Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
  • C07K16/40—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against enzymes
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • 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
  • 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
  • C07K14/52—Cytokines; Lymphokines; Interferons
  • C07K14/525—Tumour necrosis factor [TNF]
• • 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
  • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
  • C07K14/7151—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for tumor necrosis factor [TNF], for lymphotoxin [LT]
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
  • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2317/00—Immunoglobulins specific features
  • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
  • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
  • C07K2317/622—Single chain antibody (scFv)
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K2319/00—Fusion polypeptide

Definitions

• the present invention relates to a polypeptide with preferably antitumor and / or immunomodulating cytokine properties, which can be activated by processing in vivo, comprising a central region with specific biological activity, at the C-terminal end of which there is a region with a processing unit and an inhibitor domain, while there is a region at the N-terminal end of the central region which selectively recognizes a macromolecule on a cell surface or a component of the extracellular matrix.
• tumor necrosis factor and other active substances for the treatment of, for example, tumor diseases has hitherto only been possible under very limited treatment protocols (for example by means of "isolated limb perfusion") because of the strong systemic side effects which are to be regarded as therapy-limiting Indications (melanoma / sarcoma metastases of the extremities) can be carried out successfully. From these clinical data it can be estimated that a 10 to 100 times higher TNF dose than the MTD (“Maximum Tolerated Dose”) would be required for the anti-tumor efficacy than the massive systemic side effects would allow.
• the present invention is therefore based on the object of avoiding or reducing the undesirable consequences of treatment with therapeutically active polypeptide active substances, such as TNF-containing substances, while at the same time maintaining the therapeutically active, for example antitumor, properties of the active substance, such as TNF or even be reinforced.
• therapeutically active polypeptide active substances such as TNF-containing substances
• therapeutically active for example antitumor, properties of the active substance, such as TNF or even be reinforced.
• polypeptide with an amino acid sequence comprising from N- to C-terminal
• the polypeptide according to the invention is a modular active substance, according to a particularly preferred embodiment a preferably homotrimeric fusion protein with a cytokine, preferably TNF or a biologically active derivative or a biologically active mutant thereof, as an antitumor substance or region (3), which releases its biological effect in a targeted manner in the diseased tissue, for example a tumor area, by linking with four further functional modules.
• a cytokine preferably TNF or a biologically active derivative or a biologically active mutant thereof
• the N-terminal linkage of the therapeutically active substance for example the TNF molecule
• a targeting module (1) specific for the target tissue for example tumor-specific antibodies or derivatives thereof, such as scFv antibodies
• an inhibitor (5) against the therapeutically active substance in particular a peptide inhibitor, which is selectively inactivated in the target tissue, such as the tumor area, by processing the domain (4), preferably removed from the fusion protein by targeted proteolytic cleavage, and so on bioactive agent bound to the selective targeting module, for example TNF, is formed.
• a peptide linker domain (2) is located between the targeting module (eg the scFv antibody fragment) and the module with therapeutic function (eg TNF) Trimerization domain, which ensures the formation of covalent disulfide bridges and thus a regular and stable homotrimerization of the fusion protein
• the construct according to the invention it is possible to locally high active concentrations of the therapeutically active substance, e.g. of the TNF, without resulting in systemically elevated therapeutic levels (e.g. TNF in the serum) and thus therapy-limiting side effects.
• the targeting module e.g. antibody
• the targeting module mediated presentation of the locally activated TNF achieves an effect which corresponds to that of the natural membrane TNF, i.e. both types of TNF receptors are co-activated, thus potentiating the anti-tumor properties of TNF.
• the polypeptide (selectokine) provides a novel prodrug technology and is a construct which, according to a preferred embodiment, comprises a recombinant, homotrimeric fusion protein which in principle comprises a defined sequence of the following structural elements (in the monomer) (N-terminal to C-terminal): (1) a murine, humanized or human single chain antibody question ent (scFv) of defined antigen specificity consisting of VH linker VL; (2) a peptide linker with intrinsic trimerization properties; (3) a TNF molecule which corresponds, for example, to the wild-type TNF or the extracellular domain of TNF (mature 17 kDa form, AA 1-157, Swissprot # P01375) or biologically active variants derived therefrom; (4) a variable linker peptide with specific protease cleavage sites, (5) a specific TNF-binding protein or peptide.
• structural elements in the monomer
• scFv human single chain antibody question
• the targeting module (1) is preferably specific for a cell surface molecule which is expressed in tumor lesions and / or proliferating endothelial cells which are associated with the process of angiogenesis. According to another preferred embodiment, the targeting module (1) is specific for a component of the extracellular matrix that is present in tumor lesions and / or angiogenic areas of pathological lesions. According to a further preferred embodiment, the targeting module (1) is specific for a component of the malignant tumor cell itself.
• the region or the module (1) preferably comprises an antibody (for example murine, humanized or human) or a fragment thereof, for example a Fab fragment or a typical single-chain antibody fragment (scFv) murine produced according to the prior art, by CDR- Grafting of humanized or completely human origin with specificity for an antigen which is preferably selectively or dominantly expressed in the tumor tissue, for example, which can 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 becomes.
• an antibody for example murine, humanized or human
• a fragment thereof for example a Fab fragment or a typical single-chain antibody fragment (scFv) murine produced according to the prior art, by CDR- Grafting of humanized or completely human origin with specificity for an antigen which is preferably selectively or dominantly expressed in the tumor tissue, for example, which can in principle be expressed on the malignant cells themselves, but preferably in the non-malignant portion of the tumor,
• Such antigens of non-malignant tissue parts of a solid tumor are on the one hand genetically invariant, on the other hand they occur in a wide variety of tumor entities and are therefore universal tumor markers.
• the VEGFR or VEGFR VEGF complex as an example for receptor-ligand complexes
• the Integ ⁇ n avß3 the endosialin and the fibronectin isoform bFn as selective target structures of the tumor endothelium
• FAP fibroblast activation protein
• Other examples of suitable targeting modules are peptides, artificial antibodies and Spiegelmers.
• the peptide linker region (2) is preferably a trimerization module and connects the targeting region (1) with the therapeutically active region (3).
• the trimerization module comprises a naturally occurring or synthetic peptide with 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 establishes the connection between the targeting module (1) (eg scFv) and the therapeutic agent (3) (eg TNF) and at the same time ensures the covalent, homotrimeric linkage of the fusion protein during biogenesis.
• the therapeutically active module (3) preferably contains an amino acid sequence of a cytokine or a therapeutically active fragment thereof.
• Region (3) preferably contains the amino acid sequence of TNF, more preferably a TNF precursor protein and most preferably of a protein identical to the processed, mature wild-type TNF molecule (AA 1-157, Swissprot # P01375), or derivatives derived therefrom or mutants with selective receptor binding properties or mutants or derivatives which have been optimized with regard to their specific bioactivity or other properties (stability, protease resistance).
• the processing module (4) is, for example, protease-sensitive (ie the processing site corresponds to the recognition sequence of a protease) and is preferably of an amino acid composition and overall length such that it allows the fusion protein to be homotrimerized by the trimerization module and TNF itself, but at the same time also has a high affinity, stable binding of the TNF inhibitor located in the C-terminal molecule (eg the extracellular TNF receptor domain) to the TNF portion is permitted, thereby preventing the binding of the TNF module to cell-expressed TNF receptors.
• the linker is preferably such that it contains at least one, preferably several, selective cleavage sites for such extracellular or cell-associated proteases, which are preferably detected selectively in the tumor tissue.
• Suitable cleavage sites are those for urokinase-type plasminogen activator (uPA), tissue plasminogen activator (tPA), the activated coagulation factor VIIa, matrix metalloproteases, such as MMP-2 and MMP-9, and for the highly selective expression of the membrane in the stroma of tumors FAP protease.
• uPA urokinase-type plasminogen activator
• tPA tissue plasminogen activator
• matrix metalloproteases such as MMP-2 and MMP-9
• MMP-9 matrix metalloproteases
• the structure of the linker is chosen such that the protease recognition sequence is freely accessible, ie effective processing by specific proteases is possible, and after cleavage of the fusion protein, amino acids of the linker that may remain on the TNF molecule do not negatively influence the bioactivity of the therapeutically active region ,
• the inhibitor module (5) is a receptor for a cytokine or a fragment thereof. Furthermore, the inhibitor module preferably has at least one binding site for the therapeutically active region (3). Preferably, in the case of using TNF in region (3), the inhibitor module comprises the complete or partial extracellular domain of a human TNF receptor, e.g. of the hu TNFR1 (synomym p55 / 60TNFR; Swissprot # P19438, AA 1-190; or fragments of this molecule, for example AA 1-157 or AA 60-120).
• hu TNFR1 e.g. of the hu TNFR1 (synomym p55 / 60TNFR; Swissprot # P19438, AA 1-190; or fragments of this molecule, for example AA 1-157 or AA 60-120).
• TNF TNF binding proteins
• huTNFR2 extracellular domain of huTNFR2
• proteins of viral origin such as e.g. the T2 protein, as well as synthetic peptides derived therefrom, which have TNF binding properties and interfere with TNF binding to TNF receptors which are located in the cell membrane
• the fusion protein according to the invention is biologically inactive in this state, i.e. it is in the Proform (Prodrug).
• the polypeptide according to the invention can comprise further domains.
• suitable labeling sequences can be added to simplify the purification of the recombinantly produced protein and the in vitro analysis.
• a myc-hise tag derived from the vector POPE can be added to the region (5), preferably the TNFR fragment, at the C-terminal. Further labeling sequences are known to a person skilled in the art.
• the TNF selectokine preferred according to the invention is a covalently linked, homotrimeric molecule, consisting of the fusion of three functional domains explained in detail above, the tumor-specific antibody module, TNF and the blocking TNF binding protein (extracellular receptor domain or peptide derived therefrom) and functional linkers in between Trimerization properties or specific protease cleavage sites, which is inactive in this complete state with regard to the TNF effect.
• the selectokin is initially specifically enriched by the antibody portion in the tumor area and processed there by the proteases formed by the tumor itself or the reactive tumor stroma / tumor vascular system (eg FAP, uPA, tPA, MMP2, factor VIIa), ie the inhibitory one Peptide (5) is split off.
• the TNFR fragment / inhibitor peptide dissociates from the trimeric TNF molecule, the latter thus becoming bioactive (ie the biological activity of the region is released by processing the processing site in Reion (4)).
• the TNF processed in this way now binds preferentially to cellular TNF receptors, since these, as homomultimeric molecules, have a substantially higher affinity than the monomeric, soluble receptor fragments.
• the selectivity of the TNF effect is thus achieved with the selectokin according to the invention by two measures: on the one hand via the scFv-mediated selective enrichment of the inactive prodrug in the tumor and its retention even after proteolytic activation, and on the other hand via the site-specific conversion of the prodrug by proteases, which exclusively or preferably can be detected in the tumor area in significant activity.
• scFv-mediated fixation of the TNF shifts the dissociation equilibrium at the TNFR2 towards a more stable binding, thereby achieving its activation. It is known that the simultaneous activation of both TNFRs leads to a cooperative signaling mechanism and the resulting increased cellular reactions, in particular the activation of endothelial cells and the induction of apoptosis in tumor cells are resistant to conventionally used (soluble) TNF.
• polypeptide according to the invention have the amino acid sequences shown in FIGS. 1 (SEQ ID NO 1) and 5 (SEQ ID NO 3).
• the present invention further relates to a nucleic acid comprising a nucleotide sequence which codes for the polypeptide according to the invention.
• nucleic acid means a native, semi-synthetic, synthetic or modified nucleic acid molecule made from deoxyribonucleotides and / or ribonucleotides and / or modified nucleotides.
• Preferred embodiments of the nucleic acid according to the invention contain the nucleotide sequence shown in FIG. 1 (SEQ ID NO 2) and FIG. 5 (SEQ ID NO 4).
• a vector containing the nucleic acid defined above is provided.
• the vector is preferably capable of expression and / or amplification in a prokaryotic and / or eukaryotic cell.
• the vector preferably contains suitable regulatory elements, such as promoters, enhancers, termination sequences, etc.
• the vector can also be used for the stable integration of the nucleic acid according to the invention into the genetic material of a host cell.
• Another object of the invention relates to a host cell containing the above nucleic acid and / or the above vector.
• Suitable host cells are, for example, all mammalian cells, such as COS or CHO cells.
• the present invention also provides a method for producing the polypeptide of the invention comprising the steps
• the polypeptide according to the invention is preferably produced by expression with the aid of suitable expression systems, preferably stable, selectable transfectants of the Zeil line CHO DG44, or after transient expression in COS7 cells.
• suitable expression systems preferably stable, selectable transfectants of the Zeil line CHO DG44, or after transient expression in COS7 cells.
• Other state-of-the-art eukaryotic expression systems for example Pichia pastoris, insect or mammalian cells, with the expression vectors suitable for the respective cell system for secretion, for example as in Brooks et al. (Immunotechnology 3: 173-184, 1997) for mammals and insect cells.
• pPICZalpha vectors for expression and secretion in the yeast Pichia pastoris are also suitable.
• polypeptide according to the invention, the nucleic acid and / or the vector can advantageously be used for the production of pharmaceutical compositions for the treatment of pathological disorders.
• a further embodiment of the present invention therefore relates to a pharmaceutical composition containing, in pharmaceutically effective amount, the polypeptide according to the invention and / or the nucleic acid according to the invention and / or the vector according to the invention, optionally in conjunction with one or more pharmaceutically acceptable auxiliaries, diluents and / or carriers.
• the pharmaceutical composition is preferably used for the therapeutic treatment of cancer and / or infectious diseases and / or metabolic diseases. Particularly preferred areas of application for the pharmaceutical composition are the treatment of solid tumors and angiogenesis in pathological lesions.
• the pharmaceutical composition of the invention may take any form known to be suitable in the art. It is preferably solid, liquid or aerosol-like.
• the present invention thus also includes a treatment method which includes the administration of a therapeutically sufficient amount of the pharmaceutical composition according to the invention to a patient in need of treatment.
• Suitable routes of administration of pharmaceutical Zeutician composition are known to a person skilled in the art and include, for example, oral, intravenous, intraarterial, intramuscular, nasal, rectal and topical application.
• Intravenous administration can be carried out, for example, in the form of a bolus injection with subsequent injection intervals and / or in the form of an infusion. Both the human and animal patients can be treated with the pharmaceutical composition of the present invention.
• the treatment method is preferably used in patients with the diseases mentioned above.
• SEQ ID NO 1 shows the amino acid sequence (SEQ ID NO 1, top) and the corresponding cDNA nucleotide sequence (SEQ ID NO 2, bottom) of the selectokine Prodrug W24 according to the invention.
• FIG. 2 shows photographic representations of an SDS-PAGE gel stained with Coomassie and the corresponding Western blot after incubation with anti-c-myc-mAb 9E10.
• the Prodrug W24 was expressed in CH0-DG44 cells and purified using IMAC. The purified protein was applied under reducing (red.) As well as non-reducing conditions.
• Figure 3 is a photographic representation of a Western BIot analysis of a 12% SDS gel after detection with anti-c-myc-mAb 9E10. Lane 1: purified prodrug W24 after incubation with PBS. Lane 2: purified prodrug W24 after incubation with PBS plus tPA.
• 5 shows the amino acid sequence (SEQ ID NO 3, top) and the corresponding cDNA nucleotide sequence (SEQ ID NO 4, bottom) of the selectokine Prodrug W33 according to the invention.
• FIG. 6 shows photographic representations of a SDS-PAGE gel stained with Coomassie and the corresponding Western blot after incubation with anti-c-myc-mAb 9E10.
• the prodrug W32 was expressed in CHO-DG44 cells and purified using IMAC. The purified protein was applied under both reducing and non-reducing conditions.
• (B) is a graphical representation of the results for the determination of K D, apP . of the prodrug W32 with respect to the FAP binding by means of FACS analysis.
• FAP-positive HT1080 # 33 cells (O) and FAP-negative HT1080 control cells (•) were incubated with serial dilutions of prodrug W32 and the cell-bound portion was detected using indirect immunofluorescence intensity.
• the prodrug concentration used is shown against the mean fluorescence intensity (mfi).
• Figure 7 (A) is a graphical representation of the results of an apoptosis induction test on Kym-1 cells with non-activated prodrug W32 ( ⁇ ), trypsin-activated prodrug W32 (D) or wild-type TNF (•). A representative of three experiments is shown. Inserted is the photographic representation of a SDS-PAGE gel stained with Coomassie under reducing conditions of IMAC-cleaned prodrug W32 (left lane) and IMAC-cleaned prodrug W32 after trypsin activation (right lane). The arrow corresponds to the expected MW of the activated prodrug W32.
• (B) is a graphical representation of the results of an apoptosis induction test of Kym-1 cells in coculture with prodrug-presenting, FAP-positive cells (HT1080 # 33) and with FAP-negative control cells (HT1080).
• HT1080 + non-activated prodrug W32 ( ⁇ ) HT1080 + trypsin-activated prodrug W32 (D)
• (C) is a graphical representation of an experiment corresponding to that shown in (B), but the trypsin activation only occurred after binding to the HT cells and subsequent fixation.
• Example 1 Examples of the sequence of TNF selectokines
• potential endogenous interfaces in the huTNF molecule are removed by amino acid exchange (TNFmut I83F, R131Q) while maintaining the scFV, linker and receptor sequence as exemplified above.
• the co-coil domain of Tenascin-C (AS 110-139), which is highly conserved in various species, is used as the trimerization domain:
• a processing sequence according to the invention is, for example, a linker with the protease cleavage sites for thrombin, tPA, factor VIIa and uPA (amino acid sequence below, SEQ ID NO 7; cDNA nucleotide sequence above, SEQ ID NO 8):
• Example 3 Expression, purification and functional characterization of the TNF selectokine Prodrug W24
• the TNF selectokin prodrug W24 consists of the following components (from N- to C-terminal, amino acid residues (AA) are related to SEQ ID NO 1):
• AA 322-486 mutated form of the natural, human TNF precursor protein
• AA 513-639 human TNFR1 fragment containing the extracellular
• the amino acid sequence (SEQ ID NO 1) and the corresponding coding DNA sequence (SEQ ID NO 2) are shown in FIG. 1.
• the calculated MW of the protein portion is 70.3 kDa.
• the Prodrug W24 was purified from CHO supernatant using IMAC according to the manufacturer's instructions (Pharmacia). In the SDS-PAGE gel stained with Coomassie, 400 ng (20 ⁇ l) of it was applied under reducing and non-reducing conditions; in the Western blot with an anti-c-myc mAb 9E10, 2 ⁇ l were used; see. Fig. 2. The expression of the monomeric, dimeric and trimeric construct is detected.
• Prodrug W24 is cleaved by tPA
• the purified W24 prodrug 600 ng was incubated in PBS (50 ⁇ l) or in PBS + tPA (5 ⁇ g tPA in 50 ⁇ l PBS) at 37 ° C. for 16 h. After 12% SDS-PAGE (reducing) and Western blot, detection was carried out with anti-c-myc mAb 9E10, followed by alkaline phosphatase-conjugated goat anti-mouse IgG serum. The appearance of a band below 33 kDa in the amount of the expected size of the cleaved TNFR fragment (approx. 17 kDa), which carries a myc tag at the C-terminal, in the approach with tPA shows the partial digestion of prodrug W24; see. Fig. 3. The activated TNF selectokine cannot be represented with this detection method.
• the determination of the LD 50 results in an approximately 4000-fold increase in activity through the processing.
• Prodrug W33 has the same functional properties as the Prodrug W24 from Example 3, but differs from it by an extended protease-sensitive linker (AA 487-520) and a shortened TNFR fragment (AA 521-582; Swissprot # P19438, AA 54-115 of human TNFR1; see Himmler et al. (1990) DNA and Cell Biology 9, 705-715).
• Amino Acid (SEQ ID NO 3) and coding cDNA sequence (SEQ ID NO 4) of prodrug W33 are shown in FIG. 5.
• Example 5 Expression, purification and functional characterization of the TNF selectokine Prodrug W32
• Prodrug W32 Another construct (Prodrug W32) was produced, which corresponds functionally to the Prodrug W24 from Example 3, but contains as targeting module (1) another antibody fragment (scFv M036), which was isolated independently from a murine Ig gene library and for cross-reactivity human / murine FAP was selected.
• targeting module (1) another antibody fragment (scFv M036), which was isolated independently from a murine Ig gene library and for cross-reactivity human / murine FAP was selected.
• the targeting specificity of the prodrug W24 of Example 3 is based on scFv OS4, which only recognizes human FAP.
• Prodrug W32 was expressed like construct W24 (Example 3), purified by IMAC and analyzed by SDS-PAGE Western Blot; see. Figure 6A.
• the KD, app. Of the prodrug W32 for FAP binding was determined by means of FACS analysis; see. Figure 6B.
• the Ko, a pp. was calculated from the concentration at which the half-maximum signal was obtained. The result was a value of 2.4 x 10 "10 M.
• the activated prodrug W32 has an effect comparable to that of the naturally occurring TNF in the Kym-1 apoptosis test (for the implementation, see Example 3), while the unprocessed construct only develops an apoptotic effect at much higher concentrations; see. Figure 7A. Furthermore, a juxtatropic apoptosis induction of the activated prodrug W32 was investigated in co-culture with prodrug-presenting cells.
• FAP-negative HT1080 control cells or FAP-positive HT1080 # 33 cells were incubated with serial dilutions of the prodrug or of the trypsin-activated prodrug, washed, fixed, co-cultivated with Kym-1 and the vitality of the cells was determined after 16 h; see. Figure 7B.
• the trypsin activation of the prodrug was carried out only after the binding to the cells and subsequent fixation of the cells; see. Figure 7C. In both cases there was a significant juxtatropic apoptosis induction due to the activated prodrug.
• the fusion proteins are produced as follows:
• the single chain antibody fragment (scFv) OS4 (hereinafter referred to as OS4) is the CDR grafting humanized version of the FAP-specific mAb F19 (Rettig et al. 1988) and in Rippmann J.F. (Dissertation Uni Stuttgart, Verlag Grauer, Stuttgart, 1999) and Rippmann et al (Appl EnvMicrobiol 64: 4862-4869, 1998).
• trimerization domain for example AA 110-139 of chicken tenascin
• primers 1 SEQ ID NO 10
• 2 SEQ ID NO 11
• the human TNF fragment was primed 3 (SEQ ID NO 12) and 4 (SEQ ID NO 13) from a non-cleavable, membrane-bound TNF mutant (membrane TNF, TNFdelta1-12, Grell et al, Cell 83: 793-802, 1995), whereby the Kpn1 interface at the 5 'end and the Acc3 and BamH1 interfaces at the 3' end and between the sequence segments coding for the tenascin and TNF domains, a sequence coding for the peptide linker TyrGlyGlyGlySer (SEQ ID NO 9) were introduced. The two fragments were inserted into the Not1 / BamH1 digested cloning intermediate using the Not1, Kpn1 and BamH1 interfaces.
• the protease-sensitive linker and the human TNF receptor 1 fragment were cloned via several intermediates.
• the TNF receptor 1 fragment (cysteine-rich domains 1-3; AS 12-138; SwissProt # 10039) with the primers 5 (SEQ ID NO 14) and 6 (SEQ ID NO 15) from the plasmid pADBTNF-R (Himmler et al. DNA Cell Biol 9: 705-715, 1990) PCR amplified, with the primers 7 (SEQ ID NO 16) and 6 (SEQ ID NO 15) reamplified, whereby the interfaces Acc3, BamH1 and a linker 5 'from the TNFR Fragment was introduced which encodes the protease interfaces. This fragment was cloned into the intermediate described in 3 via the Acc3 / BamH1 interfaces.
• a TNF receptor with the primers 9 and 10 PCR is amplified, resulting in one for AA 54-115 of the human TNFR1 coding sequence with 5 'lying linker. This fragment is inserted into pW24 via the Sah and BamH1 interfaces and replaces the linker TNFR1 fragment contained therein.
• the resulting expression plasmid pW33 allows expression of the TNF selectokine prodrug W33.
• TNF selectokine prodrug (W24 and W33)
• CHO-DG44 cells were transfected with the constructs described under 5 and 6 according to the manufacturer's instructions with lipofectamine (Gibco-BRL) and then free using hypoxanthine and thymidine ( HT ⁇ ) CHO-S-SFM Medium (Life Technologies) selected for stable integration of the constructs into the genome.
• An increase in expression was obtained by gradually increasing the selection reagent methotrexate (0.1; 1; 10 ⁇ M).
• Both W24 and W33 were analyzed using Immobilized Metal Affinity Chromatography (IMAC) as described in Rippmann et al. (Appl EnvMicrobiol 64: 4862- 4869, 1998) described under sterile conditions, purified from the culture supernatants and stored at 4 ° C. until further use.
• IMAC Immobilized Metal Affinity Chromatography

Landscapes

• Health & Medical Sciences (AREA)
• Chemical & Material Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Organic Chemistry (AREA)
• Medicinal Chemistry (AREA)
• General Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Molecular Biology (AREA)
• Genetics & Genomics (AREA)
• Biophysics (AREA)
• Immunology (AREA)
• Toxicology (AREA)
• Cell Biology (AREA)
• Gastroenterology & Hepatology (AREA)
• Zoology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Animal Behavior & Ethology (AREA)
• Pharmacology & Pharmacy (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Epidemiology (AREA)
• Engineering & Computer Science (AREA)
• General Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Peptides Or Proteins (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Micro-Organisms Or Cultivation Processes Thereof (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=7656260

Family Applications (1)

Country Status (22)

Families Citing this family (23)

Family Cites Families (5)

• 2000
  • 2000-09-15 DE DE10045592A patent/DE10045592A1/de not_active Withdrawn
• 2001
  • 2001-09-17 CA CA002422759A patent/CA2422759A1/en not_active Abandoned
  • 2001-09-17 WO PCT/EP2001/010730 patent/WO2002022833A1/de active Application Filing
  • 2001-09-17 US US10/380,438 patent/US20040053829A1/en not_active Abandoned
  • 2001-09-17 YU YU18903A patent/YU18903A/sh unknown
  • 2001-09-17 IL IL15418501A patent/IL154185A0/xx unknown
  • 2001-09-17 EE EEP200300100A patent/EE200300100A/xx unknown
  • 2001-09-17 AU AU2001293819A patent/AU2001293819A1/en not_active Abandoned
  • 2001-09-17 PL PL36054001A patent/PL360540A1/xx not_active Application Discontinuation
  • 2001-09-17 HU HU0301693A patent/HUP0301693A3/hu unknown
  • 2001-09-17 EP EP01974261A patent/EP1317556A1/de not_active Withdrawn
  • 2001-09-17 BR BR0113928-2A patent/BR0113928A/pt not_active IP Right Cessation
  • 2001-09-17 MX MXPA03002229A patent/MXPA03002229A/es unknown
  • 2001-09-17 KR KR10-2003-7003738A patent/KR20030048041A/ko not_active Application Discontinuation
  • 2001-09-17 SK SK281-2003A patent/SK2812003A3/sk unknown
  • 2001-09-17 JP JP2002527275A patent/JP2004508828A/ja active Pending
  • 2001-09-17 CN CNB018157645A patent/CN1214115C/zh not_active Expired - Fee Related
  • 2001-09-17 RU RU2003106429/13A patent/RU2003106429A/ru not_active Application Discontinuation
• 2003
  • 2003-03-06 BG BG107613A patent/BG107613A/bg unknown
  • 2003-03-12 ZA ZA200302008A patent/ZA200302008B/en unknown
  • 2003-03-14 HR HR20030192A patent/HRP20030192A2/hr not_active Application Discontinuation
  • 2003-03-14 NO NO20031185A patent/NO20031185L/no unknown

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events