WO1991006560A1 - Cross-linked tumour necrosis factors - Google Patents

Cross-linked tumour necrosis factors Download PDF

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Publication number
WO1991006560A1
WO1991006560A1 PCT/EP1990/001754 EP9001754W WO9106560A1 WO 1991006560 A1 WO1991006560 A1 WO 1991006560A1 EP 9001754 W EP9001754 W EP 9001754W WO 9106560 A1 WO9106560 A1 WO 9106560A1
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tumor necrosis
necrosis factor
linked
cross
tnfα
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PCT/EP1990/001754
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German (de)
French (fr)
Inventor
Heinz Hillen
Thomas Subkowski
Lothar Daum
Franz Emling
Michael Kluge
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Basf Aktiengesellschaft
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Publication of WO1991006560A1 publication Critical patent/WO1991006560A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1075—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of amino acids or peptide residues
    • 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]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to cross-linked tumor necrosis factors, their production and their use for combating diseases.
  • Tumor necrosis factor ⁇ is a known protein that plays a role in many biological processes (Proc. Natl. Acad. Sei USA 72, 3666 (1975), Ann. Rev. Biochem. 57, 505 (1988) ). The same applies to the structurally very similar lymphotoxin (TNFß) (Nature 312, 721 (1984)).
  • TNF ⁇ The spatial structure of TNF ⁇ is known (Nature 338, 225 (1989)). According to this, TNF ⁇ is a protein built up symmetrically from three identical subunits. This tri ere is the biologically active form of TNF ⁇ (J. Biol. Che. 262, 6951 (1987)).
  • proteins can be crosslinked with a number of reagents which can form covalent bonds with amino groups, sulfhydryl groups or aromatic groups of the protein (Tac H. Ji "Bifunctional Reagents" in Methods Enzymol. 91, Academic Press 1983 New York, pages 580-612). However, this creates little-defined substance mixtures.
  • the invention relates to covalently crosslinked derivatives of the tumor necrosis factor, characterized in that
  • TNF ⁇ tumor necrosis factors
  • TNFß TNFß and derivatives of these two substances, which differ from these by Exchange, deletion or addition of one or more amino acids distinguish without the cytotoxic properties deteriorating significantly. Derivatives of TNF ⁇ are preferred.
  • the individual tumor necrosis factor chains are cross-linked in the N-terminal area.
  • the link between the amino group of the N-terminal amino acid of the 1st chain of the amino group and the closest lysine residue of the 2nd chain from the N-terminus is preferred (e.g. Lysii for TNF ⁇ ).
  • the cross-linked tumor necrosis factors are produced by reacting the tumor necrosis factor with a linking reagent.
  • Suitable linkage reagents are compounds which can be used as crosslinking agents and which form crosslinks with a chain length of 2 to 10 bridge atoms.
  • DMA dimethyl adipinimidate • 2HC1
  • DMP dimethyl pimelinidate • 2HC1
  • DMS diethylsuberimidate • 2HC1
  • DTBP dimethyl -3,3'-dithiobispropionimidate • 2HC1
  • DTSSP disuccinimidylsuberate Bis (sulfosuccini idyl) suberate (BS), dithiobis (succinimidylpropionate (DSP), 3,3'-dithiobis (sulfosuccinimidylpropionate) (DTSSP), ethylene glycol bis (succinimidylsuccinale) (EGS), ethylene glycol bis (sulfosuccinipateimidyl ), Disuccinimidyl tartrate (DST), disulfosuccinimidyl tetrate (sulfo-DST), bis [2- (succinimidooxycarbonyloxy)
  • a glutardialdehyde solution and a BS solution are particularly suitable for linking the tumor necrosis factor molecules.
  • the chaining is carried out in a buffer at a pH of 7-11, preferably 8-9. All customary buffers which do not contain an amino group are suitable as buffers.
  • the buffers are usually 0.001-1 molar, preferably 0.02 to 0.1 molar.
  • the concentration of tumor necrosis factor in the reaction mixture is 0.01 to 10, preferably 0.4 to 0.6 mg / ml.
  • the linking reagent is used in excess. The amount is normally 0.01-1 mg / ml, preferably 0.05-0.1 mg / ml.
  • the reaction time is between 10 min and 24 h, usually between 30 min and 5 h.
  • the reaction temperature is between 4 and 50 ° C, preferably at room temperature.
  • Suitable detergents are all customary ones, e.g. ®Tween 80, «Triton etc.
  • the amount is 0.001 to 0.1, preferably about 0.05%.
  • the new compounds can be used against cancer and are characterized by a high cytotoxicity and a favorable half-life. They are also suitable for the treatment of ascites.
  • S-Sepharose® from Pharmacia
  • Reaction product (TNF ⁇ -GDA) was obtained by eluting the column with 75 ml of a 20 mM sodium phosphate buffer pH 8.0.
  • Reaction product (TNF ⁇ -GDA) was obtained by eluting the column with 75 ml of a 20 mM sodium phosphate buffer pH 8.0.
  • game 2
  • the protein concentration of the product from Example 1 was determined by the method of Lowry [D.H. Lowry et al., J. Biol. Che., 193, 265-275 (1951) 3 determined at 0.98 mg against BSA as the standard.
  • the molecular weight of the reaction product was determined in a 15% SDS-PAGE in comparison with standard proteins.
  • the main product has a molecular weight of approximately 52 KD, which corresponds to a crosslinked trimeric TNF.
  • TNF ⁇ and TNF ⁇ -GDA were proteolyzed under exactly the same conditions with pepsin at pH 2.0 in phosphate buffer (substrate-enzyme weight ratio 20: 1) at 37 ° C. for 24 h.
  • TNF peptide chains can be derived from the primary structure of these peptides as follows:
  • ⁇ 25-lymphotoxin is a mutein of Ly photoxin, in which the first 25 amino acids are missing at the N-terminal.
  • MurT3-TNF is a mutein of TNF ⁇ , in which the following amino acid residues are exchanged: Asn30 »ser, Arg31 * Gln, Ilel36 * Leu, Aspl * 0 * Lys. 5 mg MurT3-TNF dissolved in 140 mM NaCl, 20 mM sodium phosphate buffer are reacted with glutardialdehyde analogously to Example A.
  • the reaction product covalently cross-linked trimeric TNF ⁇
  • Fig. 2 shows the result of the electrophoresis.
  • Lane 1 shows the separation of a protein isch that serves as the molecular weight standard.
  • the trace shows the behavior of the reaction product.

Abstract

Described are cross-linked tumour necrosis factors suitable for use in the treatment of illnesses.

Description

Quervernetzte Tumor-Nekrose-FaktorenCross-linked tumor necrosis factors
Die vorliegende Erfindung betrifft quervernetzte Tumor-Nekrose-Faktoren, deren Herstellung sowie deren Verwendung zur Bekämpfung von Krankheiten.The present invention relates to cross-linked tumor necrosis factors, their production and their use for combating diseases.
Tumor-Nekrose-Faktor α (TNFα) ist ein bekanntes Protein, welches in vielen biologischen Vorgängen eine Rolle spielt (Proc. Natl. Acad. Sei U.S.A 72, 3666 (1975), Ann. Rev. Biochem. 57, 505 (1988)). Ähnliches gilt für das strukturell sehr ähnliche Lymphotoxin (TNFß) (Nature 312, 721 (1984)).Tumor necrosis factor α (TNFα) is a known protein that plays a role in many biological processes (Proc. Natl. Acad. Sei USA 72, 3666 (1975), Ann. Rev. Biochem. 57, 505 (1988) ). The same applies to the structurally very similar lymphotoxin (TNFß) (Nature 312, 721 (1984)).
Die räumliche Struktur von TNFα ist bekannt (Nature 338, 225 (1989)). Danach ist TNFα ein aus drei identischen Untereinheiten symmetrisch auf¬ gebautes Protein. Dieses Tri ere ist die biologisch aktive Form des TNFα (J. Biol. Che . 262, 6951 (1987)).The spatial structure of TNFα is known (Nature 338, 225 (1989)). According to this, TNFα is a protein built up symmetrically from three identical subunits. This tri ere is the biologically active form of TNFα (J. Biol. Che. 262, 6951 (1987)).
Entsprechendes gilt für den TNFß.The same applies to the TNFß.
Es ist weiter bekannt, daß man Proteine mit einer Reihe von Reagentien, die mit Aminogruppen, Sulfhydrylgruppen oder aromatischen Gruppen der Protein kovalente Bindungen eingehen können, vernetzen kann (Tac H. Ji "Bifunctional Reagents" in Methods Enzymol. 91, Academic Press 1983 New York, Seiten 580-612). Hierbei entstehen jedoch wenig definierte Substanzgemisehe.It is also known that proteins can be crosslinked with a number of reagents which can form covalent bonds with amino groups, sulfhydryl groups or aromatic groups of the protein (Tac H. Ji "Bifunctional Reagents" in Methods Enzymol. 91, Academic Press 1983 New York, pages 580-612). However, this creates little-defined substance mixtures.
Es wurde nun gefunden, daß man durch Einhaltung bestimmter Reaktions¬ bedingungen molekular einheitliche vernetzte TNF-Derivate erhält, die gegenüber dem unveränderten TNF verbesserte Eigenschaften besitzen.It has now been found that by observing certain reaction conditions, molecularly uniform, crosslinked TNF derivatives are obtained which have improved properties compared to the unchanged TNF.
Gegenstand der Erfindung sind kovalent vernetzte Derivate des Tumor-Nekrose-Faktors, dadurch gekennzeichnet,The invention relates to covalently crosslinked derivatives of the tumor necrosis factor, characterized in that
daß sie durch Reaktion des Tumor-Nekrose-Faktors mit a inogruppen- wirksamen multifunktionellen Vernetzungsmolekülen hergestellt sindthat they are produced by reaction of the tumor necrosis factor with multifunctional crosslinking molecules which act in groups
- daß sie die drei gleichen Untereinheiten des Tumar-Nekrose-Faktors zu einem Molekül verknüpft enthalten- That they contain the three same subunits of the tumar necrosis factor linked to a molecule
daß sie chemisch definierte und weitgehend homogene Produkte dar¬ stellen.that they represent chemically defined and largely homogeneous products.
Unter dem Begriff "Tumor-Nekrose-Faktoren" sind TNFα, TNFß sowie Derivate dieser beiden Substanzen zu verstehen, die sich von diesen durch Aus- tausch, Deletion oder Addition einer oder mehrerer Aminosäuren unter¬ scheiden, ohne daß sich die cytotoxischen Eigenschaften wesentlich ver¬ schlechtern. Bevorzugt sind Derivate des TNFα.The term "tumor necrosis factors" is to be understood as TNFα, TNFß and derivatives of these two substances, which differ from these by Exchange, deletion or addition of one or more amino acids distinguish without the cytotoxic properties deteriorating significantly. Derivatives of TNFα are preferred.
Die einzelnen Tumor-Nekrose-Faktor-Ketten sind im N-terminalen Bereich miteinander vernetzt. Bevorzugt ist dabei die Verknüpfung zwischen der Aminogruppe der N-terminalen Aminosäure der 1. Kette der Aminogruppe am nächsten stehenden Lysinrest der 2. Kette von N-Terminus aus betrachtet (z.B. Lysii bei TNFα).The individual tumor necrosis factor chains are cross-linked in the N-terminal area. The link between the amino group of the N-terminal amino acid of the 1st chain of the amino group and the closest lysine residue of the 2nd chain from the N-terminus is preferred (e.g. Lysii for TNFα).
Analog sind auch die Verknüpfungen zwischen der 2. und 3. Kette sowie der 3. und 1. Kette.The links between the 2nd and 3rd chain and the 3rd and 1st chain are analogous.
In der Regel sind jeweils alle drei Ketten miteinander vernetzt.As a rule, all three chains are networked with each other.
Die quervernetzten Tumor-Nekrose-Faktoren werden hergestellt, indem man den Tumor-Nekrose-Faktor mit einem Verknüpfungsreagenz umsetzt.The cross-linked tumor necrosis factors are produced by reacting the tumor necrosis factor with a linking reagent.
Als Verknupfungsreagentien eignen sich Verbindungen, die als Quervernetzer eingesetzt werden können und die Querverbindungen mit einer Kettenlänge von 2 bis 10 Brückenatomen bilden.Suitable linkage reagents are compounds which can be used as crosslinking agents and which form crosslinks with a chain length of 2 to 10 bridge atoms.
Als solche sind zu nennen: Dimethyladipinimidate • 2HC1 (DMA), Dimethyl- pimelinidate • 2HC1 (DMP), Di ethylsuberimidate • 2HC1 (DMS), Dimethyl- -3,3'-dithiobispropionimidate • 2HC1 (DTBP), Disuccinimidylsuberate (DSS), Bis (sulfosuccini idyl)suberate (BS), Dithiobis(succinimidylpropionate (DSP), 3,3'-Dithiobis(sulfosuccinimidylpropionate) (DTSSP), Ethylenglycol- bis(succinimidylsuccinale) (EGS), Ethylenglycolbis(sulfosucciπimidyl- succinate) (Sulfo-EGS), Disuccinimidyltartrat (DST), Disulfosuccinimidyl- tratrat (Sulfo-DST), Bis[2-(succinimidooxycarbonyloxy)ethyl]sulfon (BSOCOES), Bis[2-(sulfosuccinimidooxycarbonyloxy)ethyl]sulfon (Sulfo-BSOCOES) und 4,4'-Diisothiocyano-2,2'-disulfonicacidstilbene (DIDS). Nähere Angaben zu diesen Reagentien finden sich im "Handbook and General Catalog" der Firme Pierce (1989) auf den Seiten 286-295.These include: dimethyl adipinimidate • 2HC1 (DMA), dimethyl pimelinidate • 2HC1 (DMP), diethylsuberimidate • 2HC1 (DMS), dimethyl -3,3'-dithiobispropionimidate • 2HC1 (DTBP), disuccinimidylsuberate Bis (sulfosuccini idyl) suberate (BS), dithiobis (succinimidylpropionate (DSP), 3,3'-dithiobis (sulfosuccinimidylpropionate) (DTSSP), ethylene glycol bis (succinimidylsuccinale) (EGS), ethylene glycol bis (sulfosuccinipateimidyl ), Disuccinimidyl tartrate (DST), disulfosuccinimidyl tetrate (sulfo-DST), bis [2- (succinimidooxycarbonyloxy) ethyl] sulfone (BSOCOES), bis [2- (sulfosuccinimidooxycarbonyloxy) ethyl] sulfone (sulfo-BSOCOES) and 4,4 '-Diisothiocyano-2,2'-disulfonicacidstilbene (DIDS) More information on these reagents can be found in the "Handbook and General Catalog" by Pierce (1989) on pages 286-295.
Besonders geeignet für die Verkettung der Tumor-Nekrose-Faktor-Moleküle sind eine Glutardialdehydlösung und eine BS-Lösung.A glutardialdehyde solution and a BS solution are particularly suitable for linking the tumor necrosis factor molecules.
Die Verkettung wird in einem Puffer bei einem pH-Wert von 7-11, vorzugs- weise 8-9, vorgenommen. Als Puffer eignen sich alle üblichen Puffer, die keine Aminogruppe enthalten. Die Puffer sind in der Regel 0,001-1 molar, vorzugsweise 0,02 bis 0,1 molar. Die Konzentration an Tumor-Nekrose-Faktor im Umsetzungsgemisch liegt bei 0,01 bis 10, vorzugsweise 0,4 bis 0,6 mg/ml. Der Verknüpfungsreagenz wird im Überschuß verwendet. Normal beträgt die Menge 0,01-1 mg/ml, vorzugs¬ weise 0,05-0,1 mg/ml.The chaining is carried out in a buffer at a pH of 7-11, preferably 8-9. All customary buffers which do not contain an amino group are suitable as buffers. The buffers are usually 0.001-1 molar, preferably 0.02 to 0.1 molar. The concentration of tumor necrosis factor in the reaction mixture is 0.01 to 10, preferably 0.4 to 0.6 mg / ml. The linking reagent is used in excess. The amount is normally 0.01-1 mg / ml, preferably 0.05-0.1 mg / ml.
Die Reaktionszeit liegt zwischen 10 min und 24 h, in der Regel zwischen 30 min und 5 h. Die Reaktionstemperatur liegt zwischen 4 und 50°C, vor¬ zugsweise bei Raumtemperatur.The reaction time is between 10 min and 24 h, usually between 30 min and 5 h. The reaction temperature is between 4 and 50 ° C, preferably at room temperature.
Es empfiehlt sich, die Umsetzung in Gegenwart eines Detergenz durchzu¬ führen. Als Detergentien eignen sich alle üblichen, wie z.B. ®Tween 80, «Triton usw. Die Menge liegt bei 0,001 bis 0,1 , vorzugsweise etwa 0,05 %.It is advisable to carry out the reaction in the presence of a detergent. Suitable detergents are all customary ones, e.g. ®Tween 80, «Triton etc. The amount is 0.001 to 0.1, preferably about 0.05%.
Nach Ende der Umsetzung wird überschüssiges Verknüpfungsreagenz mit einem A in, vorzugsweise Ethanolamin, zerstört.At the end of the reaction, excess linking reagent with an A in, preferably ethanolamine, is destroyed.
Die neuen Verbindungen lassen sich sich gegen Krebserkrankungen einsetzen und zeichnen sich dabei durch eine hohe Cytσtoxizität und eine günstige Halbwertszeit aus. Sie eignen sich auch zur Behandlung von Ascites.The new compounds can be used against cancer and are characterized by a high cytotoxicity and a favorable half-life. They are also suitable for the treatment of ascites.
Beispiel 1example 1
Umsetzung von TNFα mit Glutardialdehyd (GDA).Implementation of TNFα with glutardialdehyde (GDA).
400 ml einer 0,5 mg/ml TNFα enthaltenden wäßrigen Pufferlösung (50 mM Natriumphosphat, pH 8,5) wurden 10 min im Vakuum entgast und anschließend mit Argon begast. Zu der so erhaltenen Lösung wurden unter Rühren bei Raumtemperatur in einer Argon-Atmosphäre 40,8 μl 25 %ige Glutardialde- hydlösung gegeben und der Reaktionsansatz 3 h bei Raumtemperatur gerührt. Anschließend wurde überschüssiger Glutardialdehyd durch Zugabe von 50 μl Ethanolamin inaktiviert und 1 h bei Raumtemperatur nachgerührt. Der Reaktionsansatz wurde danach mit 10 %iger Essigsäure auf pH 4,5 ein¬ gestellt.400 ml of an aqueous buffer solution containing 0.5 mg / ml TNFα (50 mM sodium phosphate, pH 8.5) were degassed in vacuo for 10 min and then gassed with argon. 40.8 μl of 25% strength glutardialdehyde solution were added to the solution thus obtained with stirring at room temperature in an argon atmosphere, and the reaction mixture was stirred at room temperature for 3 h. Excess glutardialdehyde was then inactivated by adding 50 μl of ethanolamine and the mixture was stirred at room temperature for 1 h. The reaction mixture was then adjusted to pH 4.5 with 10% acetic acid.
Die Reinigung des entstandenen Produkts (TNFα-GDA) erfolgte durch Chromatographie an S-Sepharose® (Fa. Pharmacia). Dazu wurde eine mit S-Sepharose® gefüllte Säule (0 = 5 cm, 1 = 5 cm) mit 300 ml 20 mM Natrium- acetatpuffer pH 4,75 äquilibriert. Nach Auftragen des Reaktionsansatzes wurde mit weiteren 400 ml des Äquilibrierpuffers nachgewaschen. DasThe resulting product (TNFα-GDA) was purified by chromatography on S-Sepharose® (from Pharmacia). For this purpose, a column filled with S-Sepharose® (0 = 5 cm, 1 = 5 cm) was equilibrated with 300 ml of 20 mM sodium acetate buffer pH 4.75. After the reaction mixture had been applied, the mixture was washed with a further 400 ml of the equilibration buffer. The
Reaktionsprodukt (TNFα-GDA) wurde durch Elution der Säule mit 75 ml eines 20 mM Natriumphosphatpuffers pH 8,0 gewonnen. Bei spiel 2Reaction product (TNFα-GDA) was obtained by eluting the column with 75 ml of a 20 mM sodium phosphate buffer pH 8.0. In game 2
Charakterisierung des Reaktionsproduktes TNFα-GDA aus Beispiel 1Characterization of the reaction product TNFα-GDA from Example 1
a) Proteinkonzentrationa) Protein concentration
Die Proteinkonzentration des Produkts aus Beispiel 1 wurde nach der Methode von Lowry [D.H. Lowry et al., J. Biol. Che ., 193, 265-275 (1951)3 zu 0,98 mg gegen BSA als Standard bestimmt.The protein concentration of the product from Example 1 was determined by the method of Lowry [D.H. Lowry et al., J. Biol. Che., 193, 265-275 (1951) 3 determined at 0.98 mg against BSA as the standard.
b) SDS-Polyacrylamidgelektrophorese (SDS-PAGE)b) SDS polyacrylamide gel electrophoresis (SDS-PAGE)
In einer 15 %igen SDS-PAGE wurde das Molekulargewicht des Reaktions¬ produkts im Vergleich mit Standardproteinen bestimmt. Das Hauptprodukt hat ein Molekulargewicht von ca. 52 KD, was einem vernetzten trimeren TNF entspricht.The molecular weight of the reaction product was determined in a 15% SDS-PAGE in comparison with standard proteins. The main product has a molecular weight of approximately 52 KD, which corresponds to a crosslinked trimeric TNF.
c) Vergleichendes Peptide- appingc) Comparative peptide apping
TNFα und TNFα-GDA wurden unter exakt gleichen Bedingungen mit Pepsin bei pH 2,0 in Phosphatpuffer (Substrat-Enzym-Gewichtsverhältnis 20 : 1) 24 h bei 37°C proteolysiert.TNFα and TNFα-GDA were proteolyzed under exactly the same conditions with pepsin at pH 2.0 in phosphate buffer (substrate-enzyme weight ratio 20: 1) at 37 ° C. for 24 h.
Die entstehenden Gemische wurden über eine RP-Vydac Ciβ-Säule mit einem Gradienten von 0, 1 % TFA in H2O nach 0, 1 % TFA in Acetolnitril vergleichend durch HPLC analysiert (UV-Detektion bei 210 nm) .The resulting mixtures were compared on a RP-Vydac Ciβ column with a gradient of 0.1% TFA in H2O to 0.1% TFA in acetonitrile by HPLC (UV detection at 210 nm).
Der vergleich der beiden Chromatogramme (Fig. 1) zeigte die durch die Vernetzung veränderten Peptide an. Die Peptidmuster sind nahezu identisch bis auf die Peptide (1) und (2), die nach der Vernetzungs- reaktion in b fehlen.The comparison of the two chromatograms (FIG. 1) indicated the peptides changed by the crosslinking. The peptide patterns are almost identical except for peptides (1) and (2), which are missing in b after the cross-linking reaction.
Aus der Primärstruktur dieser Peptide läßt sich die Verknüpfung der TNF-Peptidketten wie folgt ableiten:The linkage of the TNF peptide chains can be derived from the primary structure of these peptides as follows:
NH2-VaH ( 1 . Kette) -GDA-NH2-Lysl i (2. Kette) NH2-Val i (2. Kette )-GDA-NH2-Lysl l (3. Kette) NH2-VaU (3. Kette)-GDA-NH2-Lysll (1. Kette) Bei spiel 3NH 2 -VaH (1st chain) -GDA-NH 2 -Lysl i (2nd chain) NH 2 -Val i (2nd chain) -GDA-NH 2 -Lysl l (3rd chain) NH 2 -VaU ( 3rd chain) -GDA-NH 2 -Lysll (1st chain) In game 3
Selektive Vernetzung von Δ25-Lymphtoxin (Δ25-TNF-ß) mit GlutardialdehydSelective cross-linking of Δ25-lymph toxin (Δ25-TNF-ß) with glutardialdehyde
Δ25-Lymphotoxin ist ein Mutein des Ly photoxins, in dem N-terminal die ersten 25 Aminosäuren fehlen.Δ25-lymphotoxin is a mutein of Ly photoxin, in which the first 25 amino acids are missing at the N-terminal.
5 mg Δ25-Lymphotoxin gelöst in 140 mM NaCl, 20 mM Natriumphosphatpuffer, pH 8,5 wurden unter Vakuum entgast und anschließend mit Argon begast. Zu dieser Lösung werden unter Rühren 1 ml 25 %ige Glutardialdehydlösung gegeben und 3 h gerührt.5 mg of Δ25-lymphotoxin dissolved in 140 mM NaCl, 20 mM sodium phosphate buffer, pH 8.5 were degassed under vacuum and then gassed with argon. 1 ml of 25% glutardialdehyde solution are added to this solution with stirring and the mixture is stirred for 3 hours.
überschüssiges Glutardialdehyd wurde durch Zugabe von 1,2 μl Ethanolamin und 1 h Nachrühren inaktiviert.Excess glutardialdehyde was inactivated by adding 1.2 μl ethanolamine and stirring for 1 h.
Beispiel 4Example 4
Selektive Vernetzung des TNF α-Muteins, MurT3-TNF, mit GlutardialdehydSelective crosslinking of the TNF α mutein, MurT3-TNF, with glutardialdehyde
MurT3-TNF ist ein Mutein des TNFα, bei dem folgende Aminosäurereste ausgetauscht sind: Asn30»ser, Arg31*Gln, Ilel36*Leu, Aspl*0*Lys. 5 mg MurT3-TNF gelöst in 140 mM NaCl, 20 mM Natriumphosphatpuffer werden analog Beispiel A mit Glutardialdehyd umgesetzt.MurT3-TNF is a mutein of TNFα, in which the following amino acid residues are exchanged: Asn30 »ser, Arg31 * Gln, Ilel36 * Leu, Aspl * 0 * Lys. 5 mg MurT3-TNF dissolved in 140 mM NaCl, 20 mM sodium phosphate buffer are reacted with glutardialdehyde analogously to Example A.
Beispiel 5Example 5
Selektive Vernetzung von TNFα mit Bis(sulfosuccinimidyl)suberat (BS)Selective crosslinking of TNFα with bis (sulfosuccinimidyl) suberate (BS)
1 mg TNFα, gelöst in 1,5 ml 20 mM Natriumphosphatpuffer, pH 8,5 werden mit 96 μl einer 10 mg/ml-Lösung von BS (Fa. Piera) in demselben Puffer ver¬ setzt und die Reaktionslösung 2 h bei Raumtemperatur gerührt. Anschließend wird mit 50 μl einer 1 %igen Ethanolaminlösung versetzt und 1 h bei Raumtemperatur stehen gelassen.1 mg of TNFα, dissolved in 1.5 ml of 20 mM sodium phosphate buffer, pH 8.5, are mixed with 96 μl of a 10 mg / ml solution of BS (from Piera) in the same buffer and the reaction solution is stirred for 2 hours at room temperature . 50 μl of a 1% ethanolamine solution are then added and the mixture is left to stand at room temperature for 1 h.
Das Reaktionsprodukt, kovalent vernetzter trimerer TNFα, kann durch SDS-Polyacrylamidgelektrophorese bei - 52 000 Dalton in der Färbung mit Co assie-blau sichtbar gemacht werden. Fig. 2 zeigt das Ergebnis der Elektrophorese. Die Spur 1 zeigt die Auftrennung eines Proteinge ischs, das als Molekulargewichtsstandard dient. Die Spur zeigt das Verhalten des Reaktionsprodukts. The reaction product, covalently cross-linked trimeric TNFα, can be visualized by SDS-polyacrylamide gel electrophoresis at - 52,000 Daltons in the coloration with Co assie-blue. Fig. 2 shows the result of the electrophoresis. Lane 1 shows the separation of a protein isch that serves as the molecular weight standard. The trace shows the behavior of the reaction product.

Claims

Patentansprüche Claims
1. Kovalent vernetzte Derivate des Tumor-Nekrose-Faktors, dadurch gekennzeichnet, 5 daß sie durch Reaktion des Tumor-Nekrose-Faktors mit aminogruppenwirksamen multifunktionellen Vernetzungsmolekülen hergestellt sind1. Covalently cross-linked derivatives of the tumor necrosis factor, characterized in 5 that they are produced by reaction of the tumor necrosis factor with amino-functional multifunctional cross-linking molecules
10 - daß sie die drei gleichen Untereinheiten des10 - that they are the same three subunits of the
Tumor-Nekrose-Faktors zu einem Molekül verknüpft enthaltenTumor necrosis factor included linked to a molecule
daß sie chemisch definierte und weitgehend homogene Produkte darstellen.that they are chemically defined and largely homogeneous products.
1515
Kovalent vernetzte Derivate des Tumor-Nekrose-Faktors ß gemäß Anspruch 1.Covalently cross-linked derivatives of the tumor necrosis factor β according to claim 1.
3. Kovalent vernetzte Derivate des Tumor-Nekrose-Faktors ß gemäß 20 Anspruch 1.3. Covalently cross-linked derivatives of the tumor necrosis factor β according to claim 20.
4. Kovalent vernetzte Derivate von Muteinen des Tumor-Nekrose-Faktors α bzw. ß gemäß Anspruch 1.4. Covalently cross-linked derivatives of muteins of the tumor necrosis factor α or β according to claim 1.
255. Verfahren zur Herstellung von kovalent vernetzten Derivaten des Tumor- Nekrose-Faktors, dadurch gekennzeichnet, daß man Tumor-Nekrose-Faktor mit einem Verknüpfungsreagenz umsetzt.255. Process for the preparation of covalently cross-linked derivatives of the tumor necrosis factor, characterized in that the tumor necrosis factor is reacted with a linking reagent.
6. Kovalent vernetzte Derivate von Tumor-Nekrose-Faktorα und ß und deren 30 Muteinen zur Verwendung bei der Bekämpfung von Krankheiten.6. Covalently cross-linked derivatives of tumor necrosis factor α and β and their 30 muteins for use in combating diseases.
3535
4040
Zeichn. Sign.
PCT/EP1990/001754 1989-11-04 1990-10-17 Cross-linked tumour necrosis factors WO1991006560A1 (en)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992006995A1 (en) * 1990-10-18 1992-04-30 Board Of Regents, The University Of Texas System Preparation and characterization of liposomal formulations of tumor necrosis factor
US5607174A (en) * 1995-05-17 1997-03-04 Ambrogio; Patrick Folding wheelbarrow
US5653974A (en) * 1990-10-18 1997-08-05 Board Of Regents,The University Of Texas System Preparation and characterization of liposomal formulations of tumor necrosis factor

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0247860A2 (en) * 1986-05-29 1987-12-02 Cetus Oncology Corporation Tumor necrosis factor formulation and its preparation

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0247860A2 (en) * 1986-05-29 1987-12-02 Cetus Oncology Corporation Tumor necrosis factor formulation and its preparation

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
DIALOG INFORMATION SERVICES, File 154, Medline 83-91, NLM Accession Number 87222282, SMITH R.A. et al., "The Active Form of Tumor Necrosis Factor is a Trimer"; & J. BIOL. CHEM., 25 May 1987, 262(15), p. 6951-4. *
DIALOG INFORMATION SERVICES, File 154, Medline 83-91, NLM Accession Number 88274422, LAM K.S. et al., "Analysis of the Molecular Organization of Recombinant Human Tumor Necrosis Factor (rINF) in Solution Using Ethylene Glycolbis (succinimidylsuccinate) as the Crosslinking Reagent"; & J. BIOL. RESPONSE MOD., June 1988, 7(3), p. 267-75. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992006995A1 (en) * 1990-10-18 1992-04-30 Board Of Regents, The University Of Texas System Preparation and characterization of liposomal formulations of tumor necrosis factor
US5653974A (en) * 1990-10-18 1997-08-05 Board Of Regents,The University Of Texas System Preparation and characterization of liposomal formulations of tumor necrosis factor
US5607174A (en) * 1995-05-17 1997-03-04 Ambrogio; Patrick Folding wheelbarrow

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