WO1995010533A1

WO1995010533A1 - Peptides for tumour therapy

Info

Publication number: WO1995010533A1
Application number: PCT/DE1994/001209
Authority: WO
Inventors: Markus Grewe; Jean Krutmann
Original assignee: KLINIKUM DER ALBERT-LUDWIGS-UNIVERSITäT FREIBURG
Priority date: 1993-10-15
Filing date: 1994-10-11
Publication date: 1995-04-20
Also published as: DE4335273A1; AU7852194A

Abstract

The invention relates to peptides which are useful in the treatment of tumour conditions because they promote the destruction of tumour cells by the body's own lymphocytes.

Description

Peptides for tumor therapy

The present invention relates to peptides which can be used for the treatment of tumor diseases.

It is known that the interaction between leukocytes and their target cells is mediated by special adhesion molecules. The so-called intercellular adhesion molecule-1 (ICAM-1) is an immunoglobulin-like single chain transmembrane molecule that is functionally defined by its ability to serve as a receptor of LFA-1. Lymphocyte function antigen-1 (LFA-1) is found on the surface of leukocytes, especially on the surface of lymphokine-activated killer cells. Adhesion mediated by LFA-1 / ICAM-1 plays a role in a variety of immunological processes, e.g. in antigen presentation or the formation of an inflammatory infiltrate in human tissues. ICAM-1 also acts as a receptor for certain human pathogenic viruses, e.g. Rhinoviruses. In vitro experiments have found that the cytotoxic destruction of tumor cells, in particular melanoma cells, is mediated by the binding of LFA-1 molecules on the surface of lymphokine-activated killer cells to melanoma cells which carry the ICAM-1 molecule. If activated killer cells bind to melanoma cells, this causes the lysis and thus the destruction of the tumor cells.

r Staunton et al. have disclosed an amino acid sequence of an ICAM-1 molecule in the article "Primary Structure of ICAM-1 Demonstrates Interaction between Members of the Immunoglobulin and Integrin Supergene Families" (Cell, Vol. 82 [1988], pp. 925-933).

The ICAM-1 molecule is normally membrane bound in the cells. This form is called mICAM-1. However, there is also a soluble form of this molecule called sICAM-1. The soluble molecule sICAM-1 is functionally active, since it can bind to LFA-1 and it could be shown in in vitro experiments that the cytotoxicity against melanoma cells caused by lymphokine-activated killer cells can be inhibited by sICAM-1 (Becker et al. "Shedding of ICAM-1 from Human Melanoma Cell Lines Induced by I N- ^ and Tumor Necrosis Factor-α", Journal of Immunology, [1991], Vol. 147, pp. 4398- 4401).

By Budnik et al. (Exp. Dermatol. 1992, Vol. 1, pp. 27-30 "Human Epidermal Keratinocytes are a Source of Soluble ICAM-1 Molecules") it was disclosed that both malignant and non-malignant keratinocytes (keratinizing cells of the epidermis) after stimulation with cytokine interferon- are able to produce both membrane-bound mICAM-1 and soluble sICAM-1. The soluble sICAM-1 has a lower molecular weight than the membrane-bound mICAM-1 and is created by proteolytic cleavage of mICAM-1. These soluble molecules sICAM-1 are functionally active, i.e. they can bind to LFA-1. However, if the soluble molecules sICAM-1 are present and bind them to the LFA-1 of the lymphocytes, then there is a fear that the leukocytes will be blocked by the soluble molecular form and will no longer be able to perform their function, namely the destruction of tumor cells.

Replacement sheet The object of the present invention is to provide means which can be used successfully in the therapy of tumor diseases.

In the context of the present invention, it was found that the peptides according to the invention can advantageously be used in tumor therapy, since these peptides surprisingly inhibit the formation of sICAM-1 without reducing the expression of membrane-bound mICAM-1.

The peptides according to the invention can have the amino acid sequence REVTVNVLSPRYEIVIITWAAAVIM. The amino acid sequence is given in the 1-letter code. The meaning of the respective letters is given for example in Römpps Chemielexikon, 8th edition, 1979, Vol. 1, p. 176. The amino acid sequence given in the 3-letter code is:

Arg Glu Val Thr Val Asn Val Leu Ser Pro Arg Tyr Glu Ile Val Ile Ile Thr Val Val Ala Ala Ala Val Ile Met.

The peptides of the invention may have a deletion at one or both ends of the above sequence, each deletion comprising up to five amino acids.

In biological systems it is often the case that the molecules are still functionally active even when individual amino acids are replaced by other amino acids. The extent of the permissible deviation is given in homology values. Homology values are the proportion of amino acids that are present in both sequences. For example, if a sequence of 10 amino acids differs in three amino acids and matches in seven amino acids, then there is a homology of 70%. The peptides according to the invention also include those peptides which have at least 70% homology to the sequence NVLSPRYEIVIITWA. This means that 30%

REPLACEMENT LEAF of the amino acids can be replaced by other amino acids. However, these derivatives still have the required biological activity.

In a preferred embodiment, the derivatives have a homology of at least 80% and in a particularly preferred embodiment a homology of at least 90% to the sequence NVLSPRYEIVIITWA.

The peptides according to the invention are relatively short. In a preferred embodiment they have no more than 26 amino acids and in a particularly preferred embodiment they have no more than 16 amino acids. The peptide with the amino acid sequence: VLSPRYEIVIITVVA is very particularly preferred.

The present invention also relates to medicaments which contain at least one peptide according to one of Claims 1 to 5. These medicines are particularly suitable for the treatment of tumor diseases. In a very particularly preferred embodiment, the peptides according to the invention can be used for the treatment of tumor cells ex vivo. Thus, if cells suffering from a tumor disease are taken from cells as part of the therapy, the peptides according to the invention can be added ex vivo, possibly together with IFN- / ⁶ ', whereby the tumor cells express the membrane-bound mICAM but the expression of soluble sICAM is reduced. The activated lymphocytes can then recognize and destroy the tumor cells.

The peptides according to the invention can have a deletion at one or both ends provided that they are longer than 16 amino acids. This means that the first and / or last amino acids are deleted, i.e. are removed. However, the deletion should not be more than five amino acids per end.

REPLACEMENT LEAF Peptide in the sense of the present invention is understood to mean a molecule in which a not too large number of amino acids are linked to one another. The number of amino acids of the peptides according to the invention is at most 30, preferably not more than 26 and particularly preferably not more than 16.

The peptides according to the invention can be prepared by methods known per se either by synthetic means (for example solid phase synthesis) or by genetic engineering, in which a DNA sequence coding for the amino acid sequence of the peptide is incorporated in a suitable vector and expressed in a suitable host system becomes.

The present invention is further illustrated by the following examples.

example 1

According to methods known per se, normal human keratinocytes derived from the foreskin were cultivated and cultivated using a defined keratinocyte growth medium (KGM, Gibco, Berlin). The cells were cultured in one layer at 37 ° C. in a humid atmosphere containing 5% CO ₂ . For stimulation, the growth medium was first replaced and human interferon v was added in order to stimulate the expression of ICAM-1. To inhibit the secretion of sICAM-1, protease inhibitors were added after an incubation period with interferon- ^.

For the experiments, chemically synthesized peptides that were diluted in water to a concentration of 1 mM were added.

REPLACEMENT LEAF Example 2

Determination of sICAM-1 using ELISA

Before stimulation, the cells were seeded in 25 mm cell culture plates (Falcon, Lincoln Park, NJ, USA) with a density of 2.5 x 10 ⁵ cells / plate. At time 0 the medium was replaced with 1.0 ml medium with or without the specified concentration of rh IFN- ^ (recombinant human interferon- ^) (Genzyme, Boston) and protease inhibitors. After an incubation period of 24 hours, the supernatants were harvested, centrifuged at 10,000 xg for five minutes to remove remaining cell particles and stored at -20 ° C. until the measurement. The amount of sICAM-1 molecules within the cell-free supernatants was determined using a commercially available, highly sensitive (detection limit for sICAM-1: 0.5 ng / ml) two-side capture sICAM-1 ELISA system. The anti-sICAM-1 monoclonal antibodies used in this sICAM-1 ELISA specifically recognize the extracellular region.

The sICAM-1 ELISAs were • performed in 96-well microtiter plates according to the manufacturer's instructions (Bender MedSystems, Vienna), using 100 μl undiluted supernatant per sample. The plates were evaluated on a Titertec Multiscan MCC at 450 nm. The concentrations of sICAM-1 were calculated using a calibration curve which was prepared with standard dilutions of sICAM-1 concentrations from 0 to 10 ng / ml.

Example 3

Analysis of ICAM-1 expression by SDS gel electrophoresis and Western blot

For the Western blot analysis, the cells were cultivated in 10 cm petri dishes. The supernatants were collected and centrifuged at 10,000 x g for 10 minutes to remove any

Replacement sheet Remove cell particles. The supernatants were then concentrated 25-fold using a Centricon 30 concentrator (Amicon, USA).

To prepare lysates from the cells, the cells were washed three times with cold PBS buffer, scraped from the culture vessels and resuspended in 1.0 ml of cold lysis buffer. The lysis buffer contained 50 mM Tris-HCl, pH 8.0; 150 mM NaCl; 2mM EDTA; 2mM EGTA; 1% triton; 1 µg / ml aprotinin; 0.5 mM PMSF; 20 mM iodoacetamide; 10 mM benzamidine and 50 mM e-aminocaproic acid. The insoluble material was removed by centrifugation at 10,000 x g for two 10 minutes at 4 ° C. The protein content of each sample was determined using the Bradford method. SDS gel electrophoresis was carried out by the method of Laemmli (Laemmli, N ture [1970], 227, 680). For this purpose, 10 μg protein were suspended in loading buffer (80 mM Tri-HCl pH 6.8; 10% glycerol; 2% SDS and 0.02% bromophenol blue) and then heated at 95 ° C. for five minutes. Electrophoresis was performed on an 8% SDS polyacrylamide gel under non-reducing conditions. After electrophoresis, the size-separated proteins were electrotransferred onto a polyvinylidene difluoride membrane in a transfer buffer (25 mM Tris; 150 mM glycine and 10% methanol) (Towbin et al. PNAS [1979] Vol. 76, p. 4350).

Western blot analysis was performed using Amersham's ECL Western blot analysis system. After the transfer, non-specific bonds were made in the membrane in a solution of 0.15 M NaCl, 50 mM Tris-HCl (pH 7.5); 0.05% Tween-20 and 5% defatted dry milk blocked for 60 minutes. The membrane with the separated proteins was then incubated for 60 minutes at room temperature with the first monoclonal antibody (clone 15.2, Boehringer Mannheim) in a dilution of 1: 500 in the blocking solution. The membrane was then washed four times for 10 minutes at room temperature in 150 mM NaCl, 50 mM Tris-HCl (pH 7.5) and 0.05% Tween-20 and then incubated for 60 minutes with a 1: 300 solution of a second antibody (horseradish peroxidase-conjugated anti-mouse IgG antibody [sheep, Amersham]) in blocking solution, washed four times as described above and subjected to the color development reaction according to the manufacturer's instructions.

Example 4

Immunofluorescence flow measurement

The collected and washed cells were incubated with a 1:40 dilution of the 84H10 monoclonal antibody (Dianova) for 30 minutes at 4 ° C. The cells were then resuspended three times in PBS buffer containing 0.05% sodium azide and incubated with a 1:20 dilution of a goat anti-mouse antibody (FITC-Fab ₂ ) for 30 minutes at 4 ° C. The cells were then washed three times and immediately analyzed using a FACScan device (Becton Dickinson, USA). Before analysis, cells were routinely stained with propidium iodide to exclude dead cells. Usually 5,000 cells per sample were checked. The graphical representation shows the fluorescence intensity versus the cell number.

The results of the examples are summarized in the figures.

Figure 1 is a schematic representation of the arrangement of the various oligopeptides tested within the ICAM molecule. Although various peptides have been investigated (peptides 1-6), surprisingly only peptide 3 was found to be effective in blocking the production of soluble slCAM molecules out. Peptide 3 has the amino acid sequence NVLSPRYEIVIITWA. The peptide according to the invention spans a membrane-near extracellular portion and an intramembrane portion of the membrane-bound ICAM-1 molecule.

REPLACEMENT LEAF FIG. 2 shows that the peptide (3) according to the invention specifically and specifically inhibits the formation of soluble sICAM-1. This is shown on the left half of FIG. 2. The expression of membrane-bound ICAM-1 molecules is not inhibited by the peptide according to the invention. This is shown in the right half of FIG. 2. The production of the soluble sICAM-1 molecules was determined using the ELISA test (Example 2). The expression of mICAM-1 was determined by means of the FACS analysis (example 4).

FIG. 3 shows that only the peptide according to the invention, which was designated as peptide 3 in the illustration, is able to significantly inhibit the production of soluble sICAM-1 molecules. The other peptides show little or no inhibition of sICAM-1 formation.

REPLACEMENT LEAF

Claims

Expectations

1) Peptide, characterized in that it has the amino acid sequence REVTVNVLSPRYEIVIITWAAAVIM, which can have a deletion of up to five amino acids at one or both ends of the sequence and derivatives thereof which have at least 70% homology to the sequence NVLSPRYEIVIITWA.

2) Peptide according to claim 1, characterized in that it has no more than 26 amino acids.

3) Peptide according to claim 1 or 2, characterized in that the derivatives to the sequence NVLSPRYEIVIITWA have a homology of at least 80%.

4) Peptide according to claim 3, characterized in that the derivatives to the sequence NVLSPRYEIVIITWA have a homology of at least 90%.

5) Peptide, characterized in that it has the sequence NVLSPRYEIVIITWA.

6) Medicament, characterized in that it contains at least one peptide according to one of claims 1 to 5.

7) Medicament for the treatment of tumor diseases, characterized in that it contains at least one peptide according to one of claims 1 to 5.

8) Medicament according to claim 7, characterized in that it is used ex vivo in the treatment of tumor diseases.

9) Use of a peptide according to one of claims 1 to 5 for the treatment of tumor diseases.

REPLACEMENT LEAF