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Document AM5 
Appl. No. 09/848,616 



Europaisches Patentamt 
European Patent Office 
Office europeen des brevets 



® Publication number: 



0 385 610 

A1 



EUROPEAN PATENT APPLICATION 



© Application number: 90301589.9 
© Date of filing: 14.02.90 



© int ci.s A61K 39/295, A61K 39/29, 
A61K 39/12, A61K 39/13, 
C12N 15/62, C12N 15/36, 
C12N 15/43 



Claims for the following Contracting State: ES. 


® Applicant: THE WELLCOME FOUNDATION 




LIMITED 


@ Priority: 14,02.89 GB 8903313 


Unicorn House 160 Euston Road 


London NW1 2BP(GB) 


@ Date of publication of application: 


@ Inventor: Rowlands, David John 


05.09.90 Bulletin 90/36 




The Wellcome Foundation Limited, Lang ley 


® Designated Contracting States: 


Court 


BE OH DE OK ES FR GB IT LI NL SE 


Beckenham, Kent BR3 3BS(GB) 




Inventor: Clarke, Berwyn Ewart 




The Wellcome Foundation Limited, Langley 




Court 




Beckenham. Kent BR3 3BS(GB) 




Inventor: Francis, Michael Uames 




The Wellcome Foundation Limited, Langley 




Court 




Beckenham, Kent BR3 3BS(GB) 




0 Representative: Woods, Geoffrey Corlett et al 




J. A. KEMP & CO. 14 South Square Gray's Inn 




London WC1R 5EU(GB) 



@ Conjugates. 



@ Hepatitis 8 core antigen (HBcAg) particles, which carry a polypeptide which presents an antigenic epitope 
and which are suitable for use in vaccines, are composed of HBcAg provided with a N-terminal extension 

^incorporating a Lys residue and the said polypeptide is coupled to the particles via the side chain amino group 

^of the Lys residue. 

O 
CO 

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CO 
00 

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Xerox Copy Centre 



EP 0 385 610 A1 



CONJUGATES 

This invention relates to conjugates which comprise polypeptides incorporating an antigenic determinant 
coupled to a carrier, to their preparation and to their use in raising antibodies. 

The aims of a good vaccine should be to provide a rapid onset of immunity that is of long duration and 
provides immunological memory for a subsequent inoculation or encounter with the infectious agent. The 
5 vaccine formulation must also be easy to administer, stable, have minimal side effects and produce broad 
protection in the recipient. These aims are largely met by many existing commercial products. However, 
conventional vaccines based on inactivated infectious agents do present problems. These include the 
undefined nature of the immunizing antigen, whether the product is truely innocuous, risk associated with 
handling large amounts of infectious material, stability and limitations on the mode of presentation, generally 
10 resulting from problems of stability. 

In an attempt to produce more stable and defined vaccines scientists have been studying the immune 
response to many infectious agents in detail in order to identify the critical epitopes involved in providing 
protective immunity. Armed with this knowledge it is now possible to mimic such epitopes by producing 
short peptides and to use these as the basis of a vaccine. The advantages of such peptide based vaccines 
75 are numerous. They are chemically defined, stable indefinitely and no infectious material is involved in their 
manufacture. Furthermore, they can be designed to stimulate the appropriate immune response and provide 
the opportunity for using novel delivery systems and for targetting the antigen. From the manufacturers 
viewpoint they should also reduce the need for a large scale production plant and for complex downstream 
processing of the product. 

20 Despite these clear advantages a number of criticisms have been levelled at peptide-based vaccines. 
These include the requirements for undefined carrier proteins and the belief that the immunogenicity of a 
peptide antigen could never approach that of the native organism. It was generally assumed that due to 
their relatively small molecular size many synthetic peptides would behave like haptens and would require 
coupling to a large "foreign" protein carrier to enhance their immunogenicity. Immunization with such 

25 conjugates often resulted in the production of anti-peptide antibodies that totally failed to recognise the 
native protein or infectious agent due to the method of peptide/carrier linkage. Other problems, of particular 
relevance to vaccination, that could be encountered were hypersensitivity to the "foreign" carrier protein 
and poor batch to batch reproducibility of the conjugates. 

There has recently been interest in the use of hepatitis B core antigen (HBcAg) as a carrier protein. 

30 Clarke et al. Nature, 300, 381-384, 1987 describe a fusion protein composed of the major antigenic site of 
the VPf~capsid protein of foot-and-mouth disease virus (FMDV) fused to the amino-terminus of HBcAg. The 
fusion protein self-assembled into regular 27 nm core-like particles. These particles were almost as 
immunogenic with respect to FMDV as the native virus itself. 

EP-A-0271302 claims immunogenic polypeptide conjugates comprising HBcAg operatively linked 

35 through an amino acid residue side chain to a polypeptide immunogen. It also claims immunogenic fusion 
proteins comprising HBcAg protein operatively finked by a peptide bond to a pathogen related immunogen. 
Specific T eel! stimulating polypeptides are claimed too. 

We have investigated chemically coupling polypeptides to HBcAg particles. Each time, however, 
satisfactory results could not be obtained. In particular: 

40 1. We sought to couple hepatitis B surface antigen (HBsAg) to HBcAg via side chain amino groups 

on HBcAg by derivatising the HBcAg particles with succinimidyl-4-(N-maleimido-methyl)-cyclohexane-l- 
carboxylate (SMCC). Little or no derivatisation of the HBcAg particles occurred. 

2. The approach in 1. was repeated but using m-maleimido-benzoyl-N-hydroxysuccinimide ester 
(MBS) instead of SMCC. Again, little or no derivatisation of the HBcAg particles occurred. 

45 3. Coupling of HBsAg to HBcAg particles was tried with glutaraldehyde. Coupling occurred at a high 

concentration of glutaraldehyde. However, the glutaraldehyde badly damaged the HBsAg. There was a 
severely reduced response to HBsAg when the resulting complexes were administered to mice. 

4. We sought to couple a Cys-containing FMDV VP1 1 41 -1 60 peptide to side chain amino groups on 
HBcAg using bis(maleimide)methyl ester (BMME). The HBcAg particles were reduced, derivatised with 

50 BMME and reacted with the peptide. Although coupling occurred, only low antibody responses to FMDV 
were induced by the resulting material. 

In order to overcome these difficulties, we expressed a fusion protein composed of a short Lys- 
containing sequence fused to the amino terminus of HBcAg. We then coupled peptides to this modified 
HBcAg protein via the side chain amino group of the Lys residue. Coupling proceeded successfully. The 
resulting conjugates raised both antipeptide and neutralising antibody. The method is simple, efficient and 



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EP 0 385 610 A1 



can allow the peptide to be coupled in a defined operation. 

Accordingly, the present invention provides particles which are composed of HBcAg provided with a N- 

terminal extension incorporating a Lys residue and to which are coupled via the side chain amino group of 

the said Lys residue a polypeptide which presents an antigenic epitope. 
5 Any desired polypeptide may be coupled to the modified HBcAg particles. However, present on the 

polypeptide there needs to be a group such as -NH2 or -SH capable of being coupled to the side chain 

amino group of the Lys residue in the N-terminal extension of HBcAg. The polypeptide typically presents a 

foreign epitope, i.e. an epitope which is not an epitope of HBcAg. 

The polypeptide may comprise an antigenic epitope capable of raising neutralising antibody, for 
10 example an epitope of an infectious agent such as a virus, bacterium or protozoan. It may be an epitope of 

a non-infectious agent such as a growth hormone fragment. The polypeptide may comprise repeats of an 

epitope, for example up to eight or up to four copies of an epitope. Two copies of an epitope may therefore 

be present in the polypeptide. A polypeptide may comprise two or more different epitopes, for example 

three or four. 

15 As examples of viruses whose epitopes may be presented there may be mentioned hepatitis A virus, 
hepatitis B virus, influenza virus, foot-and-mouth disease virus, poliovirus, herpes simplex virus, rabies virus, 
human immunodeficiency virus type 1 (HIV-1), HIV-2, simian immunodeficiency virus (SIV), human 
rhinovirus (HRV), dengue virus and yellow fever virus. The polypeptide coupled to the modified HBcAg may 
be therefore HBsAg or a polypeptide comprising the major FMDV VP1 antigenic site. A protozoan whose 

20 epitopes may be provided is the malaria parasite Plasmodium falciparum . 

A peptide incorporating the major FMDV VP1 antigenic site which may be employed comprises a 
sequence starting at a residue from 137 to 142 and ending at a residue from 160 to 162 of VP1 of FMDV. 
for example of any of the FMDV serotypes. Typical sequences are VPl residues 140 to 162, 140 to 160, 
137 to 162, 137 to 160 or 141 to 160. A polypeptide comprising tandem repeats of such a sequence or 

25 mixed serotype tandem repeats of such a sequence can be coupled to the modified HBcAg particles. 

Suitable peptides comprising tandem repeats of the major FMDV VP1 antigenic site are peptides 
comprising consecutive sequences, each sequence made up of no more than 30 amino acid residues and 
comprising the same immunogenic sequence. This immunogenic sequence is: 

(i) a sequence starting at a residue of from 137 to 145 and ending at a residue from 150 to 162 of 
30 VPl of FMDV sub- type Oi , or 

(ii) a sequence of corresonding amino acid residues of another subtype of serotype 0 or of a subtype 
of a different FMDV serotype. 

The peptides may therefore be tandem repeats of an immunogenic sequence- Alternatively, a peptide 
composed of consecutive sequences may be provided in which one or more of the sequences comprises 

35 additional amino acid residues not actually part of the repeated immunogenic sequence. Such additional 
amino acid residues may be present to link each immunogenic sequence. Such a linking sequence may be 
composed of up to six amino acid residues, for example from 1 to 3. Without such linking residues, each 
innmunogenic sequence is bonded directly to the next. 

The peptides can comprise any number of repeats of the immunogenic sequence. For example, from 

40 two to eight and, more preferably, from two to four repeats may be present. The peptides may or may not 
end at the C-terminal and/or N-terminal with a non-natural cysteine residue. 

The sequence comprising the immunogenic FMDV sequence consists of no more than 30 amino acid 
residues. The length of the sequence depends on the length of the immunogenic sequence although 
additional linking amino acid residues and possibly a C-terminal and/or N-terminal non-natural cysteine may 

45 be present too. Preferably, however, each consecutive sequence is no more than 26 residues long. 

The peptides present repeats of the major FMDV VPl immunogenic site. The major FMDV epitope is 
typically defined by at least amino acid residues 142 to 160 of the VPl capsid protein. This applies in 
particular to serotype O1. An immunogenic sequence which may therefore be repeated is that defined by 
VP1 amino acid residues 142 to 160 of FMDV serotype O1. optionally extending down to amino acid 137 at 

50 the N-terminal and/or up to amino acid 162 at the C-terminal. or by corresponding amino acids of another 
serotype. Typical sequences are VPl residues 140 to 162. 140 to 160, 137 to 162 or 137 to 160 of serotype 
O or A, for example of subtypes O1 and Ai2. These typical sequences may be repeated twice, for example. 
Useful peptides are. using the one-letter code: 
(1 37-1 62,Ot HI 37-1 62,Oi )-Cys: 

55 (NRNAVPNLRGDLQVLAQKVARTLPTS)x2C: 
(1 37-1 62.O1 HI 37-1 62.O1 )-Gly : 
(NRNAVPNLRGDLQVLAQKVARTLPTS)x2G: and 
(137-162,Ai2H137-162.Ai2)-Cys: 



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EP 0 385 610 A1 



(YSASGSGVRGDLGSLAPRVARQLPAS)k2C. 

Smaller immunogenic sequences of the FMDV epitope may be presented, however. For example, the 
sequence defined by VP1 residues 145 to 150 of serotype Oi may be presented in this way. Consequently, 
the FMDV sequence which can be repeated may be defined more broadly by VP1 residues 145 to 150 of 
5 serotype Oi, optionally extending down to amino acid 137 at the N-terminal and/or up to amino acid 162 at 
the C-terminal, or by corresponding amino acids of another serotype. Useful smaller peptides are: 
(145-1 50,Oi HI 45-1 60,Oi)-Cys: 
RGDLQVRGDLQVLAQKVARTLPC: and 
( 1 45- 1 50,0 1 )-( 1 45- 1 50.0 1 )-( 1 45- 1 60 .0 1 )-Cy s: 
10 RGOLQVRGDLQVRGDLQVLAQKVARTLPC. 

Suitable peptides comprising mixed serotype tandem repeats of the major FMDV VP1 antigenic site are 
represented by the formula (I): 
C'-X-Y-Z-C" (I) 

in which X represents (i) a sequence starting at a residue from 137 to 142 and ending at a residue from 
15 160-162 of VP1 of FMDV subtype Oi or (ii) a sequence of corresponding amino acid residues of another 
subtype of serotype 0 or of a subtype of a different FMDV serotype; 
Y represents a direct bond or a linking sequence of up to six amino acid residues; 

Z represents a sequence defined as for X but of a different serotype than that of the sequence denoted by 
X: and 

20 C and c " each independently represent an optional cysteine residue. 

Each immunogenic sequence X and Z is defined by VP1 amino acid residues 142 to 160 of FMDV 
serotype Oi , optionally extending down to amino acid 1 37 at the N-terminai and/or up to amino acid 1 62 at 
the C-terminal. or by corresponding amino acids of another subtype of serotype 0 or of a subtype of a 
different FMDV serotype. Typical sequences are VPI residues 140 to 162. 140 to 160, 137 to 162 or 137 to 

25 1 60. 

The immunogenic sequences X and Z are of different serotypes. There are seven FMDV serotypes: O. 
A, 0, Asia 1, SAT 1. SAT 2 and SAT 3. A useful peptide comprises Oi and A12 sequences in either order. 
The immunogenic sequences may be linked directly or via up to six, for example from 1 to 3, amino acid 
residues. Preferably, a C-terminal non-natural cysteine residue is present. Preferred peptides are, according 
30 to the one-fetter code: 

(1 37-I62.O1 )-(1 37-1 62.A1 2)-Cys: 

NRNAVPNLRGDLQVLAQKVARTLPTS-YSASGSGVRGDLGSLAPRVARQLPAS-C: and (1 37-1 62, Ai 2 )-(l 37- 
l62,Oi)-Cys: 

YSASGSGVRGDLGSLAPRVARQLPAS-NRNAVPNLRGDLQVLAQKVARTLPTS-C. 
35 A preferred polypeptide for coupling to the modified HBcAg particles comprises the HRV Nlm-ll epitope 
of EP-A-0287395. This epitope Is defined by amino acid residues 156 to 164 of VP2 of HRV2 or equivalent 
amino acid residues of another HRV. These amino acids may be replaced by other amino acids which do 
not affect the antigenicity of the sequence. For HRV2, the Nlm-ll sequence is 
VKAETRLNP. 

40 Equivalent amino acid residues of other HRVs are the VP2 amino acid residues corresponding to the 
VP2 amino acid residues 156 to 164 of HRV2. In other words, they can be the counterpart VP2 residues of 
another HRV serotype. These can readily be determined by lining up the VP2 sequence of another HRV 
with the VP2 sequence of HRV2. This is a straightforward matter because of the homology between VP2 
sequences of different types of HRV. 

45 The polypeptides may be relatively short peptides of up to 50, for example of up to 40 or of up to 30. 
amino acid residues. Alternatively they may be longer of up to. for example 100 or 200 amino acid residues 
in length. They may be proteins. They may have been obtained by chemical synthesis or by recombinant 
DNA methodologies. 

The particles to which the polypeptides are coupled consist essentially of HBcAg provided with a N- 
50 terminal extension which incorporates a Lys residue. HBcAg self-assembles into particles 27 nm in 
diameter. The modified HBcAg's also self-assemble to form core-like particles. More than one Lys residue 
may be present in the N-terminal extension. Up to six Lys residues, for example up to four Lys residues, 
may be provided. 

The N-terminal extension may have any appropriate length provided the HBcAg with the extension can 
55 self-assemble into core-like particles and provided a Lys residue in the extension is exposed available for 
coupling- The extension may be up to 250, for example up to 200 or up to 100 amino acid residues long. A 
short extension may be up to 60. for example up to 40, 20. 10 or 5, amino acid residues long. 

A suitable extension comprises residues 95 to 102, for example 95 to 104, of the VPI capsid protein of 



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EP 0 385 610 A1 



a strain of poliovirus type 1 (PV1 ) such as the Sabm or Mahoney strain: 

Sabin PV1 VP1 95-102 : SASTKNKD 

Sabin PVI VPI 95-104 : SASTKNKDKL 

Mahoney PVI VPI 95-102 : PASTTNKD 
5 Mahoney PVI VPI 95-104 : PASTTNKDKL 

This shows the sequences according to the one-letter code (Eur. J. Biochem. 138 . 9-37, 1984) in which 

K denotes Lys. These sequences span the proposed major antigenic site on PVI VPI. Another suitable 

extension comprises residues 156 to 184, for example 156 to 170. of the VP2 capsid protein of HRV2 or 

equivalent amino acid residues of another HRV. For HRV2, these residues are: 
w 156-164: VKAETRLNP 

156-170: VKAETRLNPDLQPTE 

The PVI- or HRV- derived sequences may be provided with linker sequences at either or each end, for 

example of up to 5 or of up to 3 amino acid residues. For any N-terminat extension of HBcAg, generally 

there should be at least one Lys residue within the first fourteen N-terminal residues, for example one Lys 
/5 residue within the first five or first twelve such residues or two Lys residues within the first fourteen N- 

terminal residues. For a selected polypeptide it is which to couple, it may be appropriate to choose a 

HBcAg with a N-terminal extension comprising a Lys residue close to the N-terminus of the extension. Also, 

the N-termtnal extension may desirably be hydrophilic. 

A modified HBcAg provided with a N-terminal extension comprising one of these sequences or any 
20 other sequence incorporating a Lys residue may be obtained by genetic engineering, for example according 

to JP-A-1 96299/88. More specifically, such a modified HBcAg may be obtained by providing a gene 

encoding the modified HBcAg in an appropriate expression vector and expressing the modified HBcAg in a 

host transformed with the vector. 

A gene encoding the modified HBcAg may be prepared by synthesising a DNA sequence encoding the 
25 desired N-terminal extension and. with appropriate linkers as necessary, ligating it to the 5 -end of a DNA 

sequence encoding HBcAg, The resulting DNA sequence can thus be provided in an expression vector 

under the control of appropriate transcriptional and translationa! regulatory sequences. The modified HBcAg 

is expressed in a suitable procaryotic or eucaryotic host such as a strain of E^ coli , Salmonella or yeast and 

recovered as particles. 

30 An expression vector, such as a plasmid, capable of expressing HBcAg may be employed where there 
is an appropriate restriction site at the s'-end of the DNA sequence encoding HBcAg. A DNA sequence 
encoding the desired N-terminal extension for HBcAg is inserted at that site. A fusion protein with the N- 
terminal extension linked to the amino-terminus of HBcAg is expressed. 

A suitable expression vector is plasmid pBc404. E. coli (JM 101) harbouring this plasmid was deposited 

35 at the National Collection of Industrial and Marine Bacteria, Aberdeen, GB on 9 February 1989 under 
accession number NCIB 40111. This plasmid codes for an ampicillinase gene as selection marker and 
possesses a strong bacterial promoter, tac, located upstream of the HBcAg gene. The presence of 
restriction sites EcoRI and Bam HI allows the insertion of a synthetic oligonucleotide coding for any desired 
N-terminal extension for HBcAg. 

40 Conjugates are prepared according to the invention by coupling the polypeptide presenting an antigenic 
determinant to the modified HBcAg via the side chain amino group of the Lys residue present amongst the 
final forty N-terminal amino acid residues of the modified HBcAg. This is typically achieved by reacting the 
modified HBcAg with a bifunctional reagent capable of linking to an amino group and to a sulphydryl group. 
The thus-derivatised modified HBcAg is reacted with the polypeptide which, if not possessing a free 

45 sulphydryl group, has been modified so that it does possess such a group. 

Suitable bifunctional reagents include SMCC, MBS and N-succinimidyl-3-(2-pyridyldithio)proprionate 
(SPDP). The bifunctional reagents generally include a group capable of forming a peptide link and a group 
capable of forming a disulphide or thioether link. A sulphydryl group may be provided on the polypeptide it 
is wished to couple to the modified HBcAg by the provision of a N-terminal and/or C-terminal Cys residue 

50 or by reaction of amino functions with 2-iminothiolane or the N-hydroxysucclnimide ester of 3-(3- 
dithiopyridyl)propionate or S-acetylthioglycolic acid. After reaction with S-acetylthioglycolic acid N-hydrox- 
ysuccinimide ester (SATA) deacetylation of the SATA with, for example, hydroxylamlne produces free -SH 
groups. 

In practice, the modified HBcAg particles are generally provided in a buffer such as phosphate buffer at 
55 a pH of about 7. A molar excess of the bifunctional agent in an inert solvent, for example an aprotic organic 
solvent such as dimethyiformamide. is added. Derivatisation of the HBcAg particles occurs. Excess of the 
bifunctional agent is removed, by filtration for example. Excess of the polypeptide to be coupled to the 
HBcAg particles is then added. Coupling occurs and the resulting conjugates composed of the polypeptide 



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EP 0 385 610 A1 



linked to the modified HBcAg particles are recovered, for example by filtration. 

The modified HBcAg core particles may be carboxymethylated before use. The purpose of this is to 
block sulphydryl groups on the core particles prior to derivatisation and therefore to prevent core proteins 
from cross-linking when a bifunctional reagent is used to couple the N-terminai side chain amino group on 

5 the core particles to a -SH group on a polypeptide. Carboxymethylation can be achieved by reacting the 
core particles with iodoacetamide. Excess reagent can be separated by gel filtration or dialysis. 

The conjugates are useful for raising antibody to the antigenic determinant presented by the polypep- 
tide linked to the HBcAg particles. The conjugates therefore may be used as vaccines for humans or 
animals. Vaccination is achieved by administering to a human or animal an effective amount of the 

70 conjugate. An oral route or a parenteral route such as sub-cutaneously. intravenously or intramuscularly 
may be adopted. Typically, the conjugate is administered in an amount of 1 to 1.000 ug per dose, more 
preferably 10 to 100 ug per dose, by either the oral or the parenteral route. 

For administration, a conjugate is typically formulated with a pharmaceutically acceptable carrier or 
diluent Conventional formulations, carriers, adjuvants and diluents may be employed. These will of course 

15 be determined by the route of administration. Suitable carriers and diluents include Freund's incomplete 
adjuvant (IFA), aluminium hydroxide, saponin, DEAE-dextran. muramyl dipeptide, mineral oils, neutral oils 
such as miglycol, vegetable oils such as arachis oil. "Iscoms", liposomes, Pluronic (trade mark) polyols or 
the Ribi adjuvant system (GB-A-2189141). 

The following Examples illustrate the invention. In the accompanying Figure plasmid pBc404 is shown, 

20 B. E and P denote restriction sites for Bam HI, EcoRI and PstI respectively; tac denotes the tac promoter; ori 
denotes the origin of replication; bla denotes j8-lactamase and SO denotes the Shine-Dalgarno sequence. 



Example 1: Preparation of HBcAg provided at its N-terminus with a short extension comprising PV1 
25 Mahoriey VP1 residues 95"To 1 04 {PV core) 

An expression plasmid for PV core was prepared, based on the parent plasmid pBc404 shown in the 
Figure. Synthetic oligonucleonucleotides representing amino acids 95 to 104 of VP1 from PV1 Mahoney 
were ligated into p8c404 using T4 ligase by standard procedures. The synthetic oligonucleotides, how they 
30 anneal together and the coding sequence of the N-terminal extension are as follows: 

*!• AATTCAGATAATCCGGCTAGTACTACCAACAAAGATAAG (39) 
2 . GATCCTTATCTTTGTTGGTAGTACTAGCCGGATTATCTG ( 39 ) 

35 



AATTCAG ATAATCCAGC TAGTACTACC AACAAAGATA AG 

GTC TATTAGGTCG ATCATGATGG TTGTTTCTAT TCCTAGG 



10 20 30 40 

ATGAATTCAGATAATCCAGCTAGTACTACCAACAAAGATAAGGATCC core 

MNSDNPASTTNKDKD 

I 11 1 

LINKER POLIOVIRUS 

Bacteria (E. coli JM 101) hartX5uring the recombinant plasmid were induced for expression using 60 
ug/ml of isopropyl-iS-D-thiogalactopyranoside (IPTG) for 6 hours. Cells were harvested by centrifugation, 
lysed by lysozyme and non-ionic detergent treatment. Bacterial debris was removed by centrifugation at 
10000 rpm for 5 minutes. 



45 



50 



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EP 0 385 610 A1 



Supernatant samples were fractionated on 15-45% linear sucrose gradient at 50000 rpm for 1 hour 15 
minutes at 20* C. Particles were detected by optical density at 260 nm. The particles were concentrated by 
centrifugation at 40000 rpm for 1 hour or used directly for derivatisatlon after dialysis of sucrose. 

5 

Example 2: Coupling of the peptide composed of FMDV VP1 residues 141-160. which has also a C-terminal 
Cys residue. (FMDV Ml^OCyin o PV cores 

PV cores, purified on two consecutive sucrose gradients, were passed at a concentration of 5 mg/ml in 
10 10 mM phosphate buffer of pH 7.2 through a Sephadex (trade mark) G100 column. The PV cores in 10 mM 
phosphate buffer at a concentration of 2 mg/ml were then derivatised by adding 1/20 volume of SMCC 
dissolved in dry dimethylformamrde so that the final concentration of SMCC in the PV core sample is a 50 
times molar excess relative to the PV core protein. 

After 30 minutes at room temperature, the SMCC was removed by filtration through Sephadex G 100 in 
75 10 mM phosphate buffer, pH 7.2. Freshly dissolved FMDV 141-180 Cys was added in 1/10 volume to give a 
10 times molar excess of the peptide with respect to the derivatised PV cores. After stirring at room 
temperature for 2 hours, uncoupled peptide was separated from the derivatised PV cores by filtration 
through Sephadex G200. The PV cores with the peptide attached were recovered. 

20 

Example 3: Coupling of HRV2 peptide to PV cores 

Following the procedure described in Example 2, the HRV2 peptide which encompasses the NIm-ll 
epitope and which has the sequence: 
25 VKAETRLNPDLQPTC 

was coupled to PV cores. The PV cores with peptide attached were recovered. 



Example 4: Carboxymethylation of PV cores 
30 ~ 

PV cores after dialysis of sucrose and concentration by centrifugation were reacted in an amount of 200 
ug/ml with 10mM lodoacetamide in 0.5M Tris, pH 8.0, for 1 hour at room temperature in the dark. Excess 
reagent was separated from the thus carboxymethylated PV cores by gel filtration. 

35 

Example 5: Coupling of hepatitis B surface antigen (HBsAg) to carboxymethylated PV cores 

Carboxymethylated PV cores from Example 4 were derivatised with an equimoiar amount of SMCC in 
dimethylformamide (DMF). The amount of SMCC was one-twentieth the volume of DMF. HBsAg particles 
40 expressed in yeast were derivatised at 200 ug/ml with S-acetylthioglycolic acid N-hydroxysuccinimide ester 
(SAT A), again at equimoiar ratios in DMF. The amount of SATA was also one-twentieth the volume of DMF. 
After deacetylation of the SATA with hydroxylamine to produce free -SH groups, equal volumes of the two 
derivatised particles, each of 200 u.g/ml, were mixed. The PV cores with HBsAg attached were recovered. 

45 

Example 6: Coupling of FMDV peptide and HRV2 peptide to carboxymethylated PV cores 

The FMDV 141-160 Cys peptide was coupled to carboxymethylated PV cores in accordance with 
Example 5. In a separate experiment, the HRV2 peptide described in Example 3 was coupled in the same 
50 way to carboxymethylated PV cores. In both cases, however, derivatisatlon was effected In 50m M 
phosphate buffer. pH 7.8, followed by transfer to 50mM phosphate buffer, pH 6.8, as quickly as possible 
after derivatisatlon to stabilise the maleimide group. 

5S Example 7: Test results analysing the coupling of FMDV peptide and of HRV2 peptide to PV cores 

Tests were carried out to analyse the PV cores with FMDV peptide attached, obtained in Examples 2 
and 6, and with HRV2 peptide attached, obtained in Examples 3 and 6, as follows: 



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EP 0 385 610 A1 



(a) Polyacrylamide gel electrophoresis. This showed that all of the core protein had been shifted to a 
higher molecular weight due to the addition of peptide. 

(b) Western blotting. Following gel electrophoresis proteins were transferred to nitro cellulose and 
reacted with anticore or antipeptide antisera. The results showed that the higher molecular weight forms of 

5 the protein appearing after coupling reacted with both antisera as expected. 

(c) ELISA. 

(i) Assays were performed by a sandwich method in which anticore antiserum bound to the ELISA 
plate was used to trap derivatised core particles. The tests were developed following reaction with 
antipeptide antiserum. These showed that the derivatised particles had both core and peptide antigenicity. 

10 (ii) Titration by ELISA of anti-HRV2 peptide antiserum against fusion core particles where the HRV2 

peptide sequence was fused to the N-terminus of the core protein and against PV cores to which the HRV2 
peptide was coupled gave similar results. As equal amounts of the two particles were used in the assay, the 
antigenic activity of the N-terminal fusion particles and the chemically coupled particles was substantially 
the same. The anti-HRV2 peptide antiserum was antiserum raised against the HRV2 peptide attached in 

15 Examples 3 and 6. 

(d) Sucrose density gradients. This analysis showed that coupling of the FMDV peptide to cores 
resulted in particle aggregation. They pelleted to the bottom of the tube. PV cores coupled with HRV 
peptide sedimented to the same position as untreated cores and had both core and peptide immunogenicity 
by ELISA. 

20 (e) Electron microscopy. PV cores coupled to HRV2 peptide have been examined and are regular 

looking particles. Electron microscopy of immune complexes formed between PV cores coupled to HRV2 
peptide and anti-HRV2 peptide antiserum showed that the core particles were linked by antibody, 
(f) Immunogenicity. 

(i) FMDV peptide coupled to cores gave neutralizing activity with <20ug of coupled material (<2ug 

25 peptide). 

(ii) The immunogenic activity of the HRV2 peptide N-terminal fusion core particles and the 
chemically coupled particles was compared in guinea pigs using 20 ug doses of each antigen. The antisera 
were tested at various times post-injection by ELISA against the HRV2 peptide. The results were: 



30 





Day 


Chemically 
coupled core 


fusion 
core 




0 


<1 


<1 




14 


2.9 


2.3 




28 


3.9 


3.3 




56 


3.9 


4.0 


boost 










63 


4.1 


3.9 




70 


4.1 


3.9 




84 


4.2 


3.9 



The antigens were injected intramuscularly in incomplete Freunds adjuvant. 
Example 8: Preparation of FMDV tandem repeat peptides 



The peptides shown below were synthesised by the solid-phase method. More specifically, synthesis 
was carried out using an adaption of the Merrifield technique (Merrifield, J ACS 85 2149-2154, 1983) 
described by Houghten (Houghten. Proc. Natl, Acad. Sci. USA 82 5131-5135. 1985).'Each peptide has an 
additional non-natural cysteine residue at its C-terminus except for peptide 199 which has a C-terminal non- 
natural glycine residue. 

Each peptide was synthesised on a p-methyl-benzhydrylamine divinylbenzene resin. The alpha-amino 
protecting group on each amino acid was t-butoxycarbonyl (Boc). Each coupling cycle was as follows: 

1. Wash resin with dichloromethane - 10 minutes 

2. Wash with 5% diisopropylethylamine in dichloromethane - 2 minutes x 3 

3. Dichloromethane wash - 1 minute x 2 

4. Couple t-butoxycarbonyl amino acid dichloromethane, 0.3M diisopropylcarbodiimide - 60 minutes 



8 



EP 0 385 610 A1 



5. As 3 

6. Deprotect with 50% trifluoroacetic acid in dichloromethane - 20 minutes 

7. Dichloromethane wash - 1 minutes x 6 

8. Return to 2. 

When the coupling cycles were completed the peptide was cleaved off the resin using hydrogen 
fluoride for 1 hour with an anisole scavenger 10%. The peptide was thus obtained with a carboxy-terminal 
amide group. It was then ether washed, dried, dissolved in 15% acetic acid and lyophilized. 



VIRUS PEPTIDE 



FMDV 
FMDV 

FMDV 

0,/0, 

FMDV 



137-162-137-162-Cys 
\ 0, 

( NRNAVPNLRGDLQVLAQKVARTLPTS )^ 2 C 
137-162-137-162-Gly 

Ox Oi 

( NRNAVPNLRGDLQVLAQKVARTLPTS ) ^ ^ G 

137-162-137-162-Cys 

Ox 0, 

( NRNAVPNLRGDLQVLAQKVARTLPTS )^^C 
1 37-162-1 37-162-Cys 



A, 2 B/A, , B A 



1 2 



'1 2 



( YSASGSGVRGDLGSLAPRVARQLPAS 
FMDV 14 5-150-14 5-1 50-1 4 5-160-Cys 

Ox/Ox/Ox 0, 0, 0, 

RGDLQVRGDLQVRGDLQVLAQKVARTLPC 
14 5-150-145-160-Cys 



FMDV 
Ox/Ox 



^1 1 

RGDLQVRGDLQVLAQKVARTLPC 



REFERENCE 
NUMBER 

198 



199 



315 



318 



112 



113 



Example 9: Preparation of FMDV mixed serotype tandem repeat peptides 

The peptides shown below were synthesised by the solid-phase method described in Example 8. 



9 



EP 0 385 610 A1 



VIRUS 

FMDV 
0,/A,,B 



FMDV 
A,,B/0, 



PEPTIDE 

13 7-162-137-162-Cys 
0, A,,B 

NRNAVPNLRGDLQVLAQKVARTLPTS- ' 
YSASGSGVRGDLGSLAPRVARQLPAS-C 
137-162-1 37-162-Cys 
A,,B 0, 

YSASGSGVRGDLGSLAPRVARQLPAS- 
NRNAVPNLRGDLQVLAQKVARTLPTS-C 



REFERENCE 

NUMBER 

316 



317 



Example 10: Preparation of HBcAg provided at its N-terminus with an extension connprising HRV2 VP2 
residues 1"56 to 170 (HRV2 core) 

To construct chimaeric fusion particles coding for a specific N-terminal HRV2 VP2 epitope comprising 
VP2 residues 156 to 170, synthetic oligonucleotides with cohesive ends for EcoRI (5) and BamHI (3) 
respectively were prepared using an Applied Biosystems 381 A DNA synthesiser. These oligonucleotides 
were ligated Into EcoRI-BamHI digested pBc404 which had been purified from 1% low melting point 
agarose by standard methods (Francis and Clarke, Meth. Enzymology 178 . 659-676, 1989). This DNA was 
then transformed into E. coli strain JM1 01 , and recombinant plasmids were restriction mapped from small 
scale DNA preparationsTfhe synthetic oligonucleotides, how they anneal together and the coding sequence 
of the N-terminal extension are: 



10 



EP 0 385 610 A1 



1 . AATTCAGTTAAAGCGGAAACGCGTTTG 

2 . AACCCAGATCTGCAACCGACCGAATGCCGG 

3 . GATCCCGGCATTCGGTCGGTTGCA 

4 . GATCTGGGTTCAAACGCGTTTCCGCTTTAACTG 



AATTCAG TTAAAGCGGA AACGCGTTTG AACCCAGATC TGCAACCGAC 
GTC AATTTCGCCT TTGCGCAAAC TTGGGTCTAG ACGTTGGCTG 
CGAATGCCGG 
GCTTACGGCC CTAG 

30 

ATG AAT TCA GTT AAA GCG GAA ACG CGT TTG AAC CCA GAT 
MNSVKAETRLNPD 

I I 1 

LINKER HRV2 VP2 EPITOPE 

60 

CTG CAA CCG ACC GAA TGC CGG GAT CC core 

LQPTECRD 



Bacteria harbouring recombinant plasmids were grown overnight in L-Amp medium to high cell density 
and diluted with fresh L-broth (1:10) the following day. Expression was routinely induced by the immediate 
addition of IPTG (60 ng/ml final concentration) and the bacteria allowed to replicate for a further 6-8 hr at 
37 'C. Bacteria were then harvested and chimaeric core particles, with N-terminal peptide epitopes, purified 
and characterised as previously described (Clarke et a[, Nature 330 . 381-383. 1987; Francis and Clarke. 
1989). 

Example 11: Coupling of HBsAg to HRV2 cores 

HRV2 cores from Example 10 were derivatised with an equimolar amount of SMCC in DMF. The 
amount of SMCC was one-twentieth the volume of DMF. Yeast-expressed HBsAg were derivatised at 200 
ug/ml with SATA in equimolar ratios of DMF. The amount of SATA was one-twentieth the volume of DMF. 
After deacetylation of the SATA with hydroxylamine to produce free -SH groups, equal volumes of the two 
derivatised particles, each of 200 ug/ml, were mixed. The HRV2 cores with HBsAg attached were 
recovered. 



Claims 

1. Hepatitis B core antigen (HBcAg) particles which carry a polypeptide which presents an antigenic 
epitope, characterised in that the HBcAg particles are composed of HBcAg provided with a N-terminal 
extension incorporating a Lys residue and the said polypeptide is coupled to the particles via the side chain 
amino group of the Lys residue. 

2. Particles according to claim 1, wherein the polypeptide comprises an antigenic epitope capable of 
raising neutralising antibody. 

3. Particles according to claim 2, wherein the epitope is an epitope of a virus, bacterium or protozoan. 



11 



EP 0 385 610 A1 



4. Particles according to any one of tin© preceding claims, wherein the polypeptide is up to 50 amino 
acid residues long. 

5. Particles according to any one of the preceding claims, wherein the N-terminal extension is up to 60 
amino acid residues long. 

5 6. Particles according to any one of the preceding claims, wherein the N-terminal extension comprises 
residues 95 to 102 of the VP1 capsid protein of a type 1 poliovirus or residues 156 to 164 of the VP2 
capsid protein of human rhinovirus (HRV) type 2 or equivalent amino acid residues of another HRV. 

7. Particles according to any one of the preceding claims, wherein the HBcAg provided with the N- 
terminai extension is carboxymethylaled. 

10 8. A process for the preparation of HBcAg particles which carry a polypeptide which presents an 
antigenic epitope, characterised in that the polypeptide is coupled to HBcAg particles, which are composed 
of HBcAg provided with a N-terminal extension incorporating an exposed Lys residue, via the side chain 
amino group of the Lys residue. 

9. A process according to claim 8. in which the HBcAg provided with the N-terminal extension is 
15 carboxymethylated prior to coupling of the polypeptide. 

10. A pharmaceutical composition comprising a pharmaceutically acceptable carrier or diluent and, as 
active principle, HBcAg particles as defined in any one of claims 1 to 7 or which have been prepared by a 
process, as claimed in claim 8 or 9 which carry a polypeptide which presents an antigenic epitope. 

20 Claims for the following Contracting State: ES 

1. A process for the preparation of HBcAg particles which carry a polypeptide which presents an 
antigenic epitope, which process comprises the step of coupling the polypeptide to HBcAg particles, which 
are composed of HBcAg provided with a N-terminal extension incorporating an exposed Lys residue, via the 

25 side chain amino group of the Lys residue. 

2. A process according to claim 1 , in which the HBcAg provided with the N-terminal extension is 
carboxymethylated prior to coupling of the polypeptide. 

3. A process according to claim 1 or 2, wherein the polypeptide comprises an antigenic epitope capable 
of raising neutralising antibody. 

30 4. A process according to claim 3, wherein the epitope is an epitope selected from an epitope of a 
virus, an epitope of a bacterium and an epitope of a protozoan. 

5. A process according to any one of the preceding claims, wherein the polypeptide is up to 50 amino 
acid residues long. 

6. A process according to any one of the preceding claims, wherein the N-terminal extension is up to 
35 60 amino acid residues long. 

7. A process according to any one of the preceding claims, wherein the N-terminal extension is 
selected from an extension which comprises residues 95 to 102 of the VP1 capsid protein of a type 1 
poliovirus and an extension which comprises residues 156 to 164 of the VP2 capsid protein of human 
rhinovirus (HRV) type 2 or equivalent residues of another HRV. 

40 8. A process according to any one of the preceding claims, further comprising forming a pharmaceutical 
composition by formulating a pharmaceutically acceptable carrier or diluent with the said HBcAg particles 
carrying the polypeptide which presents an antigenic epitope which have been obtained. 



45 



SO 



55 



12 



EP 0 385 610 A1 




European Patent 
Office 



EUROPEAN SEARCH REPORT 



Application Number 



EP 90 30 1589 



DOCUMENTS CONSIDERED TO BE RELEVANT 



Category 



Citation of document with indication, where appropriate, 
of relevant passages 



Relevant 
to claim 



CLASSIFICATION OF THE 
APPUCATION Ont. CI.5) 



D,Y 



D,Y 



P,Y 



EP-A-0 271 302 (SCRIPPS CLINIC AND 
RESEARCH FOUNDATION) 
* Page 3, lines 10-19; page 6, line 56 
- page 8; page 14, lines 33-39; claims 



"A 



SCIENCE, vol. 233, 25th July 1986, 
pages 472-475; F. DELPEYROUX et al 
poliovirus neutralization epitope 
expressed on hybrid hepatitis B surface 
antigen particles" 

* The whole document * 

NATURE, vol. 330, no. 6146, 26th 
November - 2nd December 1987, pages 
381-384, London, GB; B.E. CLARKE et 
al.: "Improved immunogenici ty of a 
peptide epitope after fusion to 
hepatitis B core protein" 

* The whole document * 

EP-A-0 287 395 (THE WELLCOME 
FOUNDATION LTD) 

* Page 3, line 62 - page 4, line 4; 
claims * 



EP-A-0 174 759 
LABORATORIES) 



(CONNAUGHT 



METHODS IN ENZYMOLOGY, vol. 178, 1989, 
pages 659-676, Academic Press, Inc.; 
M.J. FRANCIS et al . : "[42] peptide 
vaccines based on enhanced 
immunogenicity of peptide epitopes 
presented with T-cell determinants or 
hepatitis B core protein" 
* The whole document * 



The present search report has been drawn up for all claims 



1-10 



6,7,10 



1-10 



1-10 



1-10 



A 
A 
A 
A 

C 
C 
C 



61 K 
61 K 
61 K 
61 K 
12 N 
12 N 
12 N 



39/295 

39/29 

39/12 

39/13 

15/62 

15/36 

15/43 



TECHNICAL FIELDS 
SEARCHED ant. C1.5) 



A 61 K 

C 12 N 



Place of search 

THE HAGUE 



Date of conqtleUm of the seardi 

06-06-1990 



SKELLY J.M. 



CATEGORY OF QTED DOCUMENTS 

X : panicularly relevant if taken alone 

Y : particalarly relevant If combined with another 

document of the same category 
A : technolo^cal badLground 
O : non-written disclosure 
P : Intermediate document 



T : theory or principle underlying the invention 
E : earlier patent document, bat published on, or 

after the filing date 
D : document cited in the application 
L : document cited for other reasons 

& : member of the same patent family, corresponding 
document