covalent, homotrimeric linkage of the Fusion protein during biogenesis and can bind the inhibitor module (1) to the therapeutically effective module (4) improve. The ligands of the TNF family, especially the FasL, have intrinsic trimerization properties. An optional trimerization region is therefore mainly used to Stabilize ligand trimer. How important stabilization for the Preservation of the trimeric, potentially active form depends on Isolated case.

The targeting module (3) preferably binds to a cell surface molecule that is expressed in tumor lesions and / or proliferating endothelial cells associated with the process of angiogenesis. In another preferred embodiment, the targeting module (3) is specific for a component of the extracellular matrix present in tumor lesions and / or angiogenesis regions of pathological lesions. According to another preferred embodiment, the targeting module (3) is specific for a component of the malignant tumor cell itself. Preferably, the region or module (3) comprises an antibody (eg murine, humanized or human) or a fragment thereof, eg a Fab fragment or a typi In other words, a single-chain antibody fragment (scFv) produced by the prior art may be murine, CDR-grafted or completely human with specificity for, for example, an antigen which is preferentially or predominantly expressed in tumor tissue, and this may in principle be expressed on the malignant cells themselves , but preferably in the non-malignant portion of the tumor, the stromal cells or the tumor endothelium. Such antigens of non-malignant tissue portions of a solid tumor (carcinoma) are on the one hand genetically invariant, on the other hand occurring in a wide variety of tumor entities and thus universal tumor markers. Examples include the VEGFR or VEGFR / VEGF complex (as an example of receptor-ligand complexes) and the integrin αvβ3, the endosialin 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 for a targeting component of the extracellular matrix present in the tumor stroma, which can be efficiently detected, for example, with specific, high affinity scFv. Further examples of suitable targeting modules are peptides or protein domains which, for example, naturally bind to a tumor or tumor stroma or tumor endothelium-typical structure, but also to artificial antibodies and spiegelmers.

As stated above, contains the therapeutically active module (4) preferably an amino acid sequence a cytokine or a therapeutically effective fragment thereof. Preferably contains the region (4) the amino acid sequence of a Ligands of the TNF family, preferably FasL, more preferred a TNF ligand precursor protein, preferably a FasL precursor protein, particularly preferably identical to the processed, mature wild-type TNF ligand molecule Protein or derived derivatives or mutants with selective Receptor binding properties or mutants or derivatives that in terms of their specific bioactivity or other properties (Stability, Protease resistance), and most preferably from identical to the processed, mature wild-type FasL molecule Protein, or derived derivatives or mutants with selective Receptor binding properties or mutants or derivatives that in terms of their specific bioactivity or other properties (Stability, Protease resistance).

The polypeptide of the invention may include other regions. For example, for simplified cleaning recombinantly produced protein and in vitro analysis appropriate labeling sequences are added. Thus, the polypeptide of the invention e.g. at the N-terminus and / or between two adjacent regions at least have a detectable label. Such a detectable Marking may be a "day", for example for cleaning, be, e.g. a flag day.

The according to the invention preferred FasL prodrug is a non-covalently linked, homotrimeric molecule or, if a corresponding trimerization region has been inserted, a covalently linked, homotrimeric molecule, or in the case of an intermolecular interaction, a homohexameric molecule from the above explained in detail Fusion of four functional regions, the tumor-specific antibody module (3), the FasL module (4) and the blocking FasL binding protein (Extracellular receptor domain or derived peptide) module (1) and the between module (1) and module (3) specific protease cleavage module (2), which in this complete state in terms of FasL effect is inactive.

The FasL prodrug becomes in vivo administration by the antibody portion first specifically enriched in the tumor area and there by the tumor or proteases formed by the reactive tumor stroma / tumor vasculature (e.g., FAP, uPA, tPA, MMP2, Factor VIIa), i. the inhibiting Module / peptide (1) is cleaved off. After selective proteolytic Cleavage dissociates the FasL receptor fragment / inhibitor peptide from the FasL module (4), the latter thus becomes bioactive (i.e. activity The region is being processed by processing the processing center in Region (2) released). The thus processed FasL now binds preferred to cellular Fas receptors, as these are not diffused from the binding site remove. The selectivity the FasL effect is thus carried out with the FasL prodrug according to the invention two measures achieved: on the one hand over the region (3) -mediated selective enrichment of the inactive Prodrug in the tumor and the retention of the biologically active fragment, consisting of region (3) and region (4), also proteolytic Activation and on the other hand over the site-specific conversion of the prodrug by proteases, which exclusively or are preferably detectable in the tumor area in significant activity.

In a preferred embodiment In the present invention, the CTL prodrug has the amino acid sequence of SEQ ID NO: 1.

Another object of the present invention relates to a nucleic acid comprising a nucleotide sequence encoding the polypeptide of the invention. The term "nucleic acid" means a native, semisynthetic, synthetic or modified nucleic acid molecule of deoxyribonucleotides and / or ribonucleotides and / or modified nucleotides In a preferred embodiment In accordance with the present invention, the nucleic acid has the nucleotide sequence of SEQ ID NO: 2.

Furthermore becomes a vector according to the invention, containing the above-defined nucleic acid. The vector according to the invention is not subject to any particular restriction, with suitable vectors are known in the art.

Of the Vector is preferably for expression and / or amplification in a prokaryotic and / or eukaryotic cell. To contains the vector preferably contains suitable regulatory elements, such as promoters, Enhancers, termination sequences, etc. The vector may also be stable Integration of the nucleic acid according to the invention in the genetic material of a host cell.

One Another subject of the invention relates to a host cell containing the aforementioned nucleic acid and / or the aforementioned vector. Suitable host cells For example, all mammalian cells, in particular cultured cell lines, e.g. COS or CHO cells.

• (a) des Züchtens der vorstehenden Wirtszelle in einem Kulturmedium unter geeigneten Bedingungen und
• (b) des Isolierens des erfindungsgemäßen Polypeptids aus den Wirtszellen und/oder dem Kulturmedium.

The present invention also provides a method of producing the polypeptide of the invention comprising the steps

• (A) growing the above host cell in a culture medium under suitable conditions and
• (b) isolating the polypeptide of the invention from the host cells and / or the culture medium.

The polypeptide of the invention is preferably expressed by expression with the aid of suitable expression systems, preferably selectable as a secreted product, more stable Transfectants of the cell line CHO DG44 or after transient expression in COS7 cells. Other, according to the prior art eukaryotic expression systems, e.g. Pichia pastoris, insect or mammalian cells, with the for the respective cell system for Secretion of suitable expression vectors, e.g. in Brocks et al. (Immunotechnology 3: 173-184, 1997) for mammalian and insect cells. pPICZalpha vectors (INVITROGEN) for expression and secretion in the yeast Pichia pastoris, are also suitable.

The polypeptide of the invention, the nucleic acid and / or the vector can (can) Advantageously for the preparation of pharmaceutical compositions for the treatment of pathological disorders for example, of tumor diseases in an individual / patient be used.

A another embodiment The present invention therefore relates to a pharmaceutical composition, containing in a pharmaceutically effective amount the polypeptide of the invention and / or the nucleic acid according to the invention and / or the vector of the invention, optionally in conjunction with one or more pharmaceutically acceptable Excipients, diluents and / or carriers. The pharmaceutical composition is preferably for therapeutic use Treatment of cancers and / or infectious diseases and / or metabolic diseases. Particularly preferred applications the pharmaceutical composition are the treatment solid Tumors and angiogenesis in pathological lesions. The inventive pharmaceutical Composition may be any suitable in the art take known form. Preferably, it is solid, liquid or of an aerosol.

The present invention thus also a treatment method, which administration a therapeutically sufficient amount of the pharmaceutical composition of the invention to a treatment requiring Including patients. Suitable routes of administration of the pharmaceutical composition are known to a person skilled in the art and include, for example, the oral, intravenous, intra-arterial, intramuscular, nasal, rectal and topical application. Intravenous administration can e.g. in the form of a bolus injection with subsequent injection intervals and / or in the form of an infusion. With the pharmaceutical Composition of the present invention may be both human and also animal patients are treated. The treatment procedure is preferred in patients with the aforementioned disorders applied.

The Figures show:

1 : Structure and activity of various FasL fusion proteins.

1 (a) shows the schematic representation of the modular structure of the monomeric subunits of the different FasL fusion proteins. L, leader peptide; Fas, extracellular domain of the Fas molecule (binds FasL); FasL, extracellular domain of FasL; F, flag-tag; PL, linker with protease interface (s); SC40, single chain fragment "40" (binds FAP); TNC, tenascin trimerization region. (1) LF-FasL, soluble flag-tagged FasL; (2) L-SC40-F FasL, FAP-binding soluble FasL (= FasL-AMAIZE); (3) L-SC40-F-FasL-PL-Fas, FasL prodrug without free FasL-C-terminus (construction analogous to TNF-selectokine); (4) L-SC40-F-FasL- PL, FAP-binding soluble FasL with C-terminal PL residue, similar to that described by Pro Zessierung remaining remainder of L-SC40-F-FasL-PL-Fas corresponds; (5) L-Fas-PL-SC40-F-FasL, FasL prodrug with free FasL-C terminus; (6) L-Fas-PL-TNC-SC40-F-FasL, FasL prodrug with FasL-C free terminus and TNC domain, variant 1; (7) L-Fas-TNC-PL-SC40-F-FasL, FasL prodrug with free FasL-C-terminus and TNC-domain, variant 2.

1 (b) Figure 2 shows a scheme of the native structure of soluble FasL (2) and FasL prodrug fusion proteins with free FasL C-terminus in trimeric arrangement (1) (intramolecular interaction of Fas and FasL modules) and in hexameric arrangement (3) (intermolecular interaction of Fas and FasL modules).

1 (c) shows the vitality in% of HT1080 cells after treatment with different prodrugs. HT1080 cells and FAP-expressing HT1080 transfectants were cultured in 96-well plates and incubated overnight with the indicated concentrations of the reagents shown in "A." The cell vitality was determined the next day to sensitize the cells for Fas mediated apoptosis was added to CHX (2.5 μg / ml) To assess the efficiency of intra / intermolecular inhibition of FasL content by the Fas region, the reagents were assayed by secondary aggregation with an anti-Flag mAb (M2). activated and analyzed on FAP-negative HT1080 cells Result: Only FasL prodrugs with free FasL-C-terminus and TNC-domain are efficiently autoinhibited and activated after FAP-dependent processing.The FAP-dependent processing of the prodrug is in 4 shown.

2 : FasL prodrugs with free FasL-C terminus and TNC domain activate FAP-dependent non-apoptotic Fas signal transduction.

2 shows the relative IL-8 production (vertical axis) as a function of the prodrug concentration [ng / ml] (horizontal axis). HT1080 cells (A) and FAP-expressing HT1080 transfectants (B) were cultured in 96-well plates and incubated with the concentrations of anti-Flag cross-linked LF-FasL, L-Fas-TNC- on the horizontal axis in ng / ml. PL-SC40-F-FasL and anti-Flag cross-linked L-Fas TNC-PL-SC40-F-FasL in stimulated for 6 h. Then, the IL8 content in the supernatant was determined by ELISA. Result: The FasL prodrug with free FasL-C terminus and TNC domain is autoinhibited and induces FAP-dependent, Fas-mediated IL8. The efficient auto-inhibition of the FasL prodrug, which distinguishes it from a FasL-AMAIZE, follows from the fact that anti-Flag cross-linking on FAP-negative cells does not lead to IL8 induction. The FAP-dependent processing of the prodrug is in 4 shown.

3 : Selective binding of L-Fas-PL-TNC-SC40-F-FasL and L-Fas-TNC-PL-SC40-F-FasL to cell membrane-expressed FAP.

3 shows the relative cell number (vertical axis) versus the logarithmic intensity (horizontal axis) of HT1080 cells (HT1080-wt) and FAP-transfected HT1080 cells (HT1080-FAP) after incubation with various prodrugs (AC). HT1080 cells and FAP-expressing HT1080 transfectants were incubated with the FasL prodrugs and L-SC40-F-FasL as positive control. After washing the cells, the remaining cell-bound molecules were detected by FITC-labeled anti-Flag M2 by FACS analyzes.

4 Endogenous matrix metalloproteases (MMPs) mediate the processing and activation of the FasL prodrug (L-Fas-TNC-PL-SC40-F-FasL) on FAP-expressing HT1080 cells.

4 (a) shows a Western blot analysis of prodrug incubated HT1080 cells. L-Fas TNC-PL-SC40-F-FasL was added to HT1080 and HT1080-FAP transfectants for 16 hours. In some of the batches, the selective MMP inhibitor Ilomastat was additionally added. Subsequently, the medium supernatant of the cells was separated by gel electrophoresis and then analyzed in the Western Blot by means of anti-Flag.

4 (b) shows the vitality in% of HT1080 cells after treatment with different prodrugs. HT1080-FAP cells were incubated overnight with the indicated combinations of the FasL prodrug (L-Fas-TNC-PL-SC40-F-FasL), with anti-FasL (NOK-1), anti-FAP (MB36) and Ilomastat , Then the vitality of the cells was determined. - = negative control, untreated tents.

5 : Coding DNA sequence (top) and amino acid sequence (one-letter code, below) of a polypeptide according to the invention "FasL prodrug" of the module structure L-Fas-TNC-PL-SC40-F-FasL.

6 : Amino acid sequence of a polypeptide according to the invention "FasL prodrug" of the module structure L-Fas-TNC-PL-SC40-F-FasL (SEQ ID NO: 1).

7 : DNA sequence of a polypeptide according to the invention "FasL prodrug" of the module structure L-Fas-TNC-PL-SC40-F-FasL (SEQ ID NO: 2).

The The present invention is illustrated by the following, non-limiting Examples explained in more detail.

Example 1 Preparation a polypeptide of the invention

The FasL module provided with an N-terminal targeting module was inhibited by genetically engineered N-terminal attachment of the ligand-binding module of the corresponding receptor Fas, which, however, can be cleaved off by tumor-associated proteases. For this purpose, the receptor fragment was attached by means of a linker which contains corresponding interfaces ( 1 ). Through its targeting module, this FasL prodrug, designated as L-Fas TNC-PL-SC40 FasL, can be enriched in the target area ( 3 . 4 (b) ). This FasL fusion protein is processed in a FAP-dependent manner and thus activated ( 1 . 3 . 4 ). The activated fusion protein induces cell death ( 1 . 4 (b) ) or non-apoptotic Fas-mediated cellular reactions, eg IL8 production ( 2 ).

Example 2: One after FasL prodrug constructed according to the scheme of TNF-Selektokins (WO 02/22833) is indeed processable but not or only very little active.

The FasL module was inhibited by genetically engineered C-terminal attachment of the ligand-binding module of the corresponding receptor Fas, which, however, can be cleaved off by tumor-associated proteases. To this end, the receptor fragment was attached by means of a linker containing appropriate interfaces. By means of an N-terminal antibody-targeting module, this FasL fusion protein, designated as L-SC40-F-FasL-PL-Fas, can be enriched in the target area. This FasL fusion protein is not activated FAP-dependent ( 1 (c) ), although processing takes place. Subsequent studies showed that the short C-terminal amino acid additions remaining after processing at FasL's native C-terminus (as exemplified in construct L-SC40-F-FasL-PL) drastically reduce the FasL activity ( 1 (c) ).

It follows a sequence listing according to WIPO St. 25. This can from the official publication platform downloaded from the DPMA.

Claims (28)

A polypeptide having an amino acid sequence comprising of N- after C-terminal (1) a region representing the biological activity of the region (4) by intramolecular or inhibits intermolecular binding and / or interaction, (2) a region that has at least one processing station, (3) a region containing a specific macromolecule on a cell surface and / or a component of the extracellular Selectively detects matrix, and (4) a region with a biological activity for a specific one Target molecule, in which the biological activity the region (4) by processing in vivo the at least one Processing center in region (2) is releasable. Polypeptide according to claim 1, wherein the region (4) an amino acid sequence a cytokine or a fragment thereof. The polypeptide of claim 2, wherein the cytokine is a Member of the TNF ligand family, a biologically active derivative or a biologically active mutant it is. The polypeptide of claim 3, wherein the cytokine is Fas ligand (FasL), a biologically active derivative or a biologically active Mutant of it is. A polypeptide according to any one of claims 1 to 4, wherein the specific target molecule Region (4) is a cell membrane-bound cytokine receptor. A polypeptide according to any one of claims 1 to 5, wherein the at least a processing site in region (2) a cleavage site for a protease having. The polypeptide of claim 6, wherein the protease is a Urokinase-type plasminogen activator (uPA), tissue plasminogen activator (tPA), activated coagulation factor VIIa, a matrix metalloprotease or a FAP protease. A polypeptide according to any one of claims 1 to 7, wherein the region (1) has at least one binding site for the region (4). Polypeptide according to claim 8, wherein the region (1) a receptor for is a cytokine or a fragment thereof. The polypeptide of claim 9, wherein the receptor is the full 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 this domain or a synthetic TNF ligand-binding compound. The polypeptide of claim 10, wherein the receptor is the complete or partial extracellular domain of a human Fas receptor and / or a FasL binding virus protein or a mutant thereof or a synthetic FasL binding compound. A polypeptide according to any one of claims 1 to 11, wherein the polypeptide between the region (1) and the region (2) or between the region (2) and the region (3) has a trimerization module. The polypeptide of claim 12, wherein the trimerization module a natural one occurring or synthetic peptide having intrinsic trimerization properties includes. A polypeptide according to any one of claims 1 to 13, wherein the polypeptide at the N-terminus and / or between two adjacent regions at least one detectable label having. The polypeptide of claim 14, wherein the detectable Mark between the region (3) and the region (4) is located. A polypeptide according to any one of claims 1 to 15, wherein the region (3) for a cell surface molecule specific which is in tumor lesions and / or proliferating endothelial cells involved in the process of angiogenesis are expressed. A polypeptide according to any one of claims 1 to 15, wherein the region (3) for a component of the extracellular Matrix specific to tumor lesions and / or angiogenesis pathological lesions is available. A polypeptide according to any one of claims 1 to 15, wherein the region (3) for a component of the malignant tumor cell itself is specific. A polypeptide according to any one of claims 1 to 18, wherein the region (3) a murine, humanized or human antibody or a fragment thereof of defined antigenic specificity. The polypeptide of claim 19, wherein the antibody fragment is an scFv or Fab fragment. Nucleic acid, which comprises a nucleotide sequence, which for the polypeptide of any one of claims 1 to 20 encoded. Vector containing the nucleic acid according to claim 21. Vector according to claim 22, for expression and / or Amplification in a prokaryotic and / or eukaryotic Cell enabled is. A host cell containing the nucleic acid of claim 21 and / or the vector according to claim 22 or 23. Process for the preparation of the polypeptide according to the claims 1 to 20, comprising the steps (a) after culturing the host cell Claim 24 in a culture medium under suitable conditions and (B) isolating the polypeptide according to any one of claims 1 to 20 from the host cells and / or the culture medium. Pharmaceutical composition containing in pharmaceutical effective amount of the polypeptide according to any one of claims 1 to 20 and / or the nucleic acid according to claim 21 and / or the vector according to claim 22 or 23, optionally in association with one or more pharmaceutically acceptable Excipients, diluents and / or Carriers. Pharmaceutical composition according to claim 26 for the therapeutic treatment of solid tumors and / or angiogenesis in pathological lesions a patient. Pharmaceutical composition according to claim 26 or 27, the solid, liquid or aerosol.