WO2001047553A1

WO2001047553A1 - Micelle-forming lipopeptides targeted at antigen presenting cells useful as vaccine adjuvants

Info

Publication number: WO2001047553A1
Application number: PCT/GB2000/004937
Authority: WO
Inventors: Jane Nicola Zuckerman; Bala Subramaniyam Ramesh
Original assignee: University College London
Priority date: 1999-12-23
Filing date: 2000-12-21
Publication date: 2001-07-05
Also published as: GB9930591D0

Abstract

A component for an adjuvant, which is capable of micelle formation and comprises a peptide head group for binding to an antigen-presenting cell, and a lipophilic tail group.

Description

MICELLE-FORMING LIPOPEPTIDES TARGETED AT ANTIGEN PRESENTING CELLS USEFUL AS VACCINE ADJUVANTS

Field of the Invention

The present invention relates to a component for an adjuvant for use in a vaccine, to an adjuvant comprising a plurality of such components and to a vaccine composition comprising an immunogen and such an adjuvant.

Background to the Invention

A vaccine is a pharmaceutical which, when given to a subject, is intended to elicit an immune response which protects a subject from subsequent infection by a pathogen such as a virus or bacterium. Typically, a vaccine comprises an antigenic component such as an attenuated version of the pathogen or some part of it, together with an adjuvant, which is an agent thar enhances antibody or cell-mediated immune response.

Currently, the only adjuvants authorised for human use are aluminium hydroxide and aluminium phosphate. Whilst sufficient for many vaccines, these adjuvants are not as effective as Freund' s complete adjuvant, which comprises heat killed mycobacterium tuberculosis, paraffin oil and mannide monooleate, or Freund' s incomplete adjuvant, which comprises paraffin oil and mannide monooleate only. However, the Freund' s adjuvants are known to have a very undesirable side effect of producing granulomas at injection sites. Accordingly, there exists a need for improved safe adjuvants which are universally applicable, especially for antigens which are non-immunogen c on their own . In a paper entitled Lipopeptide-Polyoxyethylene Conjugates as Mitogens and Adjuvants, Kleine et al investigated complex water soluble adjuvants and their ability to activate human B lymphocytes m vi tro (Immunobiology 190, 53-66, 1994). Polyoxyethylene derivatives of tripalmitoyl- S-glycerol-cysteinyl-seπne (Pam₃ Cys-Ser-POE) were compared with similar molecules containing no polyoxyethylene and were found to yield stable solutions which appear capable of activating B lymphocytes . An adjuvant comprising these conjugates would not possess detergent or micelle-forming properties and would be expected only to bind non-specifically to cells. No T-cell mediated immune response would be elicited.

The present invention aims to overcome the disadvantages of the prior art.

Summary of the Invention

The present invention provides a component for an adjuvant, which 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. 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 Ci₆ alkyl group and preferably comprises a C₈ to Cχ₂ fatty acid. A particularly useful fatty acid is lauric acid.

In a further aspect, there is provided an adjuvant comprising a micelle which comprises a plurality of components, which may be the same or different from one another, as described above.

There is further provided a vaccine composition comprising an antigen and the adjuvant.

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 Example.

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 Freund¹ s complete adjuvant. The potential exists using this construct to design immunogens for third generation vaccines.

MATERIALS AND METHODS

Laurie acid (Dodecanoic acid) was purchased from Fluka (UK) Ltd., Di ethylformamide (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 (lomlig; 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 Rmk-amide- resin derivatised with Fmoc-amide was subjected to the deprotection cycle with 20% piperidme m 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-ammo acids, rather than the naturally occuπng 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 ('a¹ 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 % piperidme m DMF. The respective resins were washed sequentially m DMF, DCM and diethyl ether and dried under vacuum. The dried amide resins were resuspended m 10% TFA in DCM and agitated with nitrogen gas m 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 cysteinyl peptide complexed in lOOμg of lauroyl-RGD in PBS. As controls non-acrylated peptides were also injected with lauroyl-RGD peptide. A combined intraperitoneal 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 Dividastrip, 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- amino 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 d 1/100 1/10

2. Balb/c d 1/100

3. Balb/c d 1/100

4. Balb/k k 1/10 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

Claim :

1. A component for an adjuvant, which is capable of micelle formation and comprises a peptide head group for binding to an antigen-presenting cell, and a lipophilic tail group.

2. A component according to claim 1, wherein the head group comprises a motif for binding to a receptor on the antigen-presenting cell.

3. A component according to claim 2, wherein the motif comprises a sequence of amino acids in the peptide head group .

4. A component according to claim 2 or claim 3, wherein the receptor comprises an integrin.

5. A component according to claim 4, wherein the motif comprises the amino acids R, G and D.

6. A component according to claim 5, wherein the motif comprises the L amino acid sequence RGD and the lipophilic tail group is N terminal of the R amino acid.

7. A component according to any one of claims 2 to 5, wherein the peptide motif comprises D amino acids.

8. A component according to claim 7, wherein the motif comprises the D amino acid sequence DDDGGGGGRRR and the lipophilic tail group is N terminal of the D amino acid.

9. A component according to any one of the preceding claims, wherein the lipophilic tail group comprises a Ce-C₁₂ fatty acid.

10. A component according to claim 9, wherein the fatty acid comprises lauric acid.

11. An adjuvant comprising a micelle which comprises a plurality of components according to any one of the preceding claims .

12. A vaccine composition comprising an antigen and an adjuvant according to claim 11.