AU676842B2

AU676842B2 - Neutrophil stimulating peptides

Info

Publication number: AU676842B2
Application number: AU44664/93A
Authority: AU
Inventors: Antonio Ferrante; Deborah Ann Rathjen; Fred Widmer
Original assignee: Peptide Technology Ltd
Current assignee: Teva Pharmaceuticals Australia Pty Ltd
Priority date: 1993-08-13
Filing date: 1993-08-13
Publication date: 1997-03-27
Anticipated expiration: 2011-03-12
Also published as: AU4466493A

Description

1-

AUSTRALIA

Patents Act 1990 PEPTIDE TECHNOLOGY LIMITED

ORIGINAL

COMPLETE SPECIFICATION STANDARD PATENT Ce..

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b*C C. CC C a C Invention Title: *4 C CC

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"Neutrophil Stimulating Peptides" The following statement is a full description of this invention including the best method of performing it known to us:- NEUTROPHIL STIMULATING PEPTIDES This is a patent of addition of Australian patent application No 74762/91 filed 12 March 1991, the disclosure of which is incorporated herein by crossreference.

The present invention relates to peptides having neutrophil stimulating activity, and to use of these peptides as therapeutic agents.

Tumour necrosis factor (TNF) was first identified as a factor found in the serum of Bacillus Calmette-Guerin treated mice which caused haemorrhagic regression of certain transplanted tumours and had cytolytic activity on several transformed cell lines in vitro (Carswell et al, PNAS 72, 3666 3670; Helson et 15 al, 1975, Nature 258, 731-732). TNF, a product of activated macrophages, has subsequently been shown to be a primary mediator in the pathology of endotoxic shock (Tracey et al 1986, Science 234, 470-474). In addition to its pathological effects TNF also has a 20 central role in host defences against viral, bacterial and parasitic pathogens.

The cellular targets of TNF important in host S..defence include neutrophils, eosinophils, monocyte/macrophages and lymphocytes. Within this S: 25 context TNF is a major mediator of neutrophil activation. TNF stimulates enhanced phagocytosis (Shalaby et al 1985, J.Immunol., 135, 2069-2073), enhanced production of superoxide anions (Teujiimoto et al, 1986, Biochem. Biophys. Res. Commun., 137, 1094-1100), release of lysozyme and hydrogen peroxide and causes neutrophil degranulation (Klebanoff et al, 1986, J.Immunol., 136, 4220-4225). Neutrophils also show enhanced microbiocidal and tumouricidal activity when stiur.lated by TNF (Shalaby et al, 1985, J.Immunol., 135, 2069-2073; Djeu et al, 1986, J.Immunol., 137, 2980-2984; Blanchard et al, 1989, a~ 9sPllRI J.Leuk. Biol., 45, 538-545). It has been hypothesized that the cytostatic effect of TNF is mediated by high local concentrations of hydrogen peroxide produced by neutrophils (Shau 1986, J.Immunol., 141, 234-240).

TNF pretreatment enhances the response of neutrophils to N-formyl-L-methionyl-L-leucyl-Lphenylalanine (F-met-leu-phe) and phorbol myristate acetate through specific receptors (Ferrante et al 1988, Int. Arch. Allergy Appl. Immunol., 86, 82-91).

Neutrophils accumulate at sites of inflammation, caused in part by the increased expression of complement receptors by TNF (Berger et al 1988, Blood 71, 151-158). Further TNF causes neutrophil emigration into skin (Cybulsky et al 1988, J. Immunol. 140, 15 3144-3149).

Neutrophil function is known to be depressed in a number of viral, bacterial and parasitic infections (Abramson and Mills, 1988, Rev. Infect. Dis., 326-341; Ferrante et al, 1989, Immunol, Letts., 22, 20 301-6). Depressed neutrophil function has, for example, been described in Acquired Immune Deficiency Syndrome (Thorsen at al, 1989, AIDS, 3, 651-653; Ellis et al, 1988, J. Infect. Dis., 158, 1268-1276; Murphy et al, 1988, J. Infect. Dis., 158, 627-630). Clearly TNF, which appears to play an important role in neutrophil activation both in vitro and in vivo as described above, given exogenously has the potential to overcome these neutrophil defects. The administration of TNF or indeed overproduction of TNF is, however, associated with severe side effects and the manifestation of pathology such as thrombocytopaenia, lymphocytopaenia, hepatotoxicity, renal impairment and hypertension.

The present inventors have identified novel peptides derived from the primary amino acid sequence of human TNF which stimulate neutrophil activity.

These peptides have indicated that the region of amino c~ _I 31 54 to 94 of human TNF has previously undiscovered neutrophil stimulating activity. This observation has important clinical applications as treatment with such peptides would be expected to restore depressed or aberrant neutrophil activity, but would not be expected to cause the severe side effects associated with the therapeutic use of the whole TNF molecule.

Accordingly, in a first aspect the present invention consists in a peptide which primes neutrophils for superoxide production and an enhanced respiratory burst following treatment with N-formyl-Lmethionyl-L-leucyl-L-phenylalanine, wherein the peptide is of the general formula:-

X

1

-X

2

-X

3

-X

4

-X

5

X

7 -X7-X8-X,-Xl 1 -X in which 15 X 1 is Pro or Nct-alkylamino acid

X

2 is Ser or Thr 0*

X

3 is Ala or Gly

X

4 is His, Lys or Arg X, is Val, Ile, Leu or Met X, is Leu, Ile Val or Met

X

7 is Ile, Leu, Val or Met 0 0 X 8 is Thr or Ser X. is His, Lys or Arg

X

10 is Thr or Ser 25 X 1 is Ile, Leu, Val or Met In a preferred embodiment of the present invention the peptide has the amino acid sequence: Pro Ser Ala His Va! Leu Leu Thr His Thr Ile.

a As will be appreciated by those skilled in the art from the description which follows the present inventors have demonstrated that the region of human TNF from amino acid 54 to amino acid 94 plays an important functional role in the stimulation of neutrophils. Further, the present inventors have produced 6 peptides namely peptides 304, 308, 309, 395, 418 and 419 (as referred to herein) which have neutrophil stimulating activity.

Armed with this information and with the aid of co-ordinates of the crystalline structure of TNF at 2.6 A as disclosed by Eck and Sprang, 1989 Biol. Chem., 264: 18795-17605), the person skilled in the art will be able to design non-peptide structures which, in 3 dimensional terms mimic 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 S" 15 included within the scope of the present invention. Changes to the TNF molecule in these regions using eg. site directed mutagenesis vould also be expected to affect neutrophil activation. A schematic representation of .the three dimensional structure of TNFa is shown in Figure 8.

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 or second aspects of the present invention, the compound being characterized in that the compound is 0$ 0 0 0 0 L- -8capable of eliciting superoxide production by neutrophils and of priming neutrophils for an enhanced respiratory burst following treatment with N-formyl-L-methionyl-L-leucyl-L-phenylalanine.

In a further aspect, the present invention consists in a method of treating a subject having depressed neutrophil function, the method comprising administering to the subject a therapeutic amount of the peptide of the first aspect of the present invention.

In a preferred embodiment of this aspect of the present invention the subject is suffering from acquired immune deficiency syndrome.

Peptide 308, through selective effects on 15 neutrophil degranulation may be administered to individuals suffering from inflammatory syndromes e.g.

rheumatoid arthritis, adult respiratory distress syndrome.

It will be appreciated by those skilled in the art S 20 that a number of modifications may be made to the peptide of the present invention without deleteriously effecting the biological activity of the peptide. This may be achieved by various changes, such as insertions, deletions and substitutions sulfation, 25 phosphorylation, nitration, halogenation), either conservative or non-conservative W-amino acids, desamino acids) in the peptide sequence where such changes do not substantially altering the overall biological activity of the peptide. By conservative substitutions the intended combinations are:- G, A; V, I, L, M; D, E; N, Q; S, T; K, R, H; F, Y, W, H; and P, Na-alkylamino acids.

It may also be possible to add various groups to the peptide of the present invention to confer advantages such as increased potency or extended ~eP1I_--I half-life in vivo, without substantially altering the overall biological activity of the peptide.

The term peptide is to be understood to embrace peptide bond replacements and/or peptide mimetics, i.e.

pseudopeptides, as recognised in the art (see for example: Proceedings of the 20th European Peptide Symposium, edt. G. Jung. E. Bayer, pp. 289-336, and references therein), as well as salts and pharmaceutical preparations and/or formulations which render the bioactive peptide(s) particularly suitable for oral, topical, nasal spray, ocular pulmonary, I.V., subcutaneous, as the case may be, delivery. Such salts, formulations, amino acid replacements and pseudopeptide structures may be necessary and desirable 15 to enhance the stability, formulation, deliverability slow release, prodrugs), or to improve the economy of production, and they are acceptable, provided they do not negatively affect the required biological activity of the peptide.

20 Apart from substitutions, three particular forms of peptide mimetic and/or analogue structures of particular relevance when designating bioactive peptides, which have to bind to a receptor while risking the degradation by proteinases and peptidases 25 in the blood, tissues and elsewhere, may be mentioned specifically, illustrated by the following examples: Firstly, the inversion of backbone chiral centres leading to D-amino acid residue structures may, particularly at the N-terminus, lead to enhanced stability for proteolytical degradation while not impairing activity. An example is given in the paper "Tritriated D-alal-Peptide T Binding", Smith, C.S. et al, Drug Development Res. 15, pp. 371-379 (1988).

Secondly, cyclic structure for stability, such as N to C interchain imides and lactames (Ede et al in Smith ,w7T> and Rivier (Eds) "Peptides: Chemistry and Biology", -d,

L)

Escom, Leiden (1991), p268-270), and sometimes also receptor binding may be enhanced by forming cyclic analogues. An example of this is given in "Confirmationally restricted thymopentin-like compounds", U.S. pat. 4,457,489 (1985), Goldstein, G.

et al. Finally, the introduction of ketomethylene, methylsulfide or retroinverse bonds to replace peptide bonds, i.e. the interchange of the CO and NH moieties may both greatly enhance stability and potency. An example of the latter type is given in the paper "Biologically active retroinverse analogues of thymopentin", Sisto A. et al in Rivier, J.E. and Marshall, G.R. (eds.) "Peptides, Chemistry, Structure and Biology", Escom, Leiden (1990), p.

722 773 The peptides of the invention can be synthesized by various methods which are known in principle, namely by chemical coupling methods (cf. Wunsch, "Methoden der organischen Chemie", Volume 15, Band 1 2, Synthese von Peptiden, Thieme Verlag, Stuttgart (1974), and Barrany, G.; Merrifield, R.B: "The Peptides", eds. E. Gross, J. Meienhofer., Volume 2, Chapter 1, pp. 1-284, Academic Press (1980)), or by enzymatic coupling methods (cf. Widmer, Johansen, Carlsberg Res.

Commun., Volume 44, pp. 37-46 (1979), and Kullmann, W.: "Enzymatic Peptide Synthesis", CRC Press Inc., Boca Raton, Florida (1987), and Widmer, Johansen, J.T.

in "Synthetic Peptides in Biology and Medicine:, eds., Alitalo, Partanen, Vatieri, pp. 79-86, Elsevier, Amsterdam (1985)), or by a combination of chemical and enzymatic methods if this is advantageous for the process design and economy.

It will be seen that one of the alternatives embraced in the general formula set out above is for a cysteine residue to be positioned at both the amino and carboxy terminals of the peptide. This will enable the r s s a 1.=,4 Hj RAZ~ 3 /V.

ro c U s~r I cyclisation of the peptide by the formation of di-sulphide bond.

It is intended that such modifications to the peptide of the present invention which do not result in a decrease in biological activity are within the scope of the present invention.

As would be recognized by those skilled in the art there are numerous examples to illustrate the ability of anti-idiotypic (anti-Ids) antibodies to an antigen to function like that antigen in its interaction with animal cells and components of cells. Thus, anti-Ids to a peptide hormone antigen can have hormone-like activity and interact specifically with the receptors to the hormone. Conversely, anti-Ids to a receptor can 15 interact specifically with a mediator in the same way as the receptor does. (For a review of these properties see: Gaulton, G.N. and Greane, M.I. 1986.

Idiotypic mimicry of biological receptors, Ann. Rev.

"Immunol. 4, 253-280; Sege, K and Peterson, 1978.

20 Use of anti-idiotypic antibodies as cell surface receptor probes. Proc. Natl. Acad. Sci. U.S.A. 2443-2447).

As might be expected from this functional similarity of anti-Id and antigen, anti-Ids bearing the 25 internal image of an antigen can induce immunity to such an antigen. (This nexus is reviewed in Hiernaux, J.R. 1988. Idiotypic vaccines and infectious diseases.

Infect. Immun. 56, 1407-1413.) As will be appreciated by persons skilled in the art from the disclosure of this application it will be possible to produce anti-idiotypic antibodies to the peptide of the present invention which will have similar biological activity. It is intended that such anti-idiotypic antibodies are included within the scope of the present invention.

-ea Accordingly, in a fourth aspect the present invention consists in an anti-idiotypic antibody to the peptide of the first aspect of the present invention, the anti-idiotypic antibody being capable of inducing macrophage and/or neutrophil activation.

The individual specificity of antibodies resides in the structures of the peptide loops making up the Complementary Determining Regions (CDRs) of the variable domains of the antibodies. Since in general, the amino acid sequences of the CDR peptide loops of an anti-Id are not identical to or even similar to the amino acid sequence of the peptide antigen from which it was originally derived, it follows that peptides whose amino acid sequence is quite dissimilar, in 15 certain contexts can take up a very similar three-dimensional structure. The concept of this type of peptide, termed a "functionally equivalent sequence" or mimotope by Geyson is familiar to those expert in the field. (Geyson, H.M. et al 1987. Strategies for 20 epitope analysis using peptide synthesis. J. Immun.

Methods. 102, 259-274).

Moreover, the three-dimensional structure and function of the biologically active peptides can be simulated by other compounds, some not even peptidic in S: 25 nature, but which mimic the activity of such peptides.

S* This field of science is summarised in a review by Goodman, M. (1990). (Synthesis, spectroscopy and computer simulations in peptide research. Proc. llth American Peptide Symposium published in Peptides-Chemistry, Structure and Biology pp 3-29. Ed Rivier, J.E. and Marshall, G.R. Publisher ESCOM.) As will be recognized by those skilled in the art, armed with the disclosure of this application, it will be possible to produce peptide and non-peptide compounds having the same three-dimensional structure as the peptide of the present invention. These r I s -L "functionally equivalent structures" or "peptide mimics" will react with antibodies raised against the peptide of the present invention and may also be capable of stimulating macrophages and/or neutrophils.

It is intended that such "peptide mimics" are included within the scope of the present invention.

Accordingly, in a fifth aspect the presunt invention consists in a compound the three-dimensional structure of which is similar as a pharmacophore to the three- dimensional structure of the peptide of the first aspect of the present invention, the compound being characterized in that it reacts with antibodies raised against the peptide of the first aspect of the present invention and that the compound is capable of 15 activating macrophages and/or neutrophils.

More detail regarding pharmacophores can be found in Bolin et al. p 150, Polinsky et al. p 287, and Smith et al. p 485 in Smith and Rivier (Eds) "Peptides: Chemistry and Biology", Escom, Leiden (1991).

S. 20 As will be appreciated by those skilled in the art the peptides of the present invention will bind to the o TNF receptor. Accordingly, the peptides of tho present invention can be used in assays for the presence of TNF receptor in samples. These samples may be either 25 biological fluids or tissue sections. When used in this manner it is preferred that the peptides are labelled with a detectable label.

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, and Figures in which:- Figure 1 shows the amino acid sequence of human

TNF;

Figure 2 shows the effects of peptides 304 0 V R,4- VW 7O 308 0 and 309 A on the fMLP induced human neutrophil response. Peptides were used at 100tg/10 6 in the 20 min pre-incubation step, Figure 3 shows the kinetics of the chemiluminescence response elicited by Peptide 395 395 (50ug) fMLP; 395 HBSS fMLP; HBSS); Figure 4 shows stimulation of superoxide production by peptides 418 and 419; Figure 5 shows inhibition of in vitr, growth of malarial parasites by neutrophils stimulated by either peptide 418 or peptide 419; Figure 6 shows the effect of peptide 419 on granuloma formation in mice infected with Bacillus 15 Calmette Geurin; :i Figure 7 shows that peptide does not induce the cell adhesion molecule ICAM or human umbilical vein endothelial cells; and Figure 8 is a representation of the TNFa monomer showing the position of the neutrophil stimulating peptides.

Production of human TNF peptides tested for neutrophils stimulatory activity.

The following peptides were synthesised and are S• 25 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 VA H V VA (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 GA (94-109) Peptide 304 s L F KG QG C PS TH V LLTHT I S RI (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 L 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 If K V N L L (73-94) Peptide 323 *T I S R I A V S Y Q T (79-89) 15 Peptide 393 0 6 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).

*Peiptide 395 P ST HV L L T H T10-80).

Peptide 396 A V S Y Q T K V N L L (84-94).

Peptide 418 25 Peptide 419 Peiptide 462 K G Q G Cys (Acm) P S T H V L L T H T These peptides were synthesised using the following general protocol.

All peptides were synthesised using the Fmoc-polyaiide 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-hydroxymethyiphenoxyacetic acid as the functionalised /3 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 15 TABLE 1 Amino acid ProteLting group Coupling Method 1' Arg Mtr or Pmc Either Asp OBut Either Cys Acm (permanent) Either S: 20 Glu OBut Either His Boc OPfp only Lys Boc Either SB ly h Ser But BOP only Thr But BOP only 25 Tyr But Either Asn none OPfp only Asn none OPfp only Gln 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, Q A Buffer B 80% ACN 20% A).

lv;r 0 Peptide 308. Peptide is cleaved from the resin with 5% 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).

Effect of TNF peptides on neutrophil function.

Chemiluminescence assay.

This assay examined the effect of TNF peptides on priming for a neutrophil F-met-leu-phe response as described by Ferrante et al, 1988, (Int. Arch. Allergy Appl. Immunol, 86, 82-91). Purified human neutrophils 15 were pretreated with peptide for 20 minutes before the addition of f-met-leu-phe. The lucigenin dependent chemiluminescence response, which reflects superoxide production, was then measured. The results obtained are set out in Table 2 and are expressed as imV of 20 lucigenin dependent chemiluminescence and represent the maximal cell activity attained.

In addition, the effects of peptide 304,308 and 309 are shown graphically in Figure 2.

This experiment was repeated with peptides 304, 25 308 and 309. The results obtained as shown in Table 3.

SThe experiment was also conducted using peptides 393, 394, 395 and 396. Of these peptides only peptide 395 was able to stimulate the neutrophil respiratory burst (Table The effect of peptide 395 was dose dependent as shown by the results of 3 experiments (Table The kinetics of the chemiluminescence response elicited by peptide 395 is shown in Figure 3.

Peptide 395 displays improved solubility over peptide 309.

bl dI 1 38 TABLE 2 Peptide Concentration [g/10 6 cells) 0 1 10 100 500 275 1.02 0.99 0.69 0.43 0.80 301 0.34 0.93 0.74 0.55 1.10 302 0.37 0.16 0.18 0.29 303 0.37 0.23 0.17 0.22 304 0.37 0.18 0.43 2.56 2.76 305 0.37 0.27 0.36 0.24 306 0.37 0.27 0.35 0.23 307 0.37 0.35 0.37 0.42 323 0.37 0.23 0.17 0.47 308 0.37 0.91 4.80 49.52 309 0.37 0.38 0.98 13.44 Results are expressed as mV of lucigenin dependent chemiluminescence and represent peak of response i.e.

20 the maximal cell activity attained.

TABLE 3

S

5 Peptide Peptide concentration 6 cells) 0 10 100 0.04 0.36 0.64 0.71 0.91 6.97 304 304 fMIP 308 308 fMLP 309 309 fMLP 0.04 0.42 0.04 0.42 1.00 2.74 0.31 2.46 11.76 28.56 2.84 11~-1 hl L~ Table 4 Comparisons of 309 and its subpeptides on neutrophil respiratory burst Treatment Chemiluminescence (mV) (100 ug peptide) Diluent 0.58 309 4.70 393 0.31 394 0.33 395 5.32 396 0.70 oo* ooo o e eooo a ouoo oooo *o e o *oo *o* *e t k Table 5 Effect of 395 on neutrophil respiratory burst Treatment Chemiluminescence (mV) Exp. 1 Exp. 2 Exp. 3 Diluent 0.58 0.68 0.38 20 fMLP 1.53 3.53 1.96 l[g 395 3.25 0.89 0.03 l1g 395 fMLP 3.36 4.55 0.29 10Lg 395 4.92 3.97 0.64 1 0 g 395 +fMLP 7.31 9.10 2.34 25 50ug 395 8.01 10.81 395 fMLP 12.58 22.09 100 g 395 2.36 19.14 5.26 100 g 395 fMLP 5.29 18.10 10.59 100 g 309 5.98 6.68 1.24 100 g 309 fMLP 27.44 22.77 6.69 Effect on Superoxide Formation The effect of peptides 308 and 309 on superoxide formation was examined by the cytochrome reduction assay, according to the procedure of Ferrante, 1989 (Infection and Immunity), 57: 2115-2122). The results, 4 ~L I 17 expressed as n moles of 0 2 /5x10 5 cells as set out in Table 6.

TABLE 6 Peptide Peptide concentration ig/5 x 105 cells) 0 10 100 308 0.270 2.78 4.892 308 fMLP 2.757 5.00 6.729 309 0.270 0.62 2.30 309 fMLP 2.757 3.87 5.14 The effects of peptides 418 and 419 or superoxide formation were also assessed in a like manner and the results shown in Figure 4.

Effect of TNF peptides on the ability of neutrophils to inhibit in vitro Qrowth o. malaria parasites It is known that neutrophils stimulated by TNF are 20 able to inhibit the in vitro growth of malaria parasites. The ability of neutrophils stimulated with peptides 418 and 419 to inhibit in vitro growth of malaria parasites was assessed.

Briefly, neutrophils were incubated with peptides 418 and 419 at 37 0 C for 30 minutes and then washed with HBSS. The neutrophils were then added to a culture of red blood cells infected with P.falciparum. The cultures were then examined microscopically for evidence of parasite death.

The results obtained with thawed normal serum, normal serum and immune serum are shown in Figure As can be seen peptide 419 is more effective than TNF.

BCG Experiment Peptide 419 was tested for the capacity to induce early activation of monocytes leading to reduced infective load and granuloma formation. C57BL/6 (H 2 I L I I I in groups of six mice were infected with M. bovis (BCG), 107cfu, by intraperitoneal injection. On days 1, 3, 5, 7 and 9, the mice receive by intravenous injection: Peptide 419, 500[ig in 0.5ml PBS, TNF, murine recombinant 104 units in PBS (TNF at this concentration has been shown to be non-toxic in mice; Longermans et al Infection Immunity, 1992, 60:5107-5112), Saline At day 11 the mice were sacrificed for enumeration of the number of granulomas in the liver. The results are shown in Figure 6.

These results are strongly indicative that peptide 15 419 may have a therapeutic role in the treatment of granulomatous diseases.

Pseudomonas Experiment Chronic lung infection by Pseudomonas aeruginosa a. (Pa) is; common in cystic fibrosis. Although the too* 20 disease is dominated by a chronic excess of neutrophils, in vitro studies (Ferrante et al.

submitted' suggest that these cells may not be of. ,receptive to activation by TNF for antibacterial activity. The present inventors reasoned that pre- 25 sensitising neutrophils with TNF intravascularly, prior to their entry into the lung, may overcome this problem. Since TNF would not be useful as a therapeutic agent if used in this manner, because of its toxic side effects such as intravascular coagulation, the 11-amino acid peptide 419 which retains the neutrophil priming properties but lacks the toxicity of TNF was used. The ability of i.v.-injected 419 to protect against chronic lung injury induced by Pseudomonas was tested. Chronic lung infections were established in 120 14 week female Swiss mice by a ,IC introducing the bacteria suspended in agar beads (<200um diameter) into the lung. Mice were anaesthetised and of bead suspension introduced via a polyethylene cannula into the right bronchus through a small incision between the tracheal rings. After recover, animals (n=20) developed a chronic infection followed for at least 10-14 days. Control animals included: sham-operated and those inoculated with sterile beads or with beads containing fresh, heat-killed Pa Eight of the 20 infected animals were treated with either the "419" peptide (or with saline carrier, receiving 100til of 0.5mg peptide (or carrier) by tail-vein injection, each day for 3 days from day 12. After sacrifice, lungs were 15 perfusion-fixed with formalin and sections (masked to treatment type) were assessed histologically for the extent of inflammation. In this continuing study, chronically-infected animals showed extensive inflammatory reactions in the bronchi and the 20 parenchyma. Control groups revealed negligible inflammation. The animals treated with peptide 419 showed significantly less percentage peripheral consolidation (mean (0.25 (se 0.25)) than the carriertreated animals (mean 5.25 (se 1.84) p<0.05 Mann- *o S: 25 Whitney). These results suggest that the TNF-derived peptide "419" may be beneficial in the treatment of chronic pseudomonal lung infection. Further, these results suggest that peptide 419 may have a therapeutic role in the treatment of lung infection in patients suffering from cystic fibrosis or who are immunosuppressed.

Effect of Peptide 419 on monocytes Peptide 419 was assayed for its ability to prime monocytes for a respiratory burst. This assay was conducted essentially as described above for o~ ~La I~r~%l ~1 a ~I neutrophils except that the cells were cultured for 24 hours prior to assay. The results are set out in Table 7.

TABLE 7 Peak mV Chemiluminescence 1 2 Mean S.D.

HBSS 1.72 1.46 1.59 0.184 fMLP 104 114.0 109.0 7.071 419 2.42 4.35 3.385 1.365 413 fMLP 263.0 217.0 240.0 32.527 TNF 2.03 1.74 1.885 0.206 :oo. TNF fMLP 104.0 163.0 133.5 +41.719 106 monocytes/tube/ml 50 U TNF 30 min pre-incubation at 37 0

C

Peptide 419 100g/10 6 monocytes 30 mins preincubation at 37 0

C.

In Socher et al., 1987 (PNAS:84:8829-8833) a peptide having some similarity to the peptides of the present invention is disclosed. The disclosed peptide is a fragment of TNF from residues 65-79. This peptide was synthesized by the present applicant and is referred to herein as "peptide 462".

25 Peptide 462 was assayed for its ability to prime neutrophils for a respiratory burst in comparison with peptide 419 using the protocol set out above. The results obtained are set out in Table 8.

TABLE 8 Treatment Chemiluminescence mean sem (mV) HBSS 4.1 0.3 462 (itg) 3.1 462 (10oig) 4.8 0.7 462 (100Lg) 4.8 419 (100g) 18.6 It is clear from these results that whilst peptide 462 may bear some superficial similarity to the peptides of the present invention it does not possess the biological activity of those peptides.

Induction of ICAM on human umbilical vein endothelial cells (HUVEC) TNF is known to induce ICAM and it is thought that this activity contributes to its toxicity in vivo.

Peptide 419 was tested for its ability to induce ICAM using the following procedure.

Confluent HUVEC, grown in M199 supplemented with foetal calf serum, endothelial growth factor and heparin were treated with either TNF (100 units) or 15 peptide 419 (1mg) for four hours at 37 C. The cells were then washed extensively with PBS containing Ca++ and Mg and fixed (100ml 0.025% guteraldehyde, overnight, 4 0 The cells were again washed, and incubated with anti-ICAM antibody (84H10) for lhr at S 20 room temperature. The cells were washed and incubated for a further lhr at room temperature in the presence of rabbit anti-mouse Ig-HRP. Surface expression of Icam was then quantitated using the ABTS substrate by absorption at OD405 rim.

25 The results of the assay on peptide 419 are shown in Figure 7. As can be seen peptide 419 does not induce the cell adhesion molecule ICAM on human umbilical vein endothelial cells. This clearly suggests that peptide 419 would be less toxic in vivo than TNF.

Effect of TNF peptides on neutrophil random migration Migration of cells is an important property by which cells reach infection sites. Their accumulation at these sites is also dependent on the capacity of inflammatory mediators to inhibit their migration out sa of the sites. The present inventors have examined TNF I I ~-a and peptide 304, 308 and 309 for their effect on the migration of neutrophils.

In these experiments neutrophils were pre-treated with the peptide or TNF and then examined their ability to migrate out of wells in agarose as described by Ferrante et al, 1988, (Arch. Allergy Appl. Immunol.

86:82-91). The results are shown in Table 8. The results show that TNF was only partially migration inhibitory at 100 units/10 6 cells. Both peptides 308 and 309 were potent migration inhibitors, however, peptide 304 was found to be chemokinetic (it stimulated cell migration).

TABLE 8 *0 s C 9 9**t 9 ss ~g1 as '4' 0's s'4' Treatment Inhibition of Migration lg/10 6 cells) 0 10 100 TNF ND ND 4% 304* -16% -43% -883% 308 0 0 100% 309 0 0 100% *Peptide 304 was found to stimulate (chemokinetic) Chemotactic properties of TNF and peptides The chemotactic properties of TNF and peptides 304, 308 and 309 were examined using the following method: 3ml of molten 2% agarose was mixed with 3ml of 2x concentrated medium 199 containing foetal calf serum and poured into Petri dishes. Sets of 30 3 wells of 2.5mm diameter, each 3mm apart, were cut in the agarose. 5til of neutrophils (2 x 10 cells) were added to the inner well, with chemotactic agent or control medium added to the outer wells. Migration at various time intervals was then measured.

I

The results of these experiments are shown in Table 9.

TABLE 9 Agent* Migration distance (mm) at 1.5h None Agent None Agent fMLP 0.50 1.46 0.66 2.45 TNF 0.50 0.48 0.66 0.69 304 0.48 0.47 0.68 0.72 308 0.50 0.66 0.63 1.41 309 0.50 0.53 0.63 0.68 e To the chemotactic well was added 5ml of 1x10 7 MfMLP, of either peptide 304; peptide 308 and peptide 309 or 103 U/ml of TNFa Effect of TNF Peptides on Neutrophil Degranulation The conditions of measuring degranulation were as described by Ferrante A, 1989, (Infect and Immunity 57, 3110-3115). In these studies 100l of neutrophils (10 7 /ml) were incubated for 20min at 370 C after which il of cytochalasin B was added. After 10 min incubation the volume of cell suspension was made up to 25 1 ml with Hanks Balanced Salt Solution (HBSS). The cell-free supernatants were collected and analysed for enzyme levels after a further incubation at 37C.

P-Glucuronidase activity was measured fluorimetrically by using 4-methylumbelliferyl-P-D-glucuronide as substrate. This involved incubating 50tl of 2.5 mM substrate in 0.1 M citric acid-sodium phosphate buffer, pH 4.5, at 37 0 C for 3 h. The reaction was stopped by adding 1.5 ml of 0.2 M glycine-sodium hydroxide buffer, pH 10.7 and the fluorescence of the liberated 4-methylumbelliferone was quantitated by using excitation and emission wavelengths of 336 and 446 nm, d s I e-I respectively. Vitamin B 12 binding protein was measured using 57 Co-vitamin B 12 This assay is based on the principle that the binding protein binds to the 7 Co-Vitamin B 12 and as a result the radioactive vitamin B 12 does not bind to charcoal. The resultant radioactivity in the supernatant can then be equated to the concentration of vitamin B 12 -binding protein in the sample. The results of these experiments are set out in Table 10 A and B.

Table 10 Effect of TNF Peptides on Neutrophil Degranulation Neutrophils were treated with 100pg/10 6 cells of 304, 305 or 308+fMLP (in the presence of CytoB).

A.

3-glucuronidase release Treatment Exp. 1 Exp. 2 Exp. 3 Exp. 4 HBSS 3.63 1.84 2.72 6.23 HBSS fMLP 23.62 41.41 40.19 27.54 304 3.63 3.14 2.52 9.82 304 fMLP 26.95 36.43 35.34 36.65 Control peptide 13.41 Control peptide fMLP 35.69 308 0.8 0.65 2.76 0.72 308 fMLP 17.57 28.86 17.86 18.20 4**b e.

S. L r II 5 I r *4 S. 9 6*85 4* o

B.

Vitamin B 12 Binding Protein Treatment Exp. 1 Exp. 2 Exp. 3 Exp. 4 HBSS 9.21 9.27 9.67 4.80 HBSS fMLP 28.85 27.91 45.31 27.33 304 11.40 10.82 13.06 8.42 304 fMLP 43.76 35.60 59.15 37.12 Control peptide 7.49 Control peptide fMLP 38.62 308 2.00 2.08 5.70 2.25 308 fMLP 35.81 27.59 26.55 21.51 The effects of TNFa peptides on stimulation of neutrophil respiratory burst, degranulation, migration inhibition, chemokinesis and chemotaxis were investigated. As can be seen from the results set out above only peptides 304, 308 and 309 were found to prime human neutrophils for the respiratory burst associated with f-met-leu-phe treatment, i.e. in a manner analogous to that of TNFa. Together these peptides comprise the primary amino acids sequence region of amino acids 54 to 94 of human TNFa. Peptide 308 is a particularly potent primer of neutrophils in this assay.

It is to be noted, however, that peptide 323 which has a sequence which corresponds to amino acids 79 to 89 of human TNF was not found to be capable of priming neutrophils for the respiratory burst associated with f-met-leu-phe treatment. The reason for the lack of neutrophil stimulating activity of this peptide has not as yet been elucidated, however, one hypothesis for the lack of activity of this peptide may be that peptide

I~

323 does not include the amino acid residues which bind to the TNF receptor on the neutrophils.

Peptides 308 and 309 have also been found to be potent inhibitors of neutrophil migration whilst peptide 304 has been found to be chemokinetic. Peptide 308 has also been found to be strongly chemotactic.

The effects of TNF peptides 304 and 308 on degranulation of neutrophils (Table 10) showed that peptide 308 decreased the release of the contents of both the specific and the azurophilic granules as measured by the release of Vitamin B 12 binding protein and P-glucuronidase release respectively. This effect of peptide 30e was still apparent following stimulation with fMLP. In contrast, peptide 304 had no effect on 15 neutrophil degranulation in the absence of fMLP. In the presence of fMLP peptide 304 enhanced release from specific granules but not azurophilic granules.

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.

Dated this 13 day of August 1993.

PEPTIDE TECHNOLOGY LIMITED PATENT ATTORNEYS FOR THE

APPLICANT

F.B. RICE CO.

o«

Claims

1. A peptide which primes neutrophils for superoxide production and an enhanced respiratory burst following treatment with N-formyl-L- methionyl-L-leucyl-L-phenylalanine, wherein the peptide is of the general formula:- X 1 -X 2 -X3-X 4 -XS-Xo-X 7 -XB-X,-Xo-Xl in which X 1 is Pro or Na-alkylamino a id X 2 is Ser or Thr X 3 is Ala or Gly X 4 is His, Lys or Arg X, is Val, Ile, Leu or Met X, is Leu, Ile Val or Met 15 X 7 is Ile, Leu, Val or Met X 8 is Thr or Ser X 9 is His, Lys or Arg Xo 1 is Thr or Ser X 1 is Ile, Leu, Val or Met

2. A peptide as claimed in claim 1 in which the peptide has the ammno acid sequence: Pro Ser Ala His Val Leu Leu Thr His Thr Ile.

3. A method of treating a subject having depressed ne :ophil function, the method comprising administering to the subject an effective 25 therapeutic amount of the peptide as claimed in claim 1 or claim 2.

4. A method as claimed in claim 3 in which the subject is suffering from acquired immune deficiency syndrome. Dated this 24th day of December 1996. PEPTIDE TECHNOLOGY LIMITED Patent Attorneys for the Applicant: F.B. RICE CO. ABSTRACT The present invention provides peptides capable of stimulating neutrophils. In particular, the peptides prime neutrophils for a respiratory burst following treatment with N-formyl-L-methionyl-L-leucyl-L- phenylalanine. The peptides have an amino acid sequence substantially corresponding to amino acids 54 to 94 of Figure 1 or a part thereof. These peptides may also be used in the treatment of a subject having depressed neutrophil function. 0* o*0o o* 4 *440 o 4 ft r