WO2001047549A2

WO2001047549A2 - Conjugate useful as building block for vaccine preparation

Info

Publication number: WO2001047549A2
Application number: PCT/GB2000/004951
Authority: WO
Inventors: Jane Nicola Zuckerman; Bala Subramaniyam Ramesh
Original assignee: University College London
Current assignee: University College London
Priority date: 1999-12-23
Filing date: 2000-12-21
Publication date: 2001-07-05
Anticipated expiration: 2002-06-23
Also published as: WO2001047549A3; GB9930585D0

Abstract

A polymerisable compound for use as a medicament, comprising a peptide; a spacer covalently linked to the peptide; and a monomer unit covalently linked to the spacer and capable of forming a polymer with at least one further monomer unit in a further polymerisable compound.

Description

COMPONENT FOR VACCINE

Field of the Invention

The present invention relates to a component comprising a polymerisable compound for use as a medicament, especially for use as an immunogenic component for a vaccine.

Background to the Invention

A vaccine is a pharmaceutical which, when given to a subject, is intended to elicit an immune response which protects the subject from subsequent infection by a pathogen such as a virus or bacterium. Typically, a vaccine comprises an attenuated version of the pathogen or some part of it. It is often possible to identify one or more proteins in a pathogen which are antigenic. Identification of the antigenic regions of these proteins can form part of a strategy for vaccine production where the antigenic regions are in some way replicated in a vaccine composition.

One such strategy involves elucidation of the amino acid sequence of the antigenic regions and the production of synthetic peptides having such sequences. Whilst these peptides can be readily produced by genetic engineering or by solid phase peptide chemistry, they suffer from severe drawbacks associated with their small size, low copy number and poor immunogenicity. It is possible to increase the immunogenicity of synthetic peptides by coupling them to carrier proteins. Formulation of a vaccine composition including a carrier protein-peptide complex and an immunostimulant or adjuvant can elicit immunity in a subject. However, this approach also has drawbacks because of difficulties in formulation and in consistencies in the complexes produced. Moreover, protein carriers are undesirable for vaccination because they can elicit immunity not relevant to protection and frequently limit the immune response to the peptide after primary immunisation .

In an attempt to overcome some of these drawbacks, (Jackson et al Vaccine Vol 15, ppl697 to 1705 (1997)) proposed a vaccine composition incorporating synthetic peptide-based polymers . After production of a peptide-containing monomer, a polymer was formed by free radical polymerisation of the monomer and the polymer was isolated and tested in vi tro and in mice. A comparison of immunogenicity of the polymers with corresponding peptide monomers incapable of polymerisation showed the polymer to be more effective. However, this approach suffers from a number of drawbacks, including the need for a complicated series of preparation and purification steps to arrive at the polymer, requiring the ue of the highly toxic acrylamide monomer.

Summary of the Invention

The present invention aims to overcome the drawbacks of the prior art and provides a polymerisable compound for use as a medicament, comprising a peptide; a spacer covalently linked to the peptide; and a monomer unit covalently linked to the spacer and capable of forming a polymer with at least one further monomer unit in a further polymerisable compound. It has been surprisingly found that a relatively simple peptide-containing monomer can be used as an immunogenic component for a vaccine in spite of its relatively small size. Without wishing to be bound by theory, it is postulated that, once the peptide-containing compound is endocytosed or phagocytosed in an antigen presenting cell such as a macrophage, the peptide is induced to polymerise by free radicals produced by the macrophage or by the redox potential in the cell. The monomeric units can polymerise together, or even copolymerise with other units, to form a larger complex which may be presented to the immune system. It is postulated that two forms of processing take place within the cell; an exogenous and an endogenous antigen processing which may occur simultaneously. Endogenous processing of the polymerised peptide-containing compound could stimulate cellular immunity, especially cytotoxic T- lymphocyte response. A good humoral response is also achieved.

Advantageously, the peptide part of the compound is substantially non-immunogenic when in unconjugated form. Such forms include those where the peptide is not conjugated or linked to a further protein or to a spacer and monomer unit, as required by the present invention. The peptide typically has from 10 to 20 amino acids, preferably around 12 amino acids. The peptide preferably comprises an amino acid sequence from a part of a target protein such as a protein from a pathogen, especially a surface protein such as a coat protein of a pathogen. The target protein is therefore generally a naturally-occurring protein and although the peptide sequence may be produced synthetically, it generally represents a sequence from a natural protein, optionally with modifications or deletions which do not substantially affect the antigenicity of the peptide in the compound. Whilst it is preferred that the amino acids making up the peptide are L amino acids, a sequence comprising D amino acids would also be effective provided that the sequence was reversed.

The pathogen is typically a microorganism such as a virus, bacterium or fungus and the compound is found to be particularly effective towards viruses such as a hepatitis virus. For example, the target protein may comprise VP1 or VP3 from hepatitis A. Alternatively, the target protein may comprise hepatitis B surface antigen. Other virus targets include retroviruses, herpes, influenza, meazles, polio and others.

The spacer and peptide are covalently linked together either by a direct covalent bond or indirectly by covalent bonds interrupted by further atoms or groups. Typically, the amino terminus of the peptide is linked to the spacer. The exact size and nature of the spacer may be determined by routine experimentation. A function of the spacer is to be linked to the peptide and monomer unit so that they are spaced apart to prevent polymerisation at the monomer unit from interfering with the immunogenic properties of the peptide. This is preferably achieved where the spacer is linked to the peptide and monomer unit such that at least 4 atoms separate the peptide from the monomer unit, typically 6 to 8 atoms . Preferably, the spacer has an SH containing side group. Such a side group may be capable of forming disulphide bridges and/or may contribute to the immunogencity of the peptide. Such a side group may be provided as part of a cysteine residue. In a preferred embodiment, the spacer group comprises a hexanoyl group, preferably linked to cysteine .

The monomer unit must be capable of forming a polymer with further monomer units either directly as a homopoly er or indirectly by polymerisation with one or more comonomers . Preferably, the monomer unit is capable of such polymer formation by redox-mediated or free radical-mediated polymerisation .

Preferably, the monomer unit comprises an olefinic group, such as one found in an acryloyl group or similar. In this way, the monomer unit is thought to be sufficiently reactive in vivo in the presence of an antigen presenting cell that a polymer is formed.

In a further aspect, the present invention provides use of a compound as described above, for the preparation of a composition for use as a vaccine.

In a further aspect, a vaccine composition is provided, which comprises a compound as described above and an adjuvant. The adjuvant may comprise any conventional adjuvant such as Freund' s adjuvant for use in certain animals or an aluminium-based adjuvant such as is typically used in humans. In a preferred embodiment, the adjuvant comprises an adjuvant of the type described in copending

PCT patent application no. , filed on the same date as the present application under reference PWF 201094 by the same applicant. This adjuvant is discussed briefly below .

In this embodiment, the adjuvant comprises a plurality of components, each of which is the same or different, is capable of micelle formation and comprises a peptide head group for binding to an antigen-presenting cell, and a lipophilic tail group.

It is found that the provision of a peptide head group capable of binding to an antigen-presenting cell such as a macrophage confers upon the adjuvant an advantage over conventional receptor-mediated endocytosis because there is no restriction on the size of particles that can be internalised by the antigen-presenting cell. Preferably, the peptide head group comprises a binding motif, especially for a cell surface receptor. The motif may comprise a plurality of amino acids in the peptide head group. It is particularly preferred that the motif comprises an integrin-binding motif.

An integrin is a known cell surface transmembrane receptor which participates in cell-cell adhesion and as a "sensor" between cells and components of the extra cellular matrix. A number of ligands are known to interact with integrins, including extracellular matrix glycoproteins such as fibronectin and vitronectin. The integrin-binding motif generally comprises the amino acids R, G and D. In one arrangement, the integrin binding motif comprises L amino acids, preferably having the sequence RGD where the lipophilic tail group is N terminal of the R amino acid. Alternatively, the peptide head group may comprise D amino acids. In this arrangement it is preferred that the integrin binding motif comprises the D amino acid sequence DDDGGGGGRRR and the lipophilic tail group is N terminal of the D amino acid.

Typically, the lipophilic tail group comprises a single tail group. In other words, there is one lipophilic tail group for each peptide head group. In this way, the component is preferably an unbranched molecule which is simpler to synthesis and will be capable of micelle formation, unlike more complex branched molecules such as Pam₃ Cys-Ser-POE.

The lipophilic tail group may be any suitable lipophilic group such as one having a sufficiently long hydrocarbyl chain as to promote micelle forming ability. For example, the lipophilic tail group may comprise a C₄ to Cie alkyl group and preferably comprises a Cs to Cι₂ fatty acid. A particularly useful fatty acid is lauric acid.

In a further aspect, the present invention provides a process for the production of a vaccine as described above, which process comprises formulating the vaccine from a compound as described above and an adjuvant as described above . Detailed Description of the Invention

The present invention will now be described in further detail, by way of example only, with reference to the following Examples .

EXAMPLE

Synthetic peptide corresponding to virtually any accessible region of a native protein may elicit antibodies reactive with that protein. Thus a synthetic peptide antigen incorporated in an immunogen may generate antibodies that cross-react with proteins containing the amino acid sequence homologous with that of the peptide. Such antibodies are thus directed against a specific region of the protein, selected in advance, and therefore possess site-specific or predetermined specificity. Such peptide antigens used to generate site-specific antibodies to proteins are of interest in the development of novel vaccines. The requirement to conjugate them to a carrier protein for optimal immunogenicity and deliver them with a suitable adjuvant can result in a number of problems.

Here we describe a new method of synthesizing an immunogenic peptide antigen refered to as Acrylated- Cysteinyl Peptide (ACP) , and an integrin-targetted lipopeptide referred to as (Lauroyl-RGD) as the adjuvant. A known immunodominant 18-residue sequence corresponding to the Hepatitis B surface antigen. The peptides were synthesized directly by the solid-phase technique. The amino terminal of the completed peptide were coupled to hexanoic acid and terminated with cysteine sequentially. The amino terminus of the cysteine was then capped with acrylic acid.

The synthesis of the adjuvant comprises a lauryl acid coupled to amino acids of which are D-amino acids. The 11- residue peptide incorporating the lauric acid was synthesized sequentially directly onto a polystyrene solid- phase .

The acrylate-cysteinyl-peptide (ACP) when complexed with the Lauryl peptide (Lauryl-RGD) as micelles was highly immunogenic in non-responder mice and randomly bred mice. The antibodies reacted with the peptides and there were no antibodies detected to the Lauryl-RGD peptides. The antibody titre were comparable to peptides conjugated to a carrier protein (Keyhole Limpet Haemocynin, KLH) and emulsified with Freund' s complete adjuvant. The potential exists using this construct to design immunogens for third generation vaccines.

MATERIALS AND METHODS

Lauric acid (Dodecanoic acid) was purchased from Fluka (UK) Ltd., Dimethylformamide (DMF) , Dichioromethane

(DCM) , Trifluoroacetic acid (TFA) , Piperidine and N- Methylmorpholine (NMP) were purchased from Rathbum

(Scotland) . Fmoc-protected amino acids with side chain protection, Fmoc-6-aminohexanoic acid, 2- ( 1 H- Benzotriazole- 1 yl) 1,3,3 -tetramethyluronium tetrafluoroborate (TB TU) and Rink-amide resin were from Calbiochem-Novabiochem (UK) . Mouse strains Balb/c(d), Balblk (k) and BlO.S(s) were purchased from Harlan UK Ltd., Polystyrene flat bottomed wells for (ELISA) were from Dynatech laboratories Inc. USA.

Assembly of the monomeric 'a' determinant of Hepatitis B surface antigen (119-137) resin sequence: Fmoc- PCKTCTTPAQGNSMFPSC-Rink-amide-resin.

The peptide was synthesized using the stepwise solid-phase procedure using the Fmoc protection strategy. The Rink- amide resin (0. 5g) derivatized with Fmoc-amide was put through normal deprotection cycle with 20% pipridine in DMF in a reaction vessel for 12 minutes. After deprotection the resin was washed with DMF (lo lig; 5x1 mm) . A 5-fold molar excess (based on the loading) of acylating species in 0.2M NMP in DMF (5ml) were added automatically in all subsequent couplings. The synthesis was carried using the batch synthesis mode on an automated peptide synthesizer (Rainin PS3, Protein Technologies, USA) with standard protocols and scale (0.1 mmol theoretical yield of crude peptide). The cycle for the addition of protected amino acid consisted of 2x6 min wash of the solid support with 20% piperidine in DMF to cleave the N^α -Fmoc group, 10x1 min DMF wash, 25 mm coupling reaction with 5 equivalents of Fmoc-amino acid activated by TBTU in 0.2M NMP in DMF as an active ester, and an 10x1 min DMF wash for a total cycle time of about 60 min. Finally 5 equivalent Fmoc-6-amino hexanoic acid was coupled as the active ester using TBTU as described to the deprotected N-terminus of the assembled protected peptide. To the deprotected amino group of the hexanoic acid, activated Fmoc-cysteine was added as decribed above for preparing active esters for coupling and allowed to react for 25 minutes under nitrogen. Finally acrylation of the N- terminus of the resin bound cysteinyl-hexanoyl-peptide was carried out manually with nitrogen agitation at 0°C in a fume cupboard.

To the peptide-resin a 10-fold molar excess NMP and 10- fold molar excess of acryloyl chloride in DMF were agitated with nitrogen in a sintered glass column cooled at 0°C for 1 hour, then for a further 1 hour at room temperature. On completion of acrylation (a negatine test is indicated by the yellow colour of the resin using 3-hydroxy-2-3-dihydro- 4-oxo-benzotriazine (ODHBT) . On completion of the synthesis, the peptide resin was washed sequentially in DMF, DCM and diethyl ether and dried under vacuum overnight .

Assembly of the integrin binding lauryl-peptide-resin sequence: Lauroyl-RRRGGGGGDDD-Rink-amide resin.

0.5g of the Rink-amide- resin derivatised with Fmoc-amide was subjected to the deprotection cycle with 20% piperidine in DMF in a glass reaction vessel for 12 minutes. The synthesis was carried out using the batch synthesis procedure as described above. Clearly it comprises a lauryl acid coupled to the D-amino acids, rather than the naturally occuring L-amino acids. On completion of the synthesis, the lauryl-peptide-resin was dried under vacuum as described above.

Detachment and deprotection of the monomeric cysteinyl- hexanoyl-peptide ('a' determinant of Hepatitis B surface antigen) and Lauroyl-peptide (Adjuvant) .

On completion of the synthesis of the HBV peptide and Lauroyl-peptide, their respective N^α-Fmoc protective groups were removed using 20 % piperidine in DMF. The respective resins were washed sequentially in DMF, DCM and diethyl ether and dried under vacuum. The dried amide resins were resuspended in 10% TFA in DCM and agitated with nitrogen gas in a sintered glass column, and collected into a round bottomed flask at 10 minute intervals. This procedure was carried over a period of 60 minute for all peptides. Following the completion of detachment and partial deprotection, the DCM completely removed by rotary evaporation and the protected peptides are usually concentrated at this stage. Finally to remove all the protecting groups completely, 88% TFA in the presence of scavengers (phenol 5%, ethanedithiol, 5% and water, 2% were added to the residual oily solution and left at room temperature with occasional swirling for 4 hours. The TFA together with the scavengers were removed by rotary evaporation at 4°C until a small volume of the fitrate is left. To this cold anhydrous ethyl ether was added to precipitate the crude peptide products. The precipitate is washed with cold ether 3 times more and dried under vacuum for 4 hours . Samples were then analysed by HPLC to establish purity of samples. All samples were finally stored as dry powder at 4°C until further use.

Immunization Procedure: Mice were eight-week old males with the following haplotypes, Balb/c (H-2d) Balb/c(h-2k) and B. 10.S(H-2s) . The mice were immunized with lOOμg of acrylated cystemyl peptide complexed m lOOμg of lauroyl-RGD m PBS. As controls non-acrylated peptides were also injected with lauroyl-RGD peptide. A combined mtrapeπtoneal and subcutaneous route was used for the primary injection. Groups of 3 of each strain were given acrylated and non- acrylated peptide complex. 3 weeks later they were boosted with the same doses and by the same routes . Blood was then collected 3 weeks later from the retro-orbital sinus and the serum collected after clotting.

Immunological Assays (ELISA)

Polystyrene flat bottom wells (Immulon 2 Dividastπp, Dynatech Laboratories Inc) were used to test antisera for the ability to react with the peptide ('a' determinant of HBV surface antigen ,119-137) . Peptides (lμg/ml) in carbonate/bicarbonate buffer pH9 was incubated at 4°C overnight in microtitre strips before being washed. Additional binding sites were blocked with 5% skimmed milk (Marvel) for 2 hours at room temperature. The plates were washed 3 times with wash buffer (PBS containing 0.05% Tween 20, and milk) . Antibodies were then added (dilutions of murine sera) to the plates and incubated for 1 hour at room temperature. Plates were washed 6 times with wash buffer. Goat anti-mouse IgG conjugated to horseradish peroxidase was diluted at 1:5000 in PBS/milk and added to the wells as secondary antibodies and incubated for 1 hour at room temperature. Plates were then washed 6 times with wash buffer. After washing with wash buffer, the bound conjugate was reacted with the chromogen (o-phenylenediamine dihydrochloride at 50μg/ml in citrate buffer, pH 5.95 for 30 minutes. The titre for first bleed was determined visually. Results :

Adding an acrylic group to the amino terminal of the peptide confers it to be 'active' because it can be initiated to polymerize in the presence of free radicals as found in professional scavenger cells i.e in macrophages. However given in combination with peptide composed of D- a ino acids coupled to a fatty acid like lauric acid by an amide bond, it was found to function as an adjuvant. The lauroyl-D-amino acid peptide forms micelles and is resistant to proteolysis and this makes it a useful adjuvant. As an adjuvant it is almost as potent as Freund 's adjuvant but with no deleterious properties. The antibody results are presented in Table 1. Where titres are low in the case some mice, this is due to low availability of blood because they were bled from the tail (which is difficult) . High titres obtained were possible because they were from animals bled from the eye. The results show that the so called non-responders have responded only after one boost. The animals will be boosted one more time and a terminal bleeding will be performed.

Table 1. The titre of anti-peptide antibody responses against the peptide (119-137) corresponding to the 'a' determinant of Hepatitis B surface antigen when complexed with lauroyl-RRRGGGGGDDD as adjuvant..

First Bleed Mouse Strain H-2 Haplotype Acrylated-cys- Non-acrylated peptide peptide

1. Balb/c dd 1 1//110000 1/10

2. Balb/c d 1/100

3. Balb/c d 1/100

4. Balb/k kk 1 1//1100 1/2

5. Balb/k k 1/100

6. Balb/k k 1/10

7. Balb/s s 1/100

8. Balb/s s 1/10

9. Balb/s s 1/5

Claims

Claims :

1. A polymerisable compound for use as a medicament, comprising a peptide; a spacer covalently linked to the peptide; and a monomer unit covalently linked to the spacer and capable of forming a polymer with at least one further monomer unit in a further polymerisable compound.

2. A compound according to claim 1, wherein the medicament comprises an immunogenic component for a vaccine .

3. A compound according to claim 2, wherein the peptide in unconjugated form is substantially non-immunogenic .

4. A compound according to claim 2 or claim 3, wherein the peptide has from 10 to 20 amino acids.

5. A compound according to any one of claims 2 to 4, wherein the peptide comprises an amino acid sequence from a part of a target protein.

6. A compound according to claim 5, wherein the target protein comprises a surface protein of a pathogen.

7. A compound according to claim 6, wherein the pathogen is a virus.

8. A compound according to claim 7, wherein the virus is a hepatitis virus.

9. A compound according to claim 8, wherein the target protein comprises hepatitis B surface antigen.

10. A compound according to any one of claims 2 to 9, wherein the spacer is linked to the peptide and monomer unit such that at least 4 atoms separate the peptide from the monomer unit.

11. A compound according to any one of claims 2 to 10, wherein the spacer has an -SH containing side group.

12. A compound according to claim 11, wherein the -SH containing side group is part of a cysteine residue.

13. A compound according to any one of claims 2 to 12, wherein the spacer group comprises a hexanoyl group.

14. A compound according to any one of claims 2 to 13, wherein the monomer unit is capable of forming a polymer with further monomer units by redox-mediated or free- radical mediated polymerisation.

15. A compound according to claim 14, wherein the monomer unit comprises an olef ic group.

16. A compound according to claim 15, wherein the monomer unit comprises a acryloyl group.

17. Use of a compound according to any one of the preceding claims, for the preparation of a composition for use as a vaccine.

18. A vaccine composition comprising a compound according to any one of claims 1 to 16 and an adjuvant.

19. A vaccine according to claim 18, wherein the adjuvant comprises a plurality of components, each of which is the same or different, is capable of micelle formation and comprises a peptide head group for binding to an antigen- presenting cell, and a lipophilic tail group.

20. A vaccine according to claim 19, wherein the head group comprises a motif for binding to a receptor on the antigen-presenting cell.

21. A vaccine according to claim 20, wherein the motif comprises a sequence of amino acids in the peptide head group .

22. A vaccine according to claim 20 or claim 21, wherein the receptor comprises an integrin.

23. A vaccine according to claim 22, wherein the motif comprises the amino acids R, G and D.

24. A vaccine according to claim 23, wherein the motif comprises the L amino acid sequence RGD and the lipophilic tail group is N terminal of the R amino acid.

25. A vaccine according to any one of claims 19 to 24, wherein the peptide motif group comprises D amino acids.

26. A vaccine according to claim 25, wherein the motif comprises the D amino acid sequence DDDGGGGGRRR and the lipophilic tail group is N terminal of the D group.

27. A vaccine according to any one of claims 19 to 26, wherein the lipophilic tail group comprises a Cs-Cι₂ fatty acid .

28. A vaccine according to claim 27, wherein the fatty acid comprises lauric acid.

29. A process for the production of a vaccine according to any one of claims 18 to 28, which comprises formulating the vaccine from a compound as defined in any one of claims 1 to 16 and an adjuvant as defined in any one of claims 18 to 28.