WO1991014702A1 - Peptides a action anti-tumorale - Google Patents

Peptides a action anti-tumorale Download PDF

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Publication number
WO1991014702A1
WO1991014702A1 PCT/AU1991/000100 AU9100100W WO9114702A1 WO 1991014702 A1 WO1991014702 A1 WO 1991014702A1 AU 9100100 W AU9100100 W AU 9100100W WO 9114702 A1 WO9114702 A1 WO 9114702A1
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WO
WIPO (PCT)
Prior art keywords
peptide
tnf
peptides
amino acids
amino acid
Prior art date
Application number
PCT/AU1991/000100
Other languages
English (en)
Inventor
Deborah Anne Rathjen
Roger Aston
Original Assignee
Peptide Technology Ltd.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Peptide Technology Ltd. filed Critical Peptide Technology Ltd.
Publication of WO1991014702A1 publication Critical patent/WO1991014702A1/fr

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Classifications

    • 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 peptides having anti-tumour activity and to use of these peptides as therapeutic agents.
  • Tumour necrosis factor alpha (TNF ⁇ ) was first described as a factor found in the serum of BCG-treated mice which caused haemorraghic regression of certain transplanted tumours and had cytolytic activity against several transformed cell lines in vitro (Carswell et al., 1975, PNAS J ⁇ 3666-3670; Helson et al, 1975, Nature 258 731-732). TNF ⁇ was subsequently found to be produced by activated macrophages. The gene encoding TNF ⁇ has been cloned and expressed (Pennica et al.
  • the present inventors have identified novel peptides derived from primary amino acid sequence of TNF ⁇ which have both cytotoxic effects on tumour cells in vitro, some of which compete with TNF ⁇ for binding to TNF ⁇ receptors on tumour cells. These peptides have indicated that the regions of amino acids 43 to 68 and 132 to 150 in the primary amino acid sequence of human TNF ⁇ have previously undiscovered anti-tumour activity. These peptides would not be expected to cause the pathology associated with both exogenous TNF ⁇ therapy and also excessive endogenous production of TNF ⁇ such as is seen in endotoxic shock and cerebral malaria (Tracy et al., 1986, Science 134 . 470-474; Clark, 1987, Ann. Trop Med Parasitol £1, 577-585).
  • the present invention consists in a peptide having an amino acid sequence substantially corresponding to amino acids 43 to 68 or 132 to 150 of Figure 1 or a part thereof, the peptide being characterised in that the peptide has cytotoxic and/or inhibition of proliferation effects on tumour cells in vitro.
  • the peptide has an amino acid sequence substantially corresponding to amino acids 43 to 58 of Figure 1. In another preferred embodiment of this aspect of the present invention the peptide has an amino acid sequence substantially corresponding to amino acids 54 to 68 of Figure 1.
  • the peptide has an amino acid sequence substantially corresponding to amino acids 132 to 150 of Figure 1.
  • the peptide has an amino acid sequence substantially corresponding to amino acids 73 to 94 of Figure 1. In yet another preferred embodiment of the present invention the peptide has an amino acid sequence substantially corresponding to amino acids 81 to 94 of Figure 1. In yet another preferred embodiment of the present invention the peptide has an amino acid sequence substantially corresponding to amino acids 70 to 80 of Figure 1.
  • the present inventors have demonstrated that the region of TNF from amino acids 43 to 68 and from amino acids 132 to 150 play an important functional role in the cytotoxic effects on tumour cells in vitro. Further, the present inventors have produced three peptides, namely peptides 302, 305 and 308 (as referred to herein), which have cytotoxic effects on tumour cells in vitro.
  • the present invention consists in a compound, the three dimensional structure of which substantially corresponds to the three dimensional structure of the peptide of the first aspect of the present invention, the compound being characterised in that the compound has cytotoxic effects on tumour cells in vitro.
  • the compound has a three dimensional structure substantially corresponding to the three dimensional structure of amino acids 43 to 58 of Figure 1.
  • the compound has a three dimensional structure substantially corresponding to the three dimensional structure of amino acids 54 to 68 of Figure 1.
  • the compound has a three dimensional structure substantially corresponding to the three dimensional structure of amino acids 132 to 150 of Figure 1.
  • the compound has a three dimensional structure substantially corresponding to the three dimensional structure of amino acids 73 to 94 of Figure 1. In another preferred embodiment of this aspect of the present invention the compound has a three dimensional structure substantially corresponding to the three dimensional structure of amino acids 81 to 94 of Figure 1. In yet another preferred embodiment of this aspect of the present invention the compound has a three dimensional structure substantially corresponding to the three dimensional structure of amino acids 70 to 80 of Figure 1.
  • the present invention consists in a method of treating a mammal carrying tumours, the method comprising administering to the animal a composition comprising the peptide of the first aspect of the present invention in conjunction with a suitable pharmaceutical carrier. In a preferred embodiment of this aspect of the present invention low doses of whole TNF ⁇ are also administered to the mammal.
  • the peptide of the aspect of the present invention is administered to the mammal together with a cytotoxic drug selected from the group consisting of vinblastin, acyclovir, interferon alpha, IL-2, actinomysin D, AZT, radiotherapy, adria ycin, mytomycin C, cytosine arabinoside, dounorubicin, cis-platin, vincistine, 5-flurouracil and bleomycin.
  • a cytotoxic drug selected from the group consisting of vinblastin, acyclovir, interferon alpha, IL-2, actinomysin D, AZT, radiotherapy, adria ycin, mytomycin C, cytosine arabinoside, dounorubicin, cis-platin, vincistine, 5-flurouracil and bleomycin.
  • the route of administration and the nature of the pharmaceutically acceptable carrier will depend on the nature of the tumour and the mammal to be treated. It is believed that the choice of pharmaceutical carrier and route of administration is within the common general knowledge of a person skilled in the art.
  • the present invention consists in the use of the peptide of the first aspect of the present invention in the preparation of a medicament for the treatment of tumours.
  • the peptide is peptide 302, 305, 308, 309, 394 or 395 as defined hereafter.
  • the use of the peptide of the present invention obviates the need to use the entire TNF molecule, it is expected that the severe side effects associated with the therapeutic use of the whole TNF molecule will be avoided. This is confirmed by preliminary evidence which indicates that the peptide of the present invention is devoid of the toxicity associated with the whole TNF molecule.
  • Figure 1 shows the amino acid sequence of human TNF
  • Figure 2 is a schematic representation of the TNF ⁇ monomer showing the regions with anti-tumour activity
  • Figure 3 shows the binding of 125I TNF ⁇ to
  • Figure 4 shows the binding of radio-labelled TNF ⁇ to MM418E cells in the presence of peptide 309
  • FIG. 5 shows the binding of radio-labelled TNF ⁇ to MDA-MB-231 cells in the presence of peptide 309;
  • Figure 6 shows the binding of radio-labelled TNF ⁇ to 1GR3 cells in the presence of peptide 309
  • Figure 7 shows the binding of radio-labelled TNF ⁇ to CaCO cells in the presence of peptide 309
  • Figure 8 shows the binding of radio-labelled TNF ⁇ to HT-29 cells in the presence of peptide 309;
  • FIG. 9 shows the binding of radio-laelled TNF ⁇ to
  • Figure 10 shows the effect of Tween 20 on peptide 309 enhanced TNF-methA sarcoma
  • Figure 11 shows the effect of DMSO on peptide 309 enhanced TNF-methA sarcoma
  • Figure 12 shows the results of an experiment on methA solid tumour regression comparing the effect of ff PBS;010 ⁇ g TNF ⁇ ⁇ ffl 10 ⁇ g TNF ⁇ plus peptide 309; and ggf peptide 309;
  • Figure 13 shows the effect of peptides 302, 305 and 309 on tumour regression in WEHI-164 tumour-bearing mice
  • Figure 14 shows the effect of peptide 308 on tumour regression in WEH1-164 tumour-bearing mice
  • Figure 15 shows the effect of peptides 309 and 394 on regression of methA tumours
  • Figure 16 shows the effect of peptides 395, 309 and 394 on WEHI-164 tumour regression
  • Figure 17 shows the effect of TNF peptides on proliferation of WEHI-164 cells; £D peptide 302; peptide 305; ⁇ peptide 308; and ⁇ peptide 309;
  • Figure 18 shows the effect of peptide 394 ( £j ) and peptide 395 ( • ) on proliferation of WEHI-164 cells; and Figure 19 shows the effect of TNF and TNF peptides on proliferation of WEHI-164 cells;
  • PepSyn KA is a polydimethyacrylamide gel on kieselguhr support with
  • the carboxy terminal amino acid is attached to the solid support by a DCC/DMAP-mediated symmetrical-anhydride esterification.
  • Peptide 304 Peptide is cleaved from the resin with 95% TFA and 5% phenol (5 h) and purified on reverse phase C4 column. (Buffer A - 0.1% aqueous TFA, Buffer B - 80% ACN 20% A) .
  • Peptide 308 Peptide is cleaved from the resin with 95% TFA and 5% water (1.5 h) and purified on reverse phase C4 column. (Buffer A - 0.1% aqueous TFA, Buffer B - 80% ACN 20% A) .
  • Peptide 309 Peptide is cleaved from the resin with 95% TFA and 5% thioanisole and purified on reverse phase C4 column. (Buffer A - 0.1% aqueous TFA, Buffer B - 80% ACN 20% A) .
  • Peptide 394 was soluble in DMSO/buffer while peptide 395 displayed greater solubility in buffer only.
  • the cytotoxic effect of the TNF peptide on tumour cells in vitro was assessed.
  • the bioassay of both recombinant human TNF and the TNF drive peptides was performed on WEHI-164 cells according to the method of Espevik and Nissen-Meyer, 1986 (J.Immunol.Methods, 95, 99-105).
  • TNF Tumour Cell Radio-Receptor Assay
  • TNF peptides The ability of TNF peptides to compete with TNF for binding to TNF receptors on tumour cells was assessed.
  • WEHI-164 cells grown to confluency were scrape harvested and washed once with Hank's Balanced
  • HBSS HBSS Salt Solution
  • BSA bovine serum albumin
  • HBSS-BSA was added and the cells spun at 16,000 rpm for
  • TNF receptor analyses were performed on the following tumour cell lines: MethA (mouse fibrosarcoma) , U937 (human monocytoid) , HT 29 (human colon carcinoma), CaCO (human carcinoma), IGR-3 (human melanoma), MDA-MB-231 (human breast), MM418E (human melanoma).
  • Adherent cells (HT 29, Ca CO, IGR-3 MDA-MB231 and MM418E) were seeded in 24-well culture dishes at a concentration of 4 x 10 4 cells per well in either RPMI-1640 (MM418E), DMEM (CaCO and IGR-3) or Iscoves modified DMEM (HT 29 and MDA-MB23) supplemented with 10% foetal calf serum, penicillin/streptomycin, L-glutamine and in the case of MDA-MB23 50 ⁇ g/ml insulin. The cells were used for receptor analyses when confluent.
  • the cells were incubated for one hour at 37 C in the presence of either unlabelled TNF (0 to 100 ng) or peptide and iodinated TNF (50,000 cpm) .
  • the cells were scrape harvested and together with the supernatant transferred to eppendorf tubes, spun at 16,000 rpm for 30 sees. The supernatant was decanted, 1ml of fresh medium added and the cell lystate washed once before the amount of bound radiolabelled TNF in the pellet was detected by counting in a gamma counter.
  • Non-adherent cells (MethA and U937) were grown to confluency, scrape harvested, and washed once with 1% BSA in Hanks Balanced Salt Solution (HBSS) .
  • HBSS Hanks Balanced Salt Solution
  • lOOul of unlabelled TNF (0-100 ng) or peptide (o-lOO ⁇ g, dissolved initially in either dimethylsulphoxide (DMSO) or Tween 20 and then diluted to the appropriate concentration in culture media) was added to 50ul radiolabelled TNF (50,000 cpm). Cells were then added (200 ⁇ l containing 2 x 10 cells). This mixture was incubated in a shaking water bath at 37 C for three hours. At the completion of this incubation lmL of HBSS was added and the cells spun at 16,000 rpm for 30 sees. The supernatant was discarded and bound 125I TNF in the cell pellet counted.
  • Peptide 309 was found to significantly enhance the uptake of radiolabelled human TNF by the human tumour cell lines examined (Figs. 4-9), with the exception of U937 and HT 29 cells, at peptide concentrations greater than 5 ⁇ g per assay tube.
  • U937 cells showed enhanced uptake of TNF in the presence of peptide 309 only at much higher peptide concentrations (10 to lOO ⁇ g) while peptide 309 was unable to enhance TNF uptake by HT 29 cells.
  • Peptide 309 was dissolved in DMSO for receptor binding analyses,however, this is not essential since dissolving the peptide in media containing Tween 20 or sonication of media containing 309 such that liposomes were formed did not reduce the effect (enhancement of TNF uptake) as shown in Figs. 10 and 11. Peptide 309 did not demonstrate significant toxicity for the tumour cells in vitro (Table 2) . Tumour Regression Studies
  • tumour size was measured using calipers. Each experimental group contained 5 tumour-bearing mice.
  • Peptides 303, 305 and 308 were found not only to be cytotoxic for TNF sensitive tumour cells in the MTT assay but also to inhibit their proliferation as measured by thymidine incorporation (Fig. 17, Fig. 18).
  • Peptide 309 which was not directly cytotoxic for TNF- sensitive tumour cells, as shown by WEHI-164 cells, was found to inhibit their proliferation. This observation may in part account for the tumour regression activity displayed by peptide 309. Part of the anti-tumour activity of 309 may also be due to neutrophil activation. Smaller peptides comprising 309 (peptides 394 and 395) were also able to inhibit the growth of WEHI-164 cells (Fig. 18 and 19).
  • tumour cells such as MethA
  • TUMOUR CELL PROLIFERATION ASSAY Tumour cells (5 x 10 4 cells/well) in RPMI-1640 medium were plated into 96 well microplates in the presence of TNF and/or peptide as indicated. The cells were cultured at 37 C for 24 hours before the addition
  • peptides 302, 305 and 308 were found to have cytolytic activity against tumour cells in vitro and were also found to inhibit the binding of TNF to tumour cell receptors. Together these peptides comprise the primary amino acid sequence region of amino acids 43 to 68 and 132 to 150 of human TNF. Whilst these sequence regions are not in close linear proximity, these sequence regions are in close proximity in the three dimensional cystalline structure of human TNF recently determined (Jones et al, 1989, Nature 338, 225 - 229; Eck and Sprang, 1989, J.Biol.Chem. 261, 17595-17605). This is shown in Figure 2.
  • peptides 308 and 309 may also mediate other TNF activities which may enable them to be considered as a an anti-viral, anti-bacterial or anti-parasite therapy particularly since it appears to devoid of the toxicity inherent in the whole TNF molecule. These activities may be manifest by peptides 308 or 309 alone or in combination with low doses of whole TNF, other TNF peptides or other therapy. The present inventors believe the peptides will not only inhibit tumour growth (and induce regression) but also inhibit metastasis.

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  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biochemistry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

On décrit des peptides à action anti-tumorale, ainsi que l'utilisation desdits peptides en tant qu'agents thérapeutiques. Lesdits peptides ont une séquence d'acides aminés qui correspond sensiblement à celles des acides aminés de 43 à 68 ou de 132 à 150 de la figure, ou une partie de celles-ci. De préférence les peptides ont une séquence d'acides aminés correspondant sensiblement à celles des acides aminés de 43 à 58, de 54 à 68, de 132 à 150, de 73 à 94, de 81 à 94 ou de 70 à 80 de la figure.
PCT/AU1991/000100 1990-03-19 1991-03-15 Peptides a action anti-tumorale WO1991014702A1 (fr)

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
AUPJ915690 1990-03-19
AUPJ9156 1990-03-19
AUPK3477 1990-11-22
AUPK347790 1990-11-22

Publications (1)

Publication Number Publication Date
WO1991014702A1 true WO1991014702A1 (fr) 1991-10-03

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EP (1) EP0521918A4 (fr)
WO (1) WO1991014702A1 (fr)

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0519976A1 (fr) * 1990-03-12 1992-12-30 Peptide Technology Ltd Peptides stimulant les polynucleaires neutrophiles.
US5587457A (en) * 1990-03-12 1996-12-24 Peptide Technology Limited Neutrophil stimulating peptides
US6375928B1 (en) * 1990-03-12 2002-04-23 Peptech Limited Neutrophil stimulating peptides
EP2314311A3 (fr) * 2002-04-10 2011-08-17 Vaxconsulting Péptides et leur application en thérapeutique

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE3841761A1 (de) * 1988-12-12 1990-06-13 Basf Ag Neue tnf-peptide
DE3841754A1 (de) * 1988-12-12 1990-06-13 Basf Ag Neue tnf-peptide
EP0486526B2 (fr) * 1989-08-07 2001-03-07 Peptech Limited Ligands de liaison du facteur de necrose de tumeurs
JP3241041B2 (ja) * 1990-03-12 2001-12-25 ペプテック リミテッド 好中球刺激ペプチド

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
Proceedings of the National Academy of Sciences USA, Volume 84, No. 21, issued 1987, December (Washington D.C. USA), S.H. SOCHER et al. "Antibodies against amino acids 1-15 of tumor necrosis factor block its binding to cell-surface receptor", see pages 8829-8833. *
See also references of EP0521918A4 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0519976A1 (fr) * 1990-03-12 1992-12-30 Peptide Technology Ltd Peptides stimulant les polynucleaires neutrophiles.
EP0519976A4 (en) * 1990-03-12 1993-06-09 Peptide Technology Ltd Neutrophil stimulating peptides
US5587457A (en) * 1990-03-12 1996-12-24 Peptide Technology Limited Neutrophil stimulating peptides
US6375928B1 (en) * 1990-03-12 2002-04-23 Peptech Limited Neutrophil stimulating peptides
EP2314311A3 (fr) * 2002-04-10 2011-08-17 Vaxconsulting Péptides et leur application en thérapeutique

Also Published As

Publication number Publication date
EP0521918A1 (fr) 1993-01-13
EP0521918A4 (en) 1993-05-05

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