WO2000021981A2

WO2000021981A2 - TNFα BINDING PEPTIDES AND THEIR UTILIZATION FOR DETECTING, DEACTIVATING AND/OR REMOVING TNFα FROM BIOLOGICAL FLUIDS

Info

Publication number: WO2000021981A2
Application number: PCT/DE1999/003256
Authority: WO
Inventors: Heinz-Jürgen WAGNER; Hans-Werner Heinrich; Udo Meyer
Original assignee: Bioserv Ag
Priority date: 1998-10-09
Filing date: 1999-10-08
Publication date: 2000-04-20
Also published as: DE19948867A1; WO2000021981A3

Abstract

The invention relates to peptides having formula NH2-IQTLHAQ-COOH and NH2-RLANTHFVY-COOH that specifically bind in vivo and ex vivo TNF alpha . Said peptides are suitable for producing TNF alpha test systems and/or for removing TNF alpha from body fluids, e.g. by means of plasmapheresis.

Description

TNFα binding peptides and their application for the detection, inactivation and / or removal of TNFα from biological fluids

The invention relates to the construction and use of peptides which are capable of specifically binding to TNFα. According to current knowledge, these peptides do not occur in nature. With their help, TNFα can be specifically bound, biologically inactivated and / or removed from mixtures of substances.

TNF is a soluble protein (relative molecular weight 17 kD), which is produced and released primarily by monocytes and macrophages in response to endotoxins or other noxae. It was first described by Carswell, E.A. et al. (PNAS USA 72 (1975), 3666), who detected it in the serum of animals which had previously been treated with endotoxin and Bacillus Calmette Guerin (BCG).

The biologically active form is the trimere from the 17 kDa subunits. It occurs in the form of the similar variants TNFα and ß.

TNF plays a central role in the regulation of the body's defense mechanisms after infections and injuries, in the maintenance of immunopathological processes and tumor regression (Buetler, B. et al., Buetler, B. Ed. Tumor Necrosis Factors: the Molecules and Their Emerging Role in Medicine (Raven Press, New York, 1992)). It intervenes in the regulation of a variety of biological phenomena, such as B. the activation of poiymo nucleated granulocytes, cell growth, inhibition of lipoprotein lipase, antiviral activity, stimulation of collagenase, stimulation of prostaglandin E, activation of T and B cells, increased expression of MHC molecules, etc.

Excessive activity of TNF is of pathogenetic importance for chronic inflammatory processes (e.g. rheumatoid arthritis), septicemic shock and cachexia. The increased TNF production causes an excessive release of interleukin (IL) 6 and granulocyte / macrophage colony stimulating factor (GM-CSF). These cytokines increase the cytotoxicity of poiymorphonuclear neutrophil granulocytes and the expression of cellular adhesin proteins. Both phenomena are jointly responsible for the maintenance of the inflammatory processes (Williams et al. PNAS USA, 98 (1992), 9784). Cachexia (anorexia in connection with progressive loss of body mass) in tumor patients or as a result of infectious diseases leads to life-threatening conditions. TNF is a crucial mediator for cachexia in these diseases. Repeated doses of TNF to mice cause anorexia, weight loss and the breakdown of body fat and protein within 7-10 days (Carami et al., Immunol. Lett. 11 (1985), 173). These effects are reduced by the simultaneous administration of antibodies against TNF.

TNF plays a central role in the pathophysiology of sepsis with gram-negative microorganisms and endotoxin shock. Endotoxins of the Gram-negative bacteria stimulate the monocytes / macrophages to release TNF (in the wake of the synthesis of further cytokines). TNF and other monocyte cytokines mediate the organism's metabolic and neurohormonal response to endotoxins (fever, nausea, anorexia and cachexia), and TNF alone is capable of causing these clinical symptoms. Chronic intravenous TNF infusions are associated with anorexia, water retention, acute phase reaction and negative nitrogen balance (Michie, H.R. et al., Ann. Surg. 209 (1989), 19).

Increased TNF values are measured in transplant patients in the acute phase of the organ rejection reaction (Maury and Teppo; J. Exp. Med. 166 (1987), 1132). The application of antibodies against TNF prevents the typical histological changes of a 'graft versus host' reaction in animal experiments.

The diverse biological effects of TNF (alpha and beta) are mediated by two transmembrane receptors. (TNF-R55 and TNF-R75). The extracellular domains of both receptor proteins have a 28% amino acid sequence homology and both have four relatively conserved subdomains. The cytoplasmic sections of both receptor proteins have no homologies, which indicates a different way of signal transduction.

Proteolysis of the extracellular receptor section releases in vivo soluble proteins which are able to bind and inactivate TNF (TNF binding protein). Such TNF inhibitors can be detected in the urine of healthy people as well as in the serum of tumor patients. Many influences that lead to the activation of TNF release also result in an increased release of soluble TNF receptors. This leads to the conclusion that they act in the sense of negative feedback on TNF synthesis. The inhibition of TNF could be of great importance for the basic or supportive therapy of many diseases.

Many methods have been described for inactivating and / or removing TNF from patients. WO 96/16666 describes a method for removing TNF by means of immunoadsorption and plasmapheresis. Antibodies against TNF are coupled to a matrix, which bind TNF as specific ligands during plasmapheresis.

Patents WO 90/06938 and WO 90/06947 (Boehm et al.) Claim a large number of peptides which are derived from the amino acid sequence of the TNF and block the TNF receptor.

Rathjen (WO 93/06128, WO 93/01211) describes peptides which have been derived from the TNF receptor structure and are able to bind specifically to the TNF and to inactivate its biological function. These peptides were able to neutralize biological effects in vitro and in vivo (tumor regression, TNF toxicity _M. , malaria _M ).

In patent WO 92/11285 Boehm et al. further peptides which were derived from the TNF amino acid sequence and which prevent the binding and thus the biological effect of the native TNF by occupying the TNF receptor.

All examples of the effectiveness of the receptor blockade were demonstrated in vitro. However, statements on pharmacological efficacy based on animal experiments are not provided.

In the claims of patent WO 97/02345 all substances are protected which are able to bind to the intracellular domain of the TNF and thereby influence the TNF effect in some form.

The invention relates to an empirical method for determining oligopeptide structures which are suitable for specifically binding to TNFα. Such peptides are conventionally constructed as a result of ligand-receptor studies or on the basis of the analysis of the ligands, their natural receptors as anti-sense peptides. On the basis of this method, the peptides NH ₂ - IQTLHAQ-COOH and NH - RLANTFHVY-COOH according to the invention were synthesized, which selectively recognize and bind TNFα and inactivate their biological action in vitro.

The invention is implemented according to claims 1-12.

The invention will be explained in more detail below by means of exemplary embodiments.

I. to IV. Exemplary embodiments for the binding of TNF-alpha by the carrier-bound oligopeptide T2

Binding system:

1. Equilibrate the carrier-bound oligopeptide in 300 μl BP for 1 h at 37 ^° C.

2. Block the carrier-bound oligopeptide carrier in 200 μl BP with 1% BSA for 3 h at 37 ^° C.

3. Wash the carrier-bound oligopeptide in 250 ul BP for 2 x 20 min at 37 ^° C.

4. Incubate ¹²⁵ J-TNF and / or unlabeled TNF with the carrier-bound oligopeptide in 100 μl BP for 90 min at 25 ^° C.

5. Wash the carrier-bound oligopeptide in 300 μl BP for 2 x 20min at 25 ^° C

6. Measurement per cpm

Each individual binding was carried out as a double determination.

Binding buffer (BP):

100mM Tris, 0.1% Tween-20, 140mM NaCl, 0.1% BSA

I. Relationship between the binding of TNF and the TNF concentration in the binding approach, determination of the binding constant

Fig. L: Binding of ^1Λ J-TNF to Oligo T2

[r! "25'Jι-TNF] = 0.227-10nM

Measured values for Fig. 1:

J-TNF HSA + BSA cpm T2 LW

227 pM 1.56 µM 3054 394

500 pM 1.62 µM 5240 672

I nM 1.74 µM 9571 875

5 nM 3.3 µM 28593 2578

10 nM 6.6 µM 59168 5536 Fig.2: Binding of ¹²⁵ J-TNF to Oligo T2 [ ^{, 25} J-TNF] = 10 - 40 nM

Measured values for Fig. 2:

"J-TNF HSA + BSA cpm T2 LW

10 nM 15 µM 6037 1680

20 nM 15 µM 25889 6021

40 nM 15 µM 31735 13785

By evaluating the curves from Fig. 1 and Fig. 2, a binding constant of 15 - 20 nM can be determined for the binding of TNF by the carrier-bound oligopeptide T2.

II. Specific inhibition of the binding of ¹²⁵ J-TNF to the carrier-bound oligopeptide T2 by unlabelled TNF (competition inhibition)

Fig. 3: Competition for the binding of 125 J, -TNF to oligo T2 by unlabeled TNF [ ¹²⁵ J-TNF] = 4.55 nM

Measured values for Fig. 3:

TNF cold ¹²⁵ J-TNF BSA + HSA cpm too

TNF cold

0 1: 0 3.00 µM 21609

4.550 nM 1: 1 3.06 µM 18487

9.100 nM 1: 2 3.12 µM 17170

18,200 nM 1: 4 3.24 µM 13762 III. Binding of ^J25 J-TNF from diluted blood plasma by carrier- ^bound oligopeptide T2

Table 1: TNF binding in the binding buffer and in plasma diluted with BP

[ ^I25 J-TNF] portion of plasma in TNF binding

Binding buffer

2 nM 0 100%

25% 52%

50% 30%

20 nM 0 100%

25% 58%

59% 33%

IV. Specific inhibition of the binding of ¹²⁵ J-TNF to the carrier-bound oligopeptide T2 in plasma diluted with BP by unlabelled TNF (competition inhibition)

Fig. 4: Competition of binding of ^I25 J-TNF to oligo T2 by unlabeled TNF in the presence of blood plasma [ ¹²⁵ J-TNF] = 0.85 nM proportion of blood plasma in the BP = 25%

Measured values for Fig. 4:

TNF cold ¹²⁵ J-TNF cpm to TNF cold

0 1: 0 8319

0.85 nM 1: 1 5117

3.40 nM 1: 4 5078 V. Embodiment - TNFα cytotoxicity test

The ability of the peptides according to the invention to bind to the TNFα and thereby neutralize its cytotoxic properties was demonstrated using mouse biofibroblasts (cell line L929).

The cells were grown in Dulbecco's modified Eagle's Medium (DMEM) using 10% fetal calf serum (FKS).

24 hours before the experiment, the cells of the closed monolayer are separated with trypsin / EDTA and taken up in DMEM / FKS. 5 × 10 ⁴ cells in 50 ml are sown into the wells of a 96-well cell culture plate. The cell culture plates are incubated for 3-5 hours at 37 ^° C. in a 5% CO ₂ atmosphere. The monolayer should be closed approximately 50% before use. About 2 mM solutions in 10 mM HEPES (pH 7.5) are prepared from the peptides to be tested. The solutions are sterile filtered (200 nm) and diluted 1: 4 in 10 mM HEPES (pH 7.5). The monoclonal antibody TA-31 (Sigma) with known inactivation activity serves as a control.

DMEM / FKS is mixed with actinomycin D (10 μg / ml) and recombinant human TNFα (1 ng / ml). 75 μl of this solution are mixed with 75 μl of the respective peptide dilution or of the antibody (control 1) or HEPES (control 2). After an incubation of 30 min at room temperature, the mixture of each peptide dilution stage or of the controls is inoculated in 3 cavities (50 μl / cavity each), in each of which 50 μl of the cell suspension was preincubated. Control 3 is a mixture of 25 μl / well DMEM / FKS with actinomycin D (10 mg / ml) and 25 l / well HEPES (pH 7.5).

The cell culture plates are incubated overnight at 37 ^° C. in a 5% CO ₂ atmosphere. After crystal violet staining of the non-lysed cells, the relative inhibition of the cytotoxic TNFα activity by the peptides is determined.

Claims

claims

1. TNFα binding peptides consisting of 4-9 amino acids.

2. TNFα-binding peptides according to claim 1, characterized by the amino acid sequence I.

I NH - I-Q-T-L-H-A-Q-COOH

3. TNFα-binding peptides according to claim 1 and 2, characterized in that they consist of fragments of the formula I which are at least 4 amino acids long.

4. TNFα-binding peptides according to claim 2 and 3, characterized in that individual amino acids are replaced by analog amino acids.

5. TNFα-binding peptides according to claim 1, characterized by the amino acid sequence II

II NH ₂ -RLANTFHVY-COOH

6. TNFα-binding peptides according to claim 1 and 5, characterized in that they consist of fragments of the formula II which are at least 4 amino acids long.

7. TNFα-binding peptides according to claim 5 and 6, characterized in that individual amino acids are replaced by analog amino acids.

8. TNFα-binding peptides according to claims 1-7, characterized in that they are blocked at the N- and / or C-terminus.

9. TNFα-binding peptides according to claim 8, characterized in that they are blocked by acetylation, alkylation, aralkylation or amidation.

10. Use of the peptides according to claims 1-9 as a diagnostic agent for the detection of TNFα.

11. Use of the peptides according to claims 1-9 for inactivating and / or removing TNFα in body fluids.

12. Use of the peptides according to claims 1-9 as a medicament for combating neoplastic and autoimmune diseases and for combating and prophylaxis of infections, inflammation and rejection reactions in transplantations or as a specific ligand for detoxification alone or in combination with suitable carriers.