AU7467391A

AU7467391A - Anti-tumour peptides

Info

Publication number: AU7467391A
Application number: AU74673/91A
Authority: AU
Inventors: Roger Aston; Deborah Anne Rathjen
Original assignee: Peptide Technology Ltd
Current assignee: Teva Pharmaceuticals Australia Pty Ltd
Priority date: 1990-03-19
Filing date: 1991-03-15
Publication date: 1991-10-21

Description

Anti-Tumour Peptides

The present invention relates to peptides having anti-tumour activity and to use of these peptides as therapeutic agents. Tumour necrosis factor alpha (TNFα) 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 Jλ 3666-3670; Helson et al, 1975, Nature 258 731-732). TNFα was subsequently found to be produced by activated macrophages. The gene encoding TNFα has been cloned and expressed (Pennica et al. , 1984, Nature 312, 724-729; Shirai et al., 1985, Nature 311, 803-806; Wang et al., 1985 Science 228, 149-154) allowing the usefulness of this monokine as a potential cancer therapy agent to be assessed. While TNFα infusion into cancer patients in stage 1 clinical trials has resulted in tumour regression, side effects such as thro bocytopaenia, lymphocytopaenia, hepatoxicity, renal impairment and hypotension have also been reported (Creaven et al., 1987, Cancer Chemother Pharmacol 20, 137-144; Kimura et al. , 1987 Cancer Chemother Pharmacol 20, 223-229; Selby et al., 1987, Brit J. Cancer 5£, 803-808). These significant problems associated with TNFα treatment will ultimately limit its therapeutic usefulness.

The present inventors have identified novel peptides derived from primary amino acid sequence of TNFα which have both cytotoxic effects on tumour cells in vitro, some of which compete with TNFα for binding to TNFα 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 TNFα have previously undiscovered anti-tumour activity. These peptides would not be expected to cause the pathology associated with both exogenous TNFα therapy and also excessive endogenous production of TNFα 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).

It should be noted that whilst the peptides found to have anti-tumour activity are not in close linear proximity, these sequence regions are in close proximity in the three dimensional crystal structure of human TNF recently determined (Jones et al, 1989, Nature 338m 225-229; Eck and Sprang, 1989, J. Biol. Chem. 264, 17595-17605) .

Accordingly, in a first aspect 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.

In a preferred embodiment of this aspect of the present invention 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.

In yet another preferred embodiment of the present invention the peptide has an amino acid sequence substantially corresponding to amino acids 132 to 150 of Figure 1.

In yet another preferred embodiment of the present invention 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.

As will be appreciated by those skilled in the art from the description which follows, 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.

Armed with this information, and with the aid of the crystalline structure of TNF at 2.6A as disclosed by Eck and Sprang, 1989 (J.Biol.Chem., 23A' 17595-17605), the person skilled in the art will be able to design non-peptide structures, which in three dimensional terms, mimics the peptides of the present invention. It is believed that these non-peptide structures will also mimic the physiological effects of the peptides of the present invention. It is intended that such non-peptide structures are included within the scope of the present invention.

Accordingly, in a second aspect 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. In a 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 43 to 58 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 54 to 68 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 132 to 150 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 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. In a third aspect 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 TNFα are also administered to the mammal.

In a preferred embodiment of the present invention 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.

As will be readily appreciated, 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.

In a third aspect 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.

In a preferred embodiment of this aspect of the present invention the peptide is peptide 302, 305, 308, 309, 394 or 395 as defined hereafter.

As 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.

In co-pending International Patent Application No. WO 91/02078 the present applicant has shown that TNF receptors on tumour cells and on endothelial cells recognise different regions of the TNF molecule and that it is possible to segregate the biological effects of TNF on these cells using monoclonal antibodies directed against TNF. Accordingly, the anti-tumour peptides described herein should not cause perturbation of the endothelium, such as procoagulant induction and fibrin deposition, which has been observed with the whole TNF molecule. In order that the nature of the present invention may be more clearly understood, preferred forms thereof will now be described with reference to the following examples.

Figure 1 shows the amino acid sequence of human TNF; Figure 2 is a schematic representation of the TNFα monomer showing the regions with anti-tumour activity; Figure 3 shows the binding of 125I TNFα to

WEHI-164 cells in the presence of either unlabelled TNFα or peptide; ^ recombinant human TNF; # peptide 302; O peptide 304; B peptide 305; A peptide 308; M peptide 309.

Figure 4 shows the binding of radio-labelled TNFα to MM418E cells in the presence of peptide 309;

Figure 5 shows the binding of radio-labelled TNFα to MDA-MB-231 cells in the presence of peptide 309;

Figure 6 shows the binding of radio-labelled TNFα to 1GR3 cells in the presence of peptide 309;

Figure 7 shows the binding of radio-labelled TNFα to CaCO cells in the presence of peptide 309; Figure 8 shows the binding of radio-labelled TNFα to HT-29 cells in the presence of peptide 309;

Figure 9 shows the binding of radio-laelled TNFα to

U937 cells in the presence of peptide 309;

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;010μg TNFα } ffl 10μg TNFα 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;

Production of Human TNF Peptides Tested for Tumour Cell Cytotoxicity and Receptor Binding

The following peptides were synthesised and are described using the I.U.P.A.C. one-letter code abbreviations for amino acid residues with the TNF sequence region indicated in brackets. peptide 275

A K P W Y E P I Y L (111-120) peptide 301

V R S S S R T P S D K P V A H V V A (1-18) peptide 302

L R D N Q L V V P S E G L Y L I (43-58) peptide 303 L S A I K S P C Q R E T P E G A (94-109) peptide 304

L F K G Q G C P S T H V L L T H T I S R I (63-83) peptide 305

L S A E I N R P D Y L D F A E S G Q V (132-150) peptide 306

V A H V V A N P Q A E G Q L (13-26) peptide 307

A E G Q Q W L N R R A N A L L A N G (22-40) peptide 308 G L Y L I Y S Q V L F K G Q G (54-68) peptide 309

H V L L T H T I S R I A V S Y Q T K V N L L (73-94) peptide 323

T I S R I A V S Y Q T (79-89) These peptides were synthesised using the following general protocol.

All peptides were synthesised using the

Fmoc-polyamide method of solid phase peptide synthesis

(Atherton et al, 1978, J. Chem. Soc. Chem. Commun. , 13, 537-539). The solid resin used was PepSyn KA which is a polydimethyacrylamide gel on kieselguhr support with

4-hydroxymethylphenoxyacetic acid as the functionalised linker (Atherton et al, 1975, J. Am. Chem. Soc, 97,

6584-6585). The carboxy terminal amino acid is attached to the solid support by a DCC/DMAP-mediated symmetrical-anhydride esterification.

All Fmoc-groups are removed by piperidine/DMF wash and peptide bonds are formed either via pentafluorophenyl active esters or directly by BOP/NMM/HOBt (Castro's reagent) except for certain amino acids as specified in

Table 1.

Side chain protection chosen for the amino acids are removed concomitantly during cleavage with the exception of Acm on cysteine which is left on after synthesis TABLE 1

Amino acid Protecting group Coupling Method

Arg Mtr or Pmc Either

Asp OBut Either

Cys Acm (permanent) Either

Glu OBut Either

His Boc OPfp only

Lys Boc Either

Ser But BOP only

Thr But BOP only

Tyr But Either

Asn none OPfp only

Gin none OPfp only

Cleavage and Purification

Peptide 302. Peptide is cleaved from the resin with 95% TFA and 5% thioanisole (1.5 h) and purified on reverse phase C4 column. (Buffer A - 0.1% aqueous TFA, Buffer B - 80% ACN 20% A)

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) .

In addition, the following synthetic fragments of peptide 309 were synthesized. These peptides had the following amino acid sequence with the TNF sequence region indicated in brackets. Peptide 393

L T H T I S R I A (76-84) . Peptide 394

S R I A V S Y Q T K V N L L (81-94) .^' Peptide 395

P S T H V L L T H T I (70-80) . Peptide 396 A V S Y Q T K V N L L (84-94) .

Peptide 394 was soluble in DMSO/buffer while peptide 395 displayed greater solubility in buffer only. Tumour Cell Cytotoxicity Assay

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).

The effect of recombinant TNF and synthetic peptides on tumour cell viability is shown in Table 2.

TABLE 2

* % Viability was determined by comparison with untreated control cells.

# TNF was at 50 units per culture, 50 units represents approximately lμg. Each peptide was tested at 50μg per culture. Tumour Cell Radio-Receptor Assay

The ability of TNF peptides to compete with TNF for binding to TNF receptors on tumour cells was assessed. In this assay, WEHI-164 cells grown to confluency were scrape harvested and washed once with Hank's Balanced

Salt Solution (HBSS) supplemented with 1% bovine serum albumin (BSA) . lOOul of unlabelled TNF (1-10,000 ng/tube) or peptide (1-100,000 ng/tube) was added to 50ul

¹²⁵I TNF (50,000 cpm) . WEHI-164 cells were then added

4 (200ul 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 1ml of

HBSS-BSA was added and the cells spun at 16,000 rpm for

30 seconds. The supernatant was discarded and bound

125I TNF counted. All dilutions were prepared in HBSS-BSA. The results of this assay are shown in Figure 3.

Radioreceptor Analyses of Radiolabelled TNF Binding to Mouse and Human Tumour Cell in the Presence of Peptide 309 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 10⁴ 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 50μg/ml insulin. The cells were used for receptor analyses when confluent. For receptor analyses 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) . At the end of this incubation 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) . In eppendorf tubes lOOul of unlabelled TNF (0-100 ng) or peptide (o-lOOμg, 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 μl 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.

Results

1. Receptor Analyses

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 μg per assay tube. U937 cells showed enhanced uptake of TNF in the presence of peptide 309 only at much higher peptide concentrations (10 to lOOμg) 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

Subcutaneous MethA tumours were induced by the

5 injection of approximately 5 x 10 MethA cells. Mice bearing tumours of between 10-15mm in diameter were injected with either phosphate buffered saline (PBS, pH7.2), human recombinant TNF (10μg) or peptide 309

(1 mg) daily throughout the course of the experiment. At the commencement of the experiment and at daily intervals tumour size was measured using calipers. Each experimental group contained 5 tumour-bearing mice. Results

When peptide 309 was injected into mice bearing MethA sarcoma tumours daily for three days the tumours were seen to regress (Fig. 12). There was no statistical difference between peptide 309-treated animals and TNF-treated animals. The difference between the PBS-treated group and peptide 309-treated was statistically significant (p< 0.022). EFFECT OF PEPTIDES ON GROWTH OF WEHI-164 TUMOURS IN MICE Peptides showing in vitro anti-tumour activity (either cytotoxicity or inhibition of tumour cell proliferation) were found to cause tumour regression in vivo following the injection of 1 mg of peptide administered i.p. (Figs. 13 and 14). In addition peptide 309 fragments 394 and 395 were also active in both MethA and WEHI-164 tumour models (Figs. 15 and 16). EFFECT OF TNF PEPTIDES ON PROLIFERATION OF TUMOUR CELLS

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).

The peptides did not have any effect on the proliferation of tumour cells, such as MethA, which are resistant to TNF toxicity in vitro. TUMOUR CELL PROLIFERATION ASSAY Tumour cells (5 x 10⁴ 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

3 of H thymidine. 0.5μCi of tritiated thymidine was added to each well and the cells cultured for a further

24 hours before being harvested and the tritiated thymidine incorporated into the DNA of proliferating cells measured in a β scintillation counter.

As can be seen from the results set out above 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.

It is clear from the above results that the present inventors have demonstrated that the region of TNF from amino acid 43 to 68 and 132 to 150 play an important role in the anti-tumour activity of TNF. It is believed that minor modifications to the peptides referred to herein as 302, 305 and 308 may result in peptides having even greater cytotoxic activity against tumour cells in vivo. Peptides 308 and 309 had previously been shown to have neutrophil stimulating activity (see Australian patent application No. PJ9065) and have now been shown to manifest in vivo TNF-like anti-tumour activity. The in vivo activity of peptides 308 and 309 correlates with enhanced uptake of TNF by certain tumour cell lines in receptor binding analyses. In addition, all peptides did not manifest any degree of toxicity in mice at a dose of lmg. Thus these peptides may form the basis of an anti-cancer therapy either alone or in combination with low doses of TNF or in combination with other peptides or other anti-cancer therapy. In addition, 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.

It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific embodiments without departing from the spirit or scope of the invention as broadly described. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive.

Claims

CLAIMS : -

1. A peptide having an amino acid sequence substantially corresponding to amino acids 43 to 68 or 132 to 150 of Figure 1 or apart thereof, wherein the peptide has cytotoxic and/or inhibition of proliferation effects on tumour cells in vitro.

2. A peptide as claimed in claim 1 in which the peptide has an amino acid sequence substantially corresponding to amino acids 43 to 58 of Figure 1.

3. A peptide as claimed in claim 1 in which the peptide has an amino acid sequence substantially corresponding to amino acids 54 to 68 of Figure 1.

4. A peptide as claimed in claim 1 in which the peptide has an amino acid sequence substantially corresponding to amino acids 132 to 150 of Figure 1.

5. A peptide as claimed in claim 1 in which the peptide has an amino acid sequence substantially corresponding to amino acids 73 to 94 of Figure 1.

6. A peptide as claimed in claim 1 in which the peptide has an amino acid sequence substantially corresponding to amino acids 81 to 94 of Figure 1.

7. A peptide as claimed in claim 1 in which the peptide has an amino acid sequence substantially corresponding to amino acids 70 to 80 of Figure 1.

8. A compound the three dimensional structure of which substantially corresponds to the three dimensional structure of the peptide as claimed in any one of claims 1 to 7 and wherein the compound has cytotoxic effects on tumour cells in vitro.

9. A method of treating a mammal carrying tumours, the method comprising administering to the animal a composition comprising the peptide as claimed in any one of claims 1 to 7 in conjunction with a suitable pharmaceutical carrier.

10. A method as claimed in claim 9 in which low doses of whole TNFα are also administered to the mammal.

11. A method as claimed in claim 9 in which a cytotoxic drug selected from the group consisting of vinblastin, acyclovir, interferon alpha, IL-2, actinomysin D, AZT, radiotherapy, adriamycin, mytomycin C, cytosine arabinoside, dounorubicin, cis-platin, vincistine, 5-flurouracil and bleomycin is co-administered to the mammal.

12. The use of a peptide as claimed in any one of claims 1 to 7 in the preparation of a medicament for the treatment of tumours.