AU636735B2

AU636735B2 - Synthetic polypeptides

Info

Publication number: AU636735B2
Application number: AU74679/91A
Authority: AU
Inventors: Robert Vincent Fishleigh; Barry Robson
Original assignee: Proteus Molecular Design Ltd
Current assignee: Protherics Medicines Development Ltd
Priority date: 1990-03-15
Filing date: 1991-03-13
Publication date: 1993-05-06
Anticipated expiration: 2011-03-13
Also published as: CS68091A2; OA09671A; BR9106159A; CA2078220A1; NZ237417A; FI923800A0; HU9202939D0; WO1991013909A1; MX24890A; FI923800A; IL97551A0; HUT63179A; CN1054772A; EP0519986A1; GB9005829D0; JPH05505188A; AP211A; ZA911886B; AU7467991A; AP9100245A0

Description

WO 91/13909 PCT/GB91/00392 Synthetic Polypeptides The present invention relates to synthetic polypeptides. It particularly relates to synthetic polypeptides which emulate the three-dimensional structures and/or electrostatic surfaces and/or other physical, chemical and structural properties of specific regions of viral envelope proteins. It is of particular interest to the design of vaccines, immunologically active therapeutic agents, diagnostics and other medical or scientific agents in relation to the Human Immunodeficiency Virus (HIV) known to be the causative agent of Acquired Immune Deficiency Syndrome (AIDS).

Within the last decade AIDS has emerged as an important medical problem throughout the world and there is currently an urgent need for agents for the study, diagnosis, treatment and/or prevention of infection by the HIV, the causative agent of the disease. With the availability of the amino acid sequences of proteins produced by the HIV I and HIV II viruses (see, for example, Ratner, L. et al., Nature 313, 277 (1985); Meusing, M.A. et al., Nature 313, 450 (1985); Wain- Hobson, S. et al., Cell 40, 9 (1985)), it has been possible to devise synthetic polypeptides which emulate the antigenic properties of the viral envelope proteins.

An object of the present invention is the development of synthetic polypeptides which can elicit the production of antibodies to the HIV virus, and most preferably neutralising antibodies, that is, antibodies which prevent infection by and/or limit the spread of the HIV virus by passive or active immunisation.

Passive immunisation with such antibodies may constitute an effective means of treatment of AIDS patients thus controlling the spread of the virus within and between individuals and hence slow or halt the progress of the disease.

Our invention provides a synthetic polypeptide L_ PCT/GB 9 1 0 0 3 9 2 1 2 02 92 2 1I 2 Februar 1992 having at least one antigenic property of the envelope protein of at least one strain of Human Immunodeficiency Virus (HIV), said polypeptide consisting substantially of an amino acid sequence of formula X-R -R 2

-R

3 -RR-Rs-R 7 -R-Trp-Gly- Cys-R 8 -R-Ro-R -R 1 -Cys-Y

(I)

wherein R 1 is Asp or Glu; R2 is an amino acid residue selected from Gly, Ala, Pro, Ser, Thr, Asn or Gin;

R

3 is an amino acid residue selected from Gly, Ala, Pro, Ser, Thr, Asp, Glu, Asn, Lys, His, Gin or Arg;

R

4

R

5 and R1 are each independently any amino acid residue;

R

6 and Rg are each independently an amino acid residue selected from Gly, Ala, Pro, Ser, Thr, or Asn;

R

7 is an amino acid residue selected from Gly, Ala, Val, Leu, Ile, Ser, Thr, Asn, Gln, Phe, Tyr, Trp, Cys, Met or Pro;

R

9 and R 2 are each independently an amino acid residue selected from Gly, Ala, Leu, Ile, Val, Met, Cys, Phe, Tyr or Trp; Rio is an amino acid residue selected from Lys, His or Arg; and X and Y may each independently be absent or independently be one or more amino acid residues with the proviso that when present they do not provide or form part of an antigenic property of the envelope protein of at least one strain of Human Immunodeficiency Virus.

Peptides according to formula I above without X and Y being present will of course be useful, for example, 4' United Kinricll P ten' t C:ff;..

V i P C T ln n, l'o A PCT/GB 91! 0 2 12 02 92 3 1 2 February 1992 in the production of antibodies to the HIV. However, when X or Y are present they may be any length but preferably less than 20 amino acids, more preferably less than 10, eg. 3 to 6. It will of course be appreciated that the sequence according to formula I may constitute a protein with X and Y being major portions of the protein with the antigenic sequence being for example, part of an exposed loop on a globular protein.

Preferably, R 2 is selected from Gln or Thr, R 3 is selected from Ser, Asn, Gln, Arg or Ala, R 4 is selected from Leu, Ile, Gln or Arg, R S is either Leu or Lys, R 6 is either Gly or Asn, R, is selected from Gly, Ala, Leu, Ile, Val, Met, Cys, Phe, Tyr, Trp or Ser, R, is either Ser or Ala, R, is either Gly or Phe, R1 0 is either Arg or Lys, R 11 is selected from Leu, His, Ile, Gln and R1 is selected from Ala, Ile or Val. The Cys residues at positions 10 and 16 may optionally be linked by an intra-molecular disulphide bridge.

One preferred form of polypeptide according to the invention consists substantially of an amino acid sequence of formula -Asp-Gln-R 3 -Leu-RS-Gly-R 7 -Trp-Gly- Cys-Ser-Gly-Lys-R 1 1

-R

12 -Cys-Y

(II)

wherein R3, R 5

R

11

R

12 X and Y are as defined above; and R7 is an amino acid residue selected from Gly, Ala, Leu, Ile, Val, Met, Cys, Phe, Tyr or Trp.

Preferably in a sequence according to formula (II),

R

3 is selected from Ser, Asn, Gln and Arg, R1 is selected from Leu, His or Ile and R 12 is Ile or Ala.

Advantageously, R7 is selected from Ile, Phe, Met, Val or Leu. More preferably R 3 is either Ser or Asn and R, is Lys.

A preferred form of polypeptide of formula (II) according to the invention consists of the sequence:u United Kincidom Fci ntC -fPCT 1 ftn PCT/G8 9 1 0 0 2 i 12 02 92 S- 4 1 2 Febnaru 1992 X-Asp-Gln-Ser-Leu-Lys-Gly-Ile-Trp- Gly-Cys-Ser-Gly-Lys-Leu-Ala-Cys-Y wherein X and Y are as defined above, and the Cys residues at positions 10 and 16 may optionally be linked by an intra-molecular disulphide bridge.

Another preferred form of polypeptide of Formula (II) comprises R 3 selected from Gin or Arg, R 5 is Leu, R 7 is selected from Ile, Phe and Met, R 11 is selected from Leu, His or Ile, and R 12 is Ala. A preferred sequence has the formula:- X-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp- Gly-Cys-Ser-Gly-Lys-Leu-Ala-Cys-Y wherein X and Y are as defined above, and the Cys residues at positions-10 and 16 may optionally be linked by an intra-molecular disulphide bridge.

Polypeptides according to formula II resemble certain (,itopic portions of HIV I envelope protein.

Another preferred form of polypeptide according to the invention consists substantially of an amino acid sequence of formula (III):- X-R -R -R-R 4 -RR-Asn-Ser-Trp-Gly-Cys-Ala- Phe-Arg-Gln-Val-Cys-Y (III) wherein R 1 is either Glu or Asp R2 is Thr or Gln;

R

3 is an amino acid selected from Ser, Asn Arg, Gln or Ala; 30 R, is an amino acid selected from Leu, Ile, Arg or Gln; R, is Lys or Leu; and wherein X and Y are as defined above, and the Cys residues at positions 10 and 16 may optionally be linked by an intra-molecular disulphide bridge.

A preferred form of a polypeptide according to Formula III consists substantially of an amino acid sequence of formula (IIIa):- 7 cj United Kinqdom P ot Cd o SPCT Intenatio 1 t I Y if WO 91/13909 PCT/GB91/00392 5 X-Glu-Thr-R 3

-R

4 -Lys-Asn-Ser-Trp-Gly- Cys-Ala-Phe-Arg-Gln-Val-Cys-Y (IIIa) wherein R 3 is an amino acid residue selected from Ser, Asn, Arg, Gin or Ala, R. is an amino acid residue selected from Leu, Ile, Arg or Gln, X and Y are as defined above, and the Cys residues at positions 10 and 16 may optionally be linked by an intra-molecular disulphide bridge. It is preferred that R 3 is Ser when

R

4 is Ile and when R 3 is Ala R 4 is either Arg or Gln but preferably Arg.

Polypeptides according to formula III are similar to certain epitopes of HIV II envelope proteins.

Preferred polypeptide sequences according to the invention were chosen on the basis of their topographical similarity to more than one antigenic determinant of the HIV envelope proteins. For example, an antigenic determinant to which a given polypeptide was originally designed to be an analogue may also show topographical similarity to one or more other regions of the HIV envelope proteins possibly due to duplication of ancestral genes, or because the polypeptide is an analogue of a discontinuous determinant, or because the polypeptides have been designed to be polyvalent. A discontinuous epitope may be viewed as being composed of closely opposed sequential epitopes which may be of antigenic significance in their own right and a polyvalent polypeptide may contain two or more (continuous or discontinuous) determinant analogues in a single polypeptide chain, thus providing a means to simultaneously elicit the production of a range of antibodies which will recognise two or more determinants on the HIV envelope proteins.

Peptides according to the invention may be synthesised for example using either standard 9fluorenyl-methoxycarbonyl (F-Moc) chemistry (see, for example, Atherton, E. and Shepr ,rd, R. C. (1985) J.

WO 91/13909 P(7r/GB91/00392 6- Chem. Soc. Chem. Comm. 165) or standard butyloxycarbonate (T-Boc) chemistry. The correctness of the structure and the level of purity, which will normally be excess of 85%, should be carefully checked, and particular attention be given to the correctness of internal disulphide bridging arrangements when present.

Various chromatographic analyses, including high performance liquid chromatography, and spectrographic analyses, including Raman spectroscopy, may for example be employed for this purpose.

All the sequences herein are stated using the standard I.U.P.A.C. three-letter-code abbreviations for amino acid residues defined as follows: Gly-Glycine, Ala-Alanine, Val-Valine, Leu-Leucine, Ile-Isoleucine, Ser-Serine, Thr-Threonine, Asp-Aspartic acid, Glu- Glutamic acid, Asn-Asparagine, Gln-Glutamine, Lys- Lysine, His-Histidine, Arg-Arginine, Phe-Phenylalanine, Tyr-Tyrosine, Trp-Tryptophan, Cys-Cysteine, Met- Methionine and Pro-Proline.

Polypeptides according to the invention or antibodies thereto may be administered on their own or with other agents such as 3'-azido-3'-deoxythymidine (AZT) (zidovudine), which acts at a different level by interfering with the replication of the genetic material of the virus, and/or HIV protease inhibitors, which block the action of an enzyme essential to the development of the virus.

Polypeptides according to the invention may be usE~.

to raise antibodies which will cross-react with envelope proteins produced by a wide range of HIV I and/or HIV II strains. Our analyses have shown that since the conformational/topographic/electrostatic properties of polypeptides according to the invention are such that they are highly likely to elicit the production of antibodies which will cross-react with HIV envelope proteins from several or many strains, further advantages may arise from combining several variant polypeptides in a larger polypeptide. Such a WO 91/13909 PCT/GB91/00392 7 polypeptide may have the general formula (IV): [La-F m- b- in- L (IV) wherein F and G may each independently be a polypeptide according to any one of Formulae I to IIIa, L is a linking sequence, a, b and c are each independently 0 or 1 and m and n are each positive numbers e.g. b~ ween 1 and 10 inclusive. L is preferably a short, conformationally flexible section of polypeptide chain such as, for example and without limit Gly-Gly-Gly-Gly- Gly, Gly-Pro-Gly-Pro-Gly-Pro or Gly-Ser-Ala-Gly-Ser-Gly- Ala. It should be clear that each repeat may optionally have a different variant of a polypeptide according to the invention.

Polyvalent determinant analogues as defined by Formula IV are referred to as pseudohomopolyvalent, wherein variants of essentially the same determinant analogue are repeated in a single polypeptide chain. In addition, simple homopolyvalent polypeptide immunogens, which contain multiple copies of the same variant of one of the determinant analogues according to any one of formulae I to IIIa, would also be expected to be effective, and are also included within the scope of the present invention.

Pseudohomopolyvalent immunogenic polypeptides are expected to be particularly valuable as vaccines, where they should elicit the production of a range of (neutralising) antibodies with a similar but non-identical underlying specificity, which between them would cross-react with envelope protein from a wider range of HIV strains, and would thus be more effective at conferring protective immunity. There would also be advantages in constructing heteropolyvalent polypeptides which contain one or more copies, in any order, of one of the polypeptides according to the present invention and one or more other polypeptide analogues of determinant analogues. Such polypeptides, which are WO 91/13909 PCT/GB91/00392 8 provided for in the present invention, have the general formula

L

d -F-[L-G]n-L e

(V)

wherein F is a polypeptide according to any one of Formulae I to IIa, G is a polypeptide according to any one of Formulae I to IIIa or other sequence, m and n are each positive numbers e.g. between 1 and 10 inclusive, and d and e are each independently 0 or 1, is preferably a short, conformationally flexible section of polypeptide chain such as, for example and without limit Gly-Gly-Gly-Gly-Gly, Gly-Pro-Gly-Pro-Gly-Pro or Gly-Ser-Ala-Gly-Ser-Gly-Ala. In preferred forms of 1- peptide V, G may comprise a polypeptide according to any one of Formula I to IIIa or the sequence: X-Gln-Gln-Glu-Lys-Asn-Gly-Gly-Glu-Leu-Y wherein each Gly may independently be replaced with any other amino acid and/or X and Y may each independently be absent or one or more e.g. three amino acid residues or G may comprise some other polypeptide sequence related to antigenic proteins from HIV.

It is to be understood that any antigenically significant subfragments and/or antigenically significant variants of the above-identified polypeptide sequences which retain the general form and function of the parent polypeptide are included within the scope of this invention. In particular, the substitution of any of the specific residues by residues having comparable conformational and/or physical properties, incluaing substitution by rare (but naturally occurring, e.g.

D-stereoisomers) or synthetic amino acid analogues, is included. For example, substitution of a residue by another in the same Set, as defined below, is included within the ambit of the invention; Set 1 Ala, Va?, Leu, Ile, Phe, Tyr, Trp and Met; Set 2 Ser, Thr, Asn S WO 91/13909 PCT/GB91/00392 9 and Gin; Set 3 Asp and Glu; Set 4 Lys, His and Arg; Set 5 Asn and Asp; Set 6 Glu and Gin; Set 7 Gly, Ala, Pro, Ser and Thr. D-stereoisomers of all amino acid types, may be substituted, for example, D-Phe, D-Tyr and D-Trp.

In preferred embodiments of the invention, X and Y if present may independently include one or more segments of protein sequence with the ability to act as a T-cell epitope. For example, segments of amino acid sequence of the general formula 1-2-2-4, where 1 is Gly or a charged amino acid Lys, His, Arg, Asp or Glu), 2 is a hydrophobic amino acid Ile, Leu, Val, Met, Tyr, Phe, Trp, Ala), 3 is either a hydrophobic amino acid (as defined above) or an uncharged polar amino acid Asn, Ser, Thr, Pro, Gin, Gly), and 4 is a polar amino acid Lys, Arg, His, Glu, Asp, Asn, Gin, Ser, Thr, Pro), appear to act as T-cell epitopes in at least some instances (Rothbard, J.B. Taylor, W.R.

(1988). A sequence pattern in common to T-cell epitopes. The EMBO Journal 93-100). Similarly segments can be of the sequence wherein 1' is equivalent to 1 as defined earlier, 2' to 2, 3' and 4' to 3, and 5' to 4 (ibid). Both forms ara included within the scope of the present invention and one or more T-cell epitopes (preferably less than five) which may be of the type defined above or may be of other structure and which may be separated by spa.cer segments of any length or composition, preferably less than five amino acid residues in length and comprising for example residues selected from Gly, Ala, Pro, Asn, Thr, Ser or polyfunctional linkers such as non-a amino acids. It is possible for a C- or N-terminal linker to represent a comp-ete protein, thus obviating the possible need for conjugation to a carrier protein.

Also included within the scope of this invention are derivatives of the polypeptide according to formula I in which X or Y are or include a "retro-inverso" amino acid, i.e. a bifunctional amine having a functional WO 91/13909 PCT/GB91/00392 10 group corresponding to an amino acid. For example an analogue according to the invention and containing a retro-inverso amino acid may have the formula:

R

Al N C N A2 I I I H H H where R is any functional group, e.g. a glycine side chain, and Al and A2 are preferably each a copy of one of the analogues defined herein (but not necessarily the same) attached by its N- or C-terminal end. T-cell epitopes may optionally be included as discussed earlier.

Retro-inverso modification of peptides involves the reversal of one or more peptide bonds to create analogues more resistant than the original molecule to enzymatic degradation and offer one convenient route to the generation of branched immunogens which contain a high concentration of epitope for a medium to large immunogen. The use of these compounds in large-scale solution synthesis of retro-inverso analogues of shortchain biologically active peptides has great potential.

It should be noted that analogues incorporating retro-inverso amino acid derivatives cannot be made directly using a recombinant DNA system. However, the basic analogues can, and they can then be purified and chemically linked to the retro-inverso amino-acids using standard peptide/organic chemistry. A practical and convenient novel procedure for the solid-phase synthesis on polyamide-type resin of retro-inverso peptides has been described recently [Gazerro, Pinori, M. Verdini, A.S. (1990). A new general procedure for the solid-phase synthesis of retro-inverso peptides. In "Innovation and Perspectives in Solid Phase Synthesis" Ed. Roger Epton. SPCC (UK) Ltd, Birmingham, UK].

The polypeptides are optionally linked to a carrier I In I I I PC/GB91/00 392 WO 91/13909 11 molecule, either through chemical groups within the polypeptiies themselves or through additional amino acids added at either the C- or N-terminus, and which may be separated from the polypeptides themselves or surrounded by one or more additional amino acids, in order to render them optimal for their immunological function. Many linkages are suitable and include for example use of the side chains of Tyr, Cys and Lys residues. Suitable carriers include, for example, purified protein derivative of tuberculin (PPD), tetanus toxoid, cholera toxin and its B subunit, ovalbumin, bovine serum albumin, soybean trypsin inhibitor, muramyl dipeptide and analogues thereof, and Braun's lipoprotein although other suitable carriers will be readily apparent to the skilled person. When using PPD as a carrier for polypeptides according to the invention, a higher titre of antibodies is achieved if the recipient of the polypeptide-PPD conjugate is already tuberculin sensitive, e.g. by virtue of earlier BCG vaccination.

In the case of a human vaccine it is worth noting that in the UK and many other countries the population is routinely offered BCG vaccination and is therefore largely PPD-sensitive. Hence PPD is expected to be a preferred carrier for use in such countries.

The mode of coupling the polypeptide to the carrier will depend on the nature of the materials to be coupled. For example, a lysine residue in the carrier may be coupled to a C-terminal or other cysteine residue in a polypeptide by treatment with N-7 -maleimidobutyryloxy-succinimide (Kitagawa, T. Ackawa, T. (1976) J. Biochem. 79, 233). Other coupling reactions and reagents have been described in the literature.

The polypeptides, either alone or linked to a carrier molecule, may be administered by any route (eg parenteral, nasal, oral, rectal, intra-vaginal), with or without the use of conventional adjuvants (such as aluminium hydroxide or Freund's complete or incomplete 0 WO 91/13909 PC/GB91/00392 12 adjuvants) and/or other immunopotentiating agents. The iinvention also includes formulation of polypeptides according to the invention in slow-release forms, such as a sub-dermal implant or depot comprising, for example, liposomes (Allison, A.C. Gregoriadis, G.

(1974) Nature (London) 252, 252) or biodegradable microcapsules manufactured from co-polymers of lactic acid and glycolic acids (Gresser, J. D. and Sanderson, J. E. (1984) in "Biopolymer Controlled Release Systems" pp 127-138, Ed. D. L. Wise).

It is to be understood that the polypeptides according to the invention may be synthesised by any conventional method, either directly using manual or automated peptide synthesis techniques as mentioned above, or indirectly by RNA or DNA synthesis and conventional techniques of molecular biology and genetic engineering. Such techniques may be used to produce hybrid proteins containing one or more of the polypeptides inserted into another polypeptide sequence.

Another aspect of the present invention therefore provides a DNA molecule coding for at least one synthetic polypeptide according to the invention, preferably incorporated into a suitable expression vector replicable in microorganisms or in mammalian cells. The DNA may also be part of the DNA sequence for a longer product e.g. the polypeptides may be expressed as parts of other proteins into which they have been inserted by genetic engineering. One practical guide to such techniques is "Molecular cloning: a laboratory manual" by Sambrook, Fritsch, E.F. and Maniatis, T. (2nd Edition, 1989).

Polypeptides according to the invention may be used either alone or linked to an appropriate carrier, as: Peptide vaccines, for use to prevent infection by one or more strains of HIV; As ligands in assays of, for example, serum from HIV positive patients; As quality control agents in testing, for example, -Mi -1 WO 91/13909 PCT/GB91/00392 13 binding levels of antibodies raised against the polypeptides; As antigenic agents for the generation of monoclonal or polyclonal antibodies by immunisation of an appropriate animal, such antibodies being of use for the scientific study of the HIV virus, (ii) as diagnostic agents, e.g. as part of histochemical reagents, (iii) for the passive immunisation of HIV patients, either as a treatment for AIDS in itself, or in combination with other agents such as, for example AZT and/or HIV protease inhibitors, and (iv) as a means of targeting other agents AZT or HIV protease inhibitors) to HIV infected cells expressing HIV envelope proteins on their surfaces, such agents either being linked covalently or otherwise associated, e.g. as in liposomes containing such agents and incorporating antibodies raised against any of the antigenic polypeptides. The invention further provides for genetically engineered forms or sub-components, especially VH regions, of antibodies raised against the polypeptides, and of humanised forms of antibodies initially raised against the polypeptides in other animals, using techniques described in the literature; and The treatment of HIV infections,' either by displacing the binding of HIV virus to human or animal cells or by disturbing the three-dimensional organisation of the virus in vivo; as well as aiding the scientific study of HIV viruses in vitro.

In respect of detection and diagnosis, of HIV or antibodies against HIV, the skilled person will be aware of a variety of immunoassay techniques known in the art, inter alia, sandwich assay, competitive and noncompetitive assays and the use of direct and indirect labelling.

A further aspect of the invention provides a kit for detecting HIV or antibodies against HIV which comprises at least one synthetic polypeptide according I .I i

~I_

PCT/GB91/00392 WO 91/13909 -14to the invention.

The preparation of polyclonal or monoclonal antibodies, humanised forms of such antibodies (see, for example, Thompson K. M. et al (1986) Immunology 58, 157- 160), single domain antibodies (see, for example, Ward, E. Gussow, Griffiths, A. Jones, P. and Winter, G. (1989) Nature 341, 544-546), and antibodies which might cross the blood-brain barrier, which bind specifically to a synthetic polypeptide according to the present invention, may be carried out by conventional means and such antibodies are considered to form part of this invention. Antibodies according to the invention are, inter alia, of use in a method of diagnosing mammalian HIV infection which comprises incubating a sample of tissue or body fluid of mammal with an effective amount of antibody as described herein and determining whether, and if desired the extent to which and/or rate at which, cross-reaction between said sample and said antibody occurs. Diagnostic kits which contain at least one of said antibodies also form part of this invention.

A further aspect of the invention provides synthetic polypeptides for use in therapy or prophylaxis of mammalian HIV infection and/or stimulating the mammalian immune system and/or blocking the cellular receptors for the HIV virus and for the preparation of medicaments suitable for such uses. Also included are pharmaceutical compositions containing, as active ingredient, at least one polypeptide or polypeptide-carrier conjugate as described herein in association with one or more pharmaceutically acceptable adjuvants, carriers and/or excipients. The compositions may be formulated for oral, rectal, nasal or especially parenteral administration (including intra-CNS administration).

The invention further provides a method of therapy or prophylaxis of mammalian HIV infection and/or of stimulating the mammalian immune system and/or of M WO 91/13909 PCF/GB91/00392 15 blocking the cellular receptors for the HIV virus, which comprises administering an effective amount of a polypeptide as hereinbefore defined, either in isolation or in combination with other agents for the treatment of AIDS such as AZT and/or inhibitors of the HIV protease.

The following examples are intended to illustrate the invention and are not limiting in any way.

Example 1 A C-terminally extended form of basic peptide (a) with sequence Asp-Gln-Ser-Leu-Lys-Gly-Ile-Trp- Gly-Cys-Ser-Gly-Lys-Leu-Ala-Cys was synthesised using standard solid-phase F-moc methodologies and an intra-molecular disulphide bridge was formed between the two Cys residues. The peptide was cleaved from the resin in the presence of trifluoroacetic acid and subsequent purification of peptide was achieved by gel filtration, ion exchange chromatography and reverse phase high performance liquid chromatography. The purity of the resultant peptide was in excess of 85%. The C-terminal alanine was included to assist the conjugation. The peptide was dissolved in phosphate-buffered saline (PBS; 5mg/ml) and mixed with an equal volume of ovalbumin (5mg/ml) prior to the addition of glutaraldehyde to a final concentration of The conjugate mixture was allowed to stand for 30 minutes prior to emulsification with Freund's adjuvant. Each sheep (5 animals/group) was immunised with 250 ig of peptide in Freund's complete adjuvant (FCA) and subsequently (14 days) challenged with a further similar quantity in Freund's incomplete adjuvant (FIA). Further challenges were performed after a period of 3-4 weeks in FIA. Blood samples were taken 7-10 days post-challenge and assayed for binding to HIV envelope protein.

M L WO 91/13909 PC/GB91/00392 16 The determination of anti-synthetic peptide antibodies which recognise HIV envelope protein was undertaken by employing a recombinant vaccinia virus (see, for example, Macket, M. and Smith, G. L. (1986) J.

gen. Virol. 67, 2067, for general methodology) which has been constructed to express the HIV envelope proteins on the surface of infected cells. This method of assay is preferred as binding to the antigen is measured on the surface of virus infected cells and hence may be more representative than binding to solution phase antigen because certain potential epitopes on the envelope proteins may be masked in vivo through interaction with membrane phospholipids). CV1 monkey kidney cells, grown in monolayer cultures in 96-well microtitre plates, were infected with the recombinant vaccinia virus. The virus construct contained the gene encoding the HIV envelope glycoprotein and this is known to be processed and expressed on the surface of the infected cells.

Antisera from sheep were assayed in duplicate. A quantity of 50 pl of antiserum at various dilutions was added to the wells and incubated for 4 hours prior to washing (twice) and adding a second peroxidase-labelled anti-sheep antiserum. Positive wells were determined by reading the optical density in a microplate reader.

The anti-peptide antisera were found to cross-react with high affinity with HIV envelope protein expressed on the surface of infected cells (see Tables 1 and 1A).

These results confirm that these anti-peptide antibodies would be valuable as research tools and as diagnostic agents.

i

I~I~

PCT/GB 91/00392 12 02 92 17 Table 1 2 February Cross-reaction between serum from sheep immunised with conjugate of peptide and recombinant HIV envelope protein expressed on the surface of monkey kidney cells. indicates no cross-reaction and indicates strongly positive cross-reaction.

Cross reaction Control monkey Monkey kidney kidney cell line cell line expressing HIV envelope protein Serum from immunised sheep Control serum Table 1A Typical titres of positive antisera from sheep immunised with a conjugate of peptide when reacted with recombinant HIV envelope protein expressed on the surface of monkey kidney cells Mean optical density value 1992 (serum dilution) 1 1 1 1 250 1250 31250 156250 Serum sample (titre) Control sheep serum Peptide (a) treated sheep serum diluted 1/5000 0.344 0.288 0.197 0.012 0.016 0.736 0.636 0.432 0.140 0.052 The above table shows that even with significant dilution of the serum from test sheep (relative to the control) there is a greatly increased titre.

Example 2 One role of neutralising antibodies is to inhibit J "United Kingdom Pat.t i- WO91/13909 PCT/GB91/00392 -18 the transmission of virus from infected to non-infected cells. Preventing or reducing the rate of transmission of virus from T-cells to macrophages is crucial since this may extend the life of AIDS patients considerably Anti-peptide antisera were assayed for their ability to inhibit syncitia formation in vitro. Syncitia are multinucleated giant cells which result from "bridge"-formation between HIV-infected cells.

The C-terminally extended form of basic peptide (a) with sequence Asp-Gln-Ser-Leu-Lys-Gly-Ile-Trp- Gly-Cys-Ser-Gly-Lys-Leu-Ala-Cys and containing an intra-molecular disulphide bridge between the two Cys residues was synthesised, purified, conjugated and used to immunise groups of sheep as described in Example 1. The anti-peptide antisera were used in an in vitro HIV neutralisation assay. The anti-peptide antibodies were introduced into an in vitro culture of human T-lymphocytes infected with a highly virulent strain of HIV, and uninfected human macrophages. The anti-peptide antisera were found to inhibit syncitia formation amongst this mixed population of T-cells and macrophages exposed to a virulent strain of HIV I, i.e. they inhibit spread of HIV infection from T-cell to macrophage.

The antibodies were found to prevent infection of the macrophages with HIV, and are therefore neutralising in this in vitro assay, whereas the macrophages became infected in a control culture, as shown in Table 2.

WO 91/13909 PCT/GB91/00392 19 Table 2 Neutralisation of virulent strain of HIV in in vitro culture of HIV-infected human T-lymphocytes and uninfected human macrophages. indicates a high level of infection and indicates effective protection against infection.

Cell culture Level of infection of macrophages HIV infected T-lymphocytes and uninfected macrophages only HIV infected T-lymphocytes, uninfected macrophages and antipeptide(a) antibodies Also, positive antisera to peptide were evaluated in a syncitium assay. This assay determines the capability of antisera to prevent spread of live HIV from infected to uninfected cells and to prevent fusion between cells mediated through reactivity between the virus glycoprotein (gpl60) and the CD4 molecule.

Measurement is carried out by syncitium detection and enumeration.

The syncitium assay is performed as follows: a known concentration of an HIV producer (CD+4) cell line supporting active virus replication is washed three times and mixed with the antiserum under test and used at specified dilutions. After incubation for 30 minutes at 37"C these cells are mixed, at specified proportions with an indicator CD4+ cell line, higrly susceptible to HIV infection and syncitium induction. The cells are WO 91/13909 PCT/GB91/00392 20 observed daily for syncitium formation.

Antibodies to peptide(a) were found to inhibit syncitium formation as shown in Table 3.

Table 3 Syncitium inhibition by antisera between HIVinfected producer cells and an indicator cell line Serum Sample Syncitium inhibition Anti-peptide antiserum (3/5 animals) Normal sheep serum Example 3 A panel of sera from HIV-1 infected individuals in various stages of progression to acquired immunodeficiency syndrome disease (AIDS), ranging from asymptomatic to full-blow symptoms were obtained in order toproduce a representative picture of the reactivity of sera from such patients with the synthetic peptides in ELISA.

The ELISA procedure determines the degree of reactivity of the synthetic peptides (analogous to regions of either gpl20 or the gp41 transmembrane protein of HIV-1) with antibody to the surface glycoprotein of HIV-1 in sera of HIV-1 positive individuals.

One hundred antiser, from HIV positive individuals were reacted with synthetic peptide Two of these sera (from asymptomatic individuals) contained HIV neutralising antibodies and cross-reacted with peptide as shown in Table 4.

WO 91/13909 WO 91/13909PCI'/GB9I /0039 2 -21 Table 4 cross reactivity of peptide with two HIV positive antisera fromt asymuptomatic individuals Sample OD (Peiptide Buffer Normal serum 1/100 1/ 1000 HIV positive 1/100 1/1000 HIV positCive 1/100 0. 056 0.476 0.140 0 0 >2 0 1.37 replicates

Claims

1. A synthetic polypeptide having at least one antigenic property of the envelope protein of at least one strain of Human Immunodeficiency Virus (HIV), said polypeptide consisting substantially of an amino acid sequence of formula X-R 1 -R 2 -R 3 -R 4 -R-R-R-R-Trp-Gly- Cys-Rg--R,-R -R -R-Cys-Y (I) wherein R 1 is Asp or Glu; R 2 is an amino acid residue selected from Gly, Ala, Pro, Ser, Thr, Asn or Gln; R, is an amino acid residue selected from Gly, Ala, Pro, Ser, Thr, Asp, Glu, Asn, Lys, His, Gln or Arg; R 4 R 5 and R 1 1 are each independently any amino acid residue; R 6 and Rs are each independently an amino acid residue selected from Gly, Ala, Pro, Ser, Thr, or Asn; R 7 is an amino acid residue selected from Gly, Ala, Val, Leu, Ile, Ser, Thr, Asn, Gln, Phe, Tyr, Trp, Cys, Met or Pro; R 9 and R 12 are each independently an amino acid residue selected from Gly. Ala, Leu, Ile, Val, Met, Cys, Phe, Tyr S 30 or Trp; Ro is an amino acid residue selected from Lys, His or Arg; and SX and Y may each independently be absent or independently be one or more amino acid residues with the proviso that when present they do not provide or form part of an antigenic property of the envelope protein of at least one strain of Human z 3 Immunodeficiency Virus. ~CC~I 23

2. A synthetic polypeptide as claimed in claim 1 wherein R 2 is selected from Gin or Thr, R 3 is selected from Ser, Asn, Gin, Arg or Ala, R 4 is selected from Leu, Ile, Gin or Arg, R 5 is either Leu or Lys, R 6 is either Gly or Asn, R 7 is selected from Gly, Ala, Leu, Ile, Val, Met, Cys, Phe, Tyr, Trp or Ser, R 8 is either Ser or Ala, R9 is either Gly or Phe, is either Arg or Lys, R 11 is selected from Leu, His, ile, Gin and R 12 is selected from Ala, Ile or Val.

3. A synthetic polypeptide as claimed in claim 2 consisting substantially of an amino acid sequence of formula (II): X-Asp-Gln-R 3 -Leu-R 5 -Gly-Rt-Trp-Gly- Cys-Ser-Gl y-Lys-R-R 1 -Ri-Cys-Y (II) wherein R 3 R 5 R 11 R 12 are as defined in claim 2; R 7 is an amino acid residue selected from Gly, Ala, Leu, Ile, Val, Met, Cys, Phe, Tyr or Trp; and X and Y are as defined in claim 1.

4. A synthetic polypeptide as claimed in claim 3 wherein R, is selected from Ser, Asn, Gin and Arg, R 11 is selected from Leu, His or Ile and R12 is lie or Ala. A synthetic polypeptide as claimed in claim 4 wherein R, is selected from Ile, Phe, Met, Val or Leu. S 30 6. A synthetic polypeptide as claimed in claim i: wherein R, is either Ser or Asn and R 5 is Lys.

7. A synthetic polypeptide as claimed in any one of claims 1 to 6 consisting of the sequence: X-Asp-Gln-Ser-Leu-Lys-Gly-Ile-Trp- Gly-Cys-Ser-Gly-Lys-Leu-Ala-Cys-Y wherein X and Y are as defined in claim 1. I 24

8. A synthetic polypeptide as claimed in claim 3 wherein R 3 is selected from Gin or Arg, RS is Leu, R 7 is selected from Ile, Phe and Met, R 11 is selected from Leu, His or Ile and R 12 is Ala.

9. A synthetic polypeptide as claimed in claim 8 consisting of the sequence: X-Asp-Gln-Gln-Leu-Leu-Gly-Ile-Trp- Gly-Cys-Ser-Gly-Lys-Leu-Ala-Cys-Y wherein X and Y are as defined in claim 1. A synthetic polypeptide as c-jimed in claim 2 consistina of substantially of an amino acid sequence of formula (liI):- X-R -R 2 -R 3 -R 4 -RS-Asn-Ser-Trp-Gly-Cys-Ala- Phe-Arg-Gln-Val-Cys-Y (III) wherein R, is either Glu or Asp; R 2 is Thr or Gin; R3 is an amino acid selected from Ser, Asn Arg, Gin or Ala; R is an amino acid selected from Leu, lie, Arg or Gln; R 5 is Lys or Leu; and wherein X and Y are as defined in claim 1.

11. A synthetic polypeptide as claimed in claim 2 or in claim 10 consisting substantially of an amino acid i sequence of formula (IIIa):- X-Glu-Thr-R 3 -R 4 -Lys-Asn-Ser-Trp-Gly- Cys-Ala-Phe-Arg-Gln-Val-Cys-Y (IIIa) wherein R3, is an amino acid residue selected from Ser, Asn, Arg, Gln or Ala; R 4 is an amino acid residue selected from Leu, Ile, 25 Arg or Gin; wherein X and Y are as defined in claim 1.

12. A synthetic polypeptide as claimed in claim 11 wherein R 3 is Ser when R 4 is Ile or R 3 is Ala when R 4 is Arg or Gin.

13. A synthetic polypeptide as claimed in any one of the preceding claims wherein Cys residues at positions 10 and 16 are linked by an intra-molecular disulphide bridge.

14. A synthetic polypeptide having the general formula (IV): -L (IV) wherein F and G may each independently be a polypeptide according to any one of Formulae I to IIIa, L is a linking sequence, each linking sequence L being the same or different, a, b and c are each independently 0 or 1 and m and n are each positive numbers. A synthetic polypeptide having the general formula Ld-[G-L] (V) wherein F is a polypeptide according to any one of Formulae I to IIIa, G is a polypeptide according to any one of Formulae I to IIIa or other sequence, m and n are 30 each positive numbers and d and e are each independently 0 or 1.

16. A synthetic polypeptide as claimed in any one of the preceding claims including a retro-inverso amino acid.

17. A synthetic polypeptide which comprises an antigenically significant subfragment or variant of a S26 polypeptide as claimed in any one of claims 1 to 13.

18. A synthetic polypeptide as claimed in any one of the preceding claims additionally comprising at least one T-cell epitope.

19. A synthetic polypeptide as claimed in any one of claims 1 to 18 linked to a vaccine carrier.

20. A vaccine comprising at least one synthetic polypeptide as claimed in any one of claims 1 to 19 effective to promote immunity against at least one strain of HIV.

21. A kit for detecting HIV or antibodies against HIV which comprises at least one synthetic polypeptide as claimed in any one of claims 1 to 18.

22. A DNA molecule coding for at least one synthetic polypeptide as claimed in any one of claims 1 to 18.

23. A pharmaceutical composition containing, as active ingredient, at least one polypeptide as claimed in any one of claims 1 to 19 in association with one or more pharmaceutically acceptable adjuvants, carriers and/or excipients.

24. A method of therapy or prophylaxis of mammalian HIV infection and/or of stimulating the mammalian immune system and/or of blocking the cellular receptors for the HIV virus, which comprises administering an effective amount of a polypeptide as claimed in any one of claims 1 to 19.

25. A method of detecting HIV or antibodies against HIV or antigen binding fragments thereof, which comprises incubating a sample with at least one polypeptide as claimed in any one of claims 1 to 19. T X j 27

26. An antibody or antigen binding fragment thereof which specifically binds to a synthetic polypeptide as claimed in any one of claims 1 to 19.

27. A diagnostic kit for detecting HIV or antibodies against HIV which contains at least one antibody as claimed in claim 26.

28. A pharmaceutical composition containing, as active ingredient an antibody as claimed in claim 26 in association with one or more pharmaceutically acceptable adjuvants, carriers and/or excipients.

29. A pharmaceutical composition as claimed in claim 23 or in claim 28 further comprising AZT and/or an HIV protease inhibitor. A method of diagnosing mammalian HIV infection which comprises incubating a sample of tissue or body fluid of a mammal with an effective amount of an antibody as claimed in claim 26 and determining whether, and if desired the extent to which and/or rate at which, cross-reaction between said sample and said antibody occurs.

31. A synthetic polypeptide as claimed in claim 1 substantially as herein described. 32 A synthetic polypeptide as claimed in claim 14 S 30 substantially as herein described.

33. A synthetic polypeptide as claimed in claim substantially as herein described.

34. A vaccine as claimed in claim 20 substantially as herein described. A kit as claimed in claim 21 or claim 27 28 substantially as herein described.

36. A DNA molecule as claimed in claim 22 substantially as herein described.

37. A pharmaceutical composition as claimed in claim 23 or claim 28 substantially as herein described.

38. A method of therapy or prophylaxis as claimed in claim 24 substantially as herein described.

39. A method of detecting HIV as claimed in claim 25 or of diagnosing mammalian HIV infection as claimed in claim 30 substantially as herein described. An antibody or antigen binding fragment thereof as claimed in claim 26 substantially as herein described.

41. Each and every novel product pi -reand method INTERNATIONAL SEARCH REPORT Wa,mrnational Application No PCT/GB 91/00392- 1. CLASSIFICATION OF SUBJECT MATTER (it aevoral Cliassification symbols aly. Indicate *10) 6 According to International ill@ nt ClagelficAtion tifC) of to both National Clissificaion and IPC C 07 K 7/ 08,A 1K 37/02,A 61 K 39/21,G 01 N 33/567, IPC C 07 H 21/04,C 07 K 3/00,C 12 P 21/00 11. FIELDS.SEARCHED Minimum Documentation Searched I Claosfication System IClaseification Symbols C 07 K,A 61 K,G 01 N,C 07 H,C 12 P Documentationt Searched COhe than Minimum DOCumentatI111101n to the Extent that such, Documents afs Included In the. Fields Seirched Ill. DOCUMENTS CONSIDERED TO BE RELEVANT' Category Citation of Document."1 with Indication. where appropriate. of the relevant passages It Reevant to Claim No."I A EP, A2, 0 298 633 1, (PROTENS BIOTECHNOLOGY 19-21 LIMITED) 11 January 1989 23,24 (11.01.89), see claims. 26-31 A EP, Al, 0 316 695 1,21, HOFFMANN-LA ROCHE CO.) 22, 24 May 1989 (24.05.89), see 26-28 claims. 31,32 52,53 57, 61 A Chemical Abstracts, Volume 110, no. 19, issued 1989, May 8 (Columbus, Ohio, USA), E.V. Barsov et al. "Construction of a novel I vector for thermo-regulated gene expression. Synthesis of a polypeptide carrying human immunodeficiency virus antigenic determinants in Speclki categors, cited documents: 46 -7 tater document published after the Internaional filing date A doumat dfinng he sneat satsci he rg h~c Isnotor priority date and not in cOnflict with the application but "A nsdoc rmentob eingth eera~l rleaefteatwihInt cited to understand the principle or theory underlying the consdefd tobe f caicuar rlevnceInvention earlier documnt but published on or after the internallonal X- document of particutir relevance; the claimed Invention iling date cannot be considered noveol or cannot be considered to docume nt which nm.y throw doubtsaon 0riori1y claim(s) Or involve an Inventive step wvhich is cited to establish the Publication date Of another document of Particular rlevan~ce;' the clalmed Invention citation of other special reason (as specified) cannot be conaidered to Involve en inventive step when the 0' document referring to en oral disclosure. use. eshlbitlon or document Is combined with one or more other such docy- other means menta. such combination being obvious to a person skilled "P document published orlor to the ig!temtational filing date but In the art. later then the priority date claimed -A document member Of the "ame patent family IV. CERTIFICATION Date of the Actual Comokbton of the interetionel Search Date of Milling ol Itita Inteoiationall Search Repo"t June 1991 1 .o.9 Internationl Searching Authort" Signature oi Authortaed Ot~tcer EURPEA PTEN ~miss T. MORTENSE t Forn PCTIISAZO (second shee"(jse~e INS) -2- PCT/GB 91/00392 International Application No. FURTHER INFORMATION CONTINUIO FROM THE SECOND SHEET Escherichia coli cells", see page 230, right column, the abstract no. 167479q, Biotekhnologiya 19 89, 5(1), 4-8 VQ OBSERVATIONS WHERE %7ERTAIN CLAIMS WERE FOUND UNSEARCHABLE'I This internatioanal "earch report has not b~en established In respect of certain claims under Article t7C2) for the flloWing reasons: IMClaim numberi...?§. becaus, they relat, to subltct matter not required to be searhed by this Authoolty, namrely: Method for treatment of human or animal body by surgery or therapy as (Rule 39.1 (iv) PCT). well as diagnostic mnethodis. claim numnber.... because they' relate to parts of the International application that do not comply with the proscribed require- manta to such an eulent that no meaningful International search can be Carried out specllcaay: 3[ Cli oor- b.iamm air dee-n claimw areM notr OrWW. In socrdancie wtt Vie second arW third sartoile COf PCT Rule GA~a), Vl. 011OBERVATIONS WHERE UNITY OF INVENTION IS LACKING I This International Searching Athority found mnutipl nvtntoi n this. internaional appliation as follows: IQf As all reqcuired additional search fees viere timely paid by the applicant, this international search report cover. all sarhabie claims of the internatlonal appolication. As only eame of the required additonal search fees wore timly paid by the applicant, thia International seercht report covert only thote claims of the International application for which loee wre paid, apecicaky clams: 3M No required additionai search lees were timely paid by the applicant. Consequently, this interational rj.arch report Is restricted to the Invention first mentioned in the claims; Kt Is coweed by claimn numbers: all searchable claImi. could be sarched wrtthout e1Ioa't luating an additilow lee, the Internatio"a $earching Authortty did not Invite payment of any additonal fee. Flamers on protest OThe additonal search fees were acconpan~ed by appilicats protest. 0No protect accompanied the paymenatl el addstional seweh fees. Form PCTIISAM2O (supplemental sheet Cr)) (.lwvJry t~113 zva internationaien Recherchen- bericht fiber die internationals Patenstanueldung Nc. In dieses Anhang sirwd die Mitiieder der Patrf.tfamiliert aer in, oenge- nannten internationalen Recnerchenbericht angef~hrten Palentdokumente anroeoecen. Diese Angafen dienen nur Zur Ltitr- richtung und er4olme ohrte Sewimr. to the International Search frepart to the Internatioinal Patent Application No. PCT/A991/00M~ SE 45493 This Annex lists the patent family memers relating to the patent docutent-s cited in the above-sentioned inter- natia s-arch report. The Office is in no way liable for these particulars which ar-e given so-rely +or the purpose of information. AEXE au rapport de recnercfe inter- national relatii a la deuae de brevet international ri" La. presente anniexe indicue les wres de la famille de brevets relatifs aux documents de brevets cit&s daits le rapport dei echer-lie inter- national vise. ci-dessus. Les reseione- tents fournis son!~ donnis A titre iFdica- t if et neangagent pas la responsibilit6 d; I-Office. It Rechercnerroericht Datu der Mitq liedler" der Datum aer Fatentdokuunt Ver6+ientlichung Pdtentfaiiie Veriffentlichuna Patent document cited Publication Patent family Publication in search report date meber date Document de orevet cit& Date de ebre(s) de la Date ae darts le rapport de rec-hercfle publication famille de brevets publication EF'-A2- 2 6 7-. iI- I _B?8 AU-A 1-1"7572/980 OK- A 6520/89 D i4::-A0- 65""(:/99 EP-A3- 29863Z EF'-A I- :771046 F 296242 GB-AC- 871 4602 JP-2 2503,79!6 NO-A 895235 WO-A-- 81027 21 -i'-29 21-12-89? 26-04-99 Z-9 2q-12-BB EFA 1 '5 24-05-29? AU-A1 -25076/88 18-05- 8 9 DK- A 0- 6379/98 15-11-88 DK-A 6379/98 1 7-05-9c9 FI-ACO- 885296 16-11-E9 FI-A 885296 17-05-6? JF- A 2- 1160998 2 3-06-895 NO-AtC- 685090 15- 11-88 No-A 885090 18-05-e7